RECOMBINANT DNA (GENETIC ENGINEERING)

 

 

 

J O Ogunranti

Professor of Anatomy

University of Jos, Jos

 

 

 

 

 


 

 

 

 

 

 

1. Introduction

Recombinant DNA is a bona fide part of molecular biology. What is molecular biology? This is the study of genes and gene products. It is the study of the chemistry of the substance that gives life. Genes are made up of deoxyribonucleic acids and hence the study of nucleic acid is central to the study of molecular biology. Recombinant DNA therefore is the study of DNA and how to manipulate it in order to provide sequence of genes which will give useful knowledge about synthesis of such genes, synthesis of complementary DNA, cloning or amplification of the synthesized gene, use of the gene for translation in vitro to produced its peptide products which may be useful therapeutically, biologically or agriculturally and development of gene probes for diagnosis of the presence of genes in embryos, fetuses and adults. It also includes the breaking of genes at sequence specific sites and the use of such for other manipulative purposes as enumerated above. Finally it includes the injection of genes previously manipulated or synthesized for injection into either somatic or germ cells for incorporation into the genome and therefore the alteration of genetic makeup of the individual cell or the entire organism. The entire goal and desire of recombinant DNA is to provide a strategy for treatment of genetic conditions, maximisation of animal production using gene injection; change in genome of agricultural products to provide more germ resistant transgenic varieties of crops or animals, production of blood products, factors or hormones (such as human insulin, somatostatin etc), design of new drugs after the synthesis of its receptors from its genes, and the exploitation of the human genome project with the use of numerous known human genes to synthesize new peptide products for treatment of diverse disease conditions in man. Hence it is called genetic engineering by the lay public.

 

Figure 1. Some of the applications of DNA technology and how these are approached (Emery 1981).

 

History of recombinant DNA

The history of recombinant DNA technology can be said to have begun with the Father of modern genetics himself, Father Gregor Mendel (1822-1884 ). This monk was the first to use the word genes and hence left the modern world a useful concept to build on for it was indeed only a concept at the tie of the holy monk. After Mendel came the medical student Sutton who proposed the chromosome theory of inheritance. Sutton suggested that the chromosomes carried the genes of Father Mendel. He proved his point by showing the methods of replication of chromosomes during cell division and insisted that only the behaviour of chromosomes can adequately explain what becomes of the genes which are passed into offsprings as was clearly shown by father Mendel.

 

 

Avery and his associates first suggested that genetic material was carried in nucleic acids which had just been discovered in the 1940s. These were acids found only in the nucleus hence the name nucleic acids. The acids were known to be of two types- deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). This ended the previously theory which had suggested that genes were carried in proteins.  The structure of the DNA and the way and manner by which it replicates itself just as the chromosomes in which it is embedded still remained a mystery until Watson and Crick (1953) suggested the double helix model of DNA structure following the Xray studies of Wilkins published in April 1955 in Nature as Molecular structure of Nucleic acids- Nature 171, 737-738.

 

Modern molecular biology therefore started with Watson and Crick in 1953. These two scientists proposed the double helix model of DNA structure and also showed the backbone of the DNA to be nucleotide bases which link to each other to form a continuous chain of nucleotide bases. The bases can be either purines or pyrimidines. They are attached to a pentose sugar, and a phosphate radical. The bases pair in definite method in what is now known as hybridisation (the formation of two chains linked together by bonded purine and pyrimidine bases). Adenine pairs with thymine (A-T) while cytosine pairs with guanine (C-G). The pentose sugar can be either ribose sugar (with hydroxyl group present) or a deoxyribose sugar (with the absence of hydroxyl group).

 

 

Table :  Nitrogenous bases and their equivalent nucleosides and the abbreviations used for their nucleotides e.g. AMP= adenosine monophosphate etc. (‘d’ represents the deoxy form in DNA). After Emery A, 1984, p 15.

Base

Nucleoside

Abbreviation of nucleotide

DNA

RNA

Adenine

Adenosine

dAMP

AMP

Guanine

Guanosine

dGMP

GMP

Cytosine

Cytidine

dCMP

CMP

Thymine

Thymidine

dTMP

-

Uracil

Uridine

-

UMP

 

Berg papers

 

The arrival of the technology of Recombinant DNA did not come without challenge from scientist and public alike. The first indication that research into molecular biology of the gene might pose problems of biohazards came from a scientific meeting at Cold Spring Harbor in 1971. In this conference an experiment was described in which portions of the DNA from a tumor virus SV40 was to be cloned in the e coli. This immediately drew  curious listeners at the scientific meeting who became interested in the effect this might have on health since E coli was a normal commensal of the human intestine and the development of a laboratory pathogenetic or tumor inducing species by this form of genetic engineering might be a fatal development. Berg from whose laboratory the proposed study was to be carried out then called attention to the biohazards of genetic engineering in his famous letter of 1974 which was signed by 10 other scientists (Berg et al 1974). Scientific committees were then set up to look into the matter of biohazards and an Ashby report was published in the United Kingdom in 1975and Asiomar communique also issued. From the Asilomar communiqué it became necessary to provide biological and physical containment of molecular biological experiments that were potentially hazardous. The idea was to develop certain microorganisms which cannot survive outside the laboratory for the various experiments on molecular biology such as E coli K12 strain 1775 which is unable to synthesize an important cell wall component that would require to be supplied to it in laboratory culture media without which it would not grow. Hence it would be impossible for an infection of this strain to occur outside the laboratory.

 

In July 1976, Mayor Welluci opened up another debate on genetic engineering in Cambridge, Mass. However, the opponents of the molecular biological technology lost to the overwhelming majority who felt the experiments and technologies were safe enough in expert hands and could therefore constitute no biohazard.

 

2. Gene

The gene forming the actual DNA is supercoiled within the chromosome . A gene is in most cases responsible for the synthesis of proteins or translation of a polypeptide.   In translation, a triplet of bases which codes for an amino acid is called a codon while the sequence of codons responsible for the synthesis of a specific polypeptide is called a cistron. The length of a gene will be much longer than that of  whole chromosome because of its supercoiling within that chromosome or its segments. There are 4 categories of coiling which are recognised

 

A gene is a sequence on a chromosome containing polynucleotide bases supercoiled on the chromosome. It therefore has sequence of polynucleotide bases specific for it. It is measure in the number of bases which is found in its entire length – either as kilobase pairs (or kilobases-kb for short) or base pairs if the gene is not too long. 1 kb is 1000 bp. For example a gene with 5 base pairs could run as follows

A-T

C-G

T-A

A-T

A-T

G-C

Some long genes may run for 300kb

A enzyme called DNA topoisomerse helps to uncoil the gene . A gene could be also circular or superhelix in structure

 

3. Types of DNA

Repititive DNA: These are sequences of varying length which occur repeatedly throughtout the  genome. All eukaryotes possess repetitive DNA. In man it is said that 40% of DNA material are repeated. Repititive DNA fall into two classes- middle repititive and highly repititive. Middle repetitives make up 30% of all DNA. It includes genes for ribosomal DNA, transfer RNA, histones and immunoglobulins. Highly repetitive DNA forms only 10% of all DNAs. It is founding satellite DNA located around the centromere of chromosomes. Best known highly repetitive DNA are Alu, Hinf and EcoRI. Each Alu sequence is about 300bp and it has over 300,000 copies interspersed at random in haploid genome while EcoRI and Hinf have about 300bp which exist in two subunits. They occur as tandem repeats.

Introns and Exons: Intervening sequences between genes are called introns. These are sequences which do not translate into any type f protein or peptide amino acids and are therefore useless to the gene. They are not included in complementary DNA formation and not also in mRNA. Exons on the other hand, are the actual sequences which help to form codons that translate into amino acids of the peptide product which the gene in question expresses. Electron microscopy can be used for the study of introns and exons. They are utilised to visualise hybrids of DNA-DNA strands and DNA-RNA strands in the technique called heteroduplex mapping.

 

Numbers of introns in various human genes

Gene

Number

Mitochondrial

0

Histones

0

F interferons

0

Insulin

2

Globins

2

Ig light chains

3

A1 antitrypsin

4

Actin

5

Ig heavy chain

7

HLA (A,B,C)

7

α fetoproteins

13

Collagen

50

 

Almost all genes in eucaryotic cells are split by introns. In man the only exceptions may be histones, mitochondrial and certain interferon genes. The functions of introns have been suggested to be gene regulation.

 

Alternative splicing

Certain genes have alternate sequences. For example genes which encode calcitonin and genes for calcitonin gene related peptide.

 

Pseudogenes

These re sequences which do not yield any recognizable gene products

 

DNA polymorphism

These are variations in nucleotide sequences of a particular gene which have no phenotypic effects on the individual . Through linkage with genes for serious inherited disorders, they are proving useful in carrier detection and prenatal diagnosis.

 

Multigene families

Certain traits are controlled by several genes with related functions which occur either on the same chromosome (e.g. the α interferon genes on chromosome 9 and the major histocompatibility gene complex on chromosome 6 or two different chromosomes (eg the α globin and β globin related gene loci on chromosomes 16 and 11 respectively on several different chromosomes (eg argininsuccinate synthetase is dispensed over at least 8 autosomes and one sex chromosome.

 

Transposons

In early 1950s Barbara McClintock (Calos MP Miller JH (1980) Cell 20, 579, reported that certain genes in maize are responsible for mottling of seed colour which moved around the genome and were referred to as controlling elements.

 

Table :  Some examples of mobile genetic elements (transposons) in various organisms

Organism

Transposon

Function

Bacteria

Insertion sequences (IS)

-

 

Transposons (Tn)

Antibiotic resistance

Yeast

‘Cassettes’

Mating types

Maize

Controlling elements

Seed colour

Drosophila

Copia elements,

P elements, etc

Various

 

 

Mammals

Provirus

Retrovirus sequences

Cellular oncogenes

 

 

Gene mapping

A given point on a chromosome is designated by the chromosome number, the arm symbol m the region number an hand number in that order (See Cytogenetics and Cell genetics 1978, 21: 313. For example the formula for the gene for Duchenne muscular dystrophy is Xp21.

 

Methods by which genes are mapped or localised on chromosomes vary and are as follows

1. The use of recombination frequencies as a result of gene linkages following form crossing over phenomenon during meiosis. During meiosis there is crossing over which occurs at pachytene of the first meiotic prophase and there is therefore  exchange of homologous materials. When two different genes are located on the same chromosome pair they are said to be linked. Crossing over is more likely to occur between genes which are far apart. Conversely, the closer the two genes are on any particular chromosome the less likelihood  that crossing over will occur. We can assess the relative distances between genes on any particular chromosome by determining the frequency with which crossing over occurs between these genes. Thus if 5% of the offspring of informative matings are of recombinant type (i.e. result from crossing over) then the two gene loci are said to be about 5 map units apart.

 

The relative distance between genes on any chromosome is therefore measured by the frequency with which crossing over occurs between the genes. These distances are measured in map units. One map unit is equivalent to 1% crossing over. It is also called centiMorgan. 1 centimorgan is roughly equal to 1000kb

2. In situ hybridisation (see below)

3. Somatic cell hybrids. Cells from a human and rodent are fused together (using a virus or polyethylene glycol) to produce somatic cell hybrids. Correlations are made between various histochemical markers exhibited by the hybrid cells in tissue culture and the remaining human chromosomes in the cells.

4. Recombinant DNA label probes used to localise gene loci in chromosomes.

 

3.  DNA or gene sequencing

DNA sequencing methods were designed independently by two groups of experimentalists working in Cambridge UK and Harvard in 1977. Sanger and Coulson are from Cambridge and Maxam and Gilbert (1977). Their methods differ but are nevertheless able to arrive at the same conclusion.

Why do we sequence genes. The following can be given as reasons why we sequence genes.

  1. To learn how to synthesize genes. For example, if eh nucleotide base sequence of a gene is known, then they can be put together to synthesize the gene in vitro.
  2. Such information can be placed in computer data banks and this can be access by anyone who may need the information for various recombinant DNA reasons.

 

Sanger’s method.

Sanger designed his method while working in Cambridge. In his method dideoxynucleotides are incorporated into DNA molecules to be sequenced. They differ from the deoxynucleotides in that they do not have the hydroxyl group in position 3. In view of the absence of OH at position 3, wherever there is incorporation of dideoxynucleotide there will be a break in the nucleotide chain since it is the position 3 hydroxyl group that makes the linkage with other nucleotide bases. Hence several fragments of the DNA are obtained by this method. These fragments are then subjected to electrophoresis using an electric current source.

 

 

The Chain Termination Method for Determination of the Nucleotide Sequence in DNA. After Gaastra Wim and Oudega Bauke, Determination of DNA sequences, In Walker JM and Gaastra Wim eds. Techniques in Molecular Biology London: Croom Helm, 1983, p.292.

 

Using template mobility patterns, mobility of fragments to be sequences are then determined and compared with the template mobility patterns. Based on the principle that similar fragments must  have similar sequences, hence sequences can be determined using the template mobilities in electrophoresis.

 

Maxam and Gilbert method

Maxam and Gilbert in Havard utilised chemical compounds to break nucleotides bonds rather than dideoxynucleotide molecules. These compounds have the property of breaking the DNA at different sites on the chain. Thus OH and (CH3)2SO4 can break at different sites in the DNA molecule. The resulting fragments are then subjected to electrophoresis from where their sequences are determined.

Note: In this case the sequence read from the autoradiogram is the actual sequence of the DNA fragment.

 

Schematic diagram of the Maxam and Gilbert Method for sequencing.  After Gaastra Wim and Oudega Bauke, Determination of DNA sequences, In Walker JM and Gaastra Wim eds. Techniques in Molecular Biology London: Croom Helm, 1983, p.302

 

 

4. Protein techniques in molecular biology

Proteins which are the products of gene expression are required to be studied in molecular biology

High performance liquid chromatography (HPLC). This is used to separate large proteins and other complex mixtures of molecules. It achieves separation very rapidly, and only very small quantities of molecule are required. Fractionated molecule are detected by the monitoring of their ultraviolet absorbance.

SDS (sodium dodecyl sulphate) -polyacrylamide gel electrophoresis: When a protein profile of a tissue is to be determined, the tissue may be subjected to SDS-PGE. The technique is dependent on the electrophoretic properties of the molecules is also dependent on the electrophoretic properties of the molecules and also on the physical properties of the protein contained in the tissue. These include isoelectric focussing with the degree of electrical charges and molecular weight.

 

The gel may be uni, multi or bi dimensional. The gel used for electrophoresis made up of 2 compounds mainly

i.                     The acrylamide compound

ii.                   Criss cross linkage compound called bis.

 

The cross linker helps in the polymerization of the acryl amide on the gel. The acrylamide molecule forms polyacrilamide after several polymerizations. The substance to be determined is the passed through the electrophoretic gel, where depending on molecular size, the mobility becomes varied- ie those with heavy molecular weight pass slower than those with lighter molecular weights. Molecular size and isoelectric focussing are taken into consideration. Isoelectrical focussing is dependent on the electrical charges of eh constituent protein molecules. Thus molecules with positive charges will move to negative regions and negative charges tot e positive regions. In order to identify the proteins in gel after mobility they will be require to be stained. They appear dark or blue with stains like Coomasie blue.

Western blot (see below).

 

 

5. Recombinant DNA techniques

 

The following are the basic recombinant DNA techniques

 

Synthesis of complimentary DNA

The gene being made up of sequences of nucleic bases can be split up fragmented and joined together in the technology now known as recombinant DNA. But before the gene can be manipulated in several ways, the gene has to be obtained. There are two main methods of obtaining the gene

 

The synthesis of cDNA involves the use of the enzyme known as reverse transcriptase. This enzyme assists in synthesizing DNA from mRNA, hence the name reverse transcriptase for it does the reverse transcription. Transcription is the process of converting DNA to mRNA. The DNA which is formed from mRNA is of a different variety than the normal DNA. It does not contain repititive DNA and its introns are not available. It is called cDNA and for all purposes treated as the gene for the particular protein in question. It is used in recombinant experiments and coned also in order to provide a means by which its protein translation can be achieved. Hence cDNA is very versatile in its use in modern recombinant DNA technology. The enzyme reverse transcriptase are found abundantly in retroviruses.

 

 

 

Figure:  Synthesis of cDNA from mRNA using reverse transcriptase

 

Gene cleavage

Genes or complimentary DNAs can be fragmented in any site using the enzymes called restriction endonucleases. These enzymes are also founding many organisms and they cleave DNA at sequence specific sites. Thus a single enzyme would have its own site for splitting the DNA using the sequence of the nucleotides bases on the actual gene. The sites which are cleaved by restriction enzymes are usually unprotected by methylation. These enzymes therefore break genes or cDNA into pieces. The units which are broken are now available for manipulation and they can be placed in a plasmid for cloning or into polymerase chain reaction technology for more rapid cloning. The main process of recombination therefore comes when a gene is split and then incorporated into another gene using DNA ligase enzyme.

 

Table: Restriction endonucleases which cleave DNA into fragments with 5’ extensions, readily end labelled with T4 polynucleotide kinase. After Gaastra Wim and Oudega Bauke, Determination of DNA sequences, In Walker JM and Gaastra Wim eds. Techniques in Molecular Biology London: Croom Helm, 1983, p.303

 

Enzyme

Sequence recognised

BamHI

5’G|GATCC3’

3’CCTAG|G5’

BgIII

5’A|GATCT3’

3’TCTAG|A5’

BstEII

5’G|GTNACC3’

3’CCANTG|G5’

EcoRI

5’G|AATTC2

3’CTTAA|G5’

HindIII

5’A|AGCTT3’

3’TTCGA|A5’

HpaII

5’C|CGG3’

3’GGC|C5’

Hinfl

5’G|ANTC3’

3’CTNA|G5’

Mbol

5’G|ATC3’

3’CTA|G5’

SaII

5’G|TCGAC3’

3’CAGCT|G5’

Sau3AI

5’|GATC3’

3’CTAG|5’

TaqI

5’T|CGA|3’

3’AGC|T5’

Xbal

5’T|CTAGA3’

3’AGATC|T5’

Xhol

5’C|TCGAG3’

3’GAGCT|C5’

Xmal

5’C|CCGGG3’

3’GGGCC|C5’

Note: In this table N is sequence stands for any of the four bases. The line indicate the points of cleavage

 

 

Gene ligation

The combination of gene sequences can occur in a bacterial plasmid (see below) or any other genetic sequence. It is catalysed by the enzyme DNA ligase. It is used extensively in gene cloning.

 

Gene Cloning

This simply means making several copies of any material to be cloned. In the case of gene cloning it simply means making several copies of the gene. It is easy to generate a gene using reverse transcriptase enzyme which gives rise to cDNA or use restriction enzyme which break a sequence that contains a particular gene to be studied. The gene is only one molecule and to be useful it has to be made into several copies in order to use them for translational purposes.

 

Table:  Enzymes used in DNA technology. After Emery A, (1984) p. 41

Enzyme

Function

Main uses

Alkaline phosphatase

Dephosphorylates 5’ ends of RNA and DNA

Prevention of self-ligation

DNA ligase

Catalyzes bonds between DNA molecules

Joining DNA molecules

DNA polymerase (e.g. ‘Klenow fragment’)

Synthesizes double stranded from single stranded DNA

(a)    Synthesis of double stranded cDNA

(b)   Nick translation

DNase I

Under appropriate conditions produced single-stranded nicks in DNA

Nick translation

Exonuclease III

Removes nucleotides from 3’ ends of DNA

DNA sequencing

γ Exonuclease

Removes nucleotides from 5’ ends of DNA

DNA sequencing

Nuclease Ba/31

Degrades both the 3’ and 5’ ends of DNA

Progressive shortening of DNA molecules

Polynucleotide kinase

Transfers phosphate from ATP to DNA or RNA

32P labelling of DNA or RNA

Restriction endonucleases (type II)

Cleave DNA at sequence-specific sites

Generation of recombinant DNA

Reverse transcriptase

Synthesizes DNA from RNA

Synthesis of cDNA from mRNA

SI nuclease

Degrades single-stranded DNA

Removal of ‘hairpin’ in synthesis of cDNA

Terminal transferase

Adds nucleotides to the 3’ ends of DNA

Homopolymer tailing

 

 

 

 

Plasmids in E coli and Yeast

 

Classical method. This involves the use of bacterial plasmid. A plasmid is a circular gene which is found in the mitochondria of procaryotes such as bacteria. They can replicate many copies of themselves in only a few minutes. This is therefore exploited in makingy several copies of genes. The strategy is to place the gene to be cloned into the bacterial plasmid. The plasmid is opened up with a restriction enzyme which breaks at sequence specific site. Then the same restriction enzyme is used to break the gene (either a restriction fragment- i.e. fragment of gene generated by cleavage by restriction enzyme) or cDNA. The restriction enzymes create sticky ends which are quite easily joined together on the plasmid using DNA ligase enzyme. The combination of plasmid and gene will now replicate at the same rate as replication of plasmid. It generates several copies of the gene which are them removed for translational purposes.

 

 

Strategy for generating plasmid-cDNA combination for cloning

 

 

 

Table  Preparation of plasmid DNA. In Arrand JE, Preparation of Nucleic acid probes. Hames B D and Higgins S J eds. Nucleic acid hybridisation; a practical approach. Oxford: IRL press, 1985, p 27.

 

  1. Grow the host bacteria in broth culture containing a suitable concentration of the appropriate antibiotic to which the plasmid confers resistance.
  2. Just before beginning he plasmid isolation, prepare lysosyme buffer:

50mM glucose

10 mM EDTA

2-10 mg/ml chicken egg-white lysozyme

25mM Tris-HCl, pH 8.0

  1. Harvest the bacteria by centrifugation at 4000g for 10 min at 4oC
  2. Resuspend the pellets in 4ml of freshly prepared lysosyme buffer per 200ml of original culture. Leave at room temperature for 10 min
  3. Add 8ml of 0.2M NaOH, 1% SDS per 200ml original culture, shake gently, and leave on ice for 10 min.
  4. Add 4ml of 3 M potassium acetate, pH 4.8, per 200ml original culture. Vortex thoroughly then leave on ice for 30 min.
  5. Centrifuge at 4000g for 10 min at 4oC
  6. Take the supernatant (which contains the supercoiled DNA), and add 0.6 volume of isopropanol. Mix well and leave at room temperature for 5 min.
  7. Harvest the precipitated DNA by centrifugation at 14 000 g for 10 min. Resuspend the pellet in 6.5ml of TE buffer (10mM Tris-HCl, pH 7.5, 1 mM EDTA) per 200ml original culture.
  8. Purify covalently closed circular plasmid DNA either by equilibrium density gradient centrifugation in caesium chloride or by chromatography on NACS resin as follows

Equilibrium density gradient centrifugation

  1. To each 6.5ml solution of DNA in TE buffer, add 7g CsCl and 0.5ml of 5mg/ml ethidium bromide solution (stored dark at 4oC).
  2. Centrifuge to equilibrium (36h) at 120 000g
  3. At this time, the RNA will have pelleted onto the bottom of the tube and two DNA bands will be visible in the upper half of the gradient. Collect the more intense, lower plasmid DNA band through the side of the centrifuge tube using a syringe and needle. (The upper band contains nicked, open circles of plasmid DNA plus residual fragments of chromosomal DNA and is not collected).
  4. Remove ethidium bromide from the plasmid solution by repeated extractions with isoamyl alcohol saturated with CsCl solution.
  5. Dialyse the plasmid DNA preparation extensively against TE buffer.

 

Purification of supercoiled DNA by NACS chromatography

The resin of choice for this purpose is NACS 37 (BRL) which is suitable for use with a peristaltic pump. It has a capacity of 0.57mg total nucleic acid per gram, and can be re-used repeatedly. RNA competes with supercoiled DNA or binding to this resin and so should be removed by RNase digestion prior to chromatography.

  1. To each 6.5ml of plasmid solution in TE buffer (from step 9 of the first part of this table) add 200units RNase T1 and incubate for 15 min at 37oC
  2. Add NaCl to a final concentration of exactly 0.5M. Mix well
  3. Prepare a NACS column by suspending NACS resin in TE buffer containing 2 M NaCl, stirring gently overnight, pouring a column of suitable size (see capacity of NACS 37 resin above), and washing extensively with TE buffer containing 0.5M NaCl.
  4. Load the plasmid DNA preparation form step 2 onto the column and wash with several column volumes of TE buffer containing 0.5 M NaCl.
  5. Elute the column with a linear gradient of 0.5-0.7 M NaCl in TE buffer and collect fractions. Monitor the A260 of the eluate. Residual RNA will be found in the loading eluate. Supercoiled plasmid DNA elutes in the gradient. Open circular plasmid DNA and genomic DNA fragments are retained on the column, which can be regenerated by extensive washing with 2 M NaCl in TE buffer followed by re-equilibration with loading buffer.
  6. Pool those fractions containing supercoiled DNA (as visualised by agarose gel electrophoresis) and add 2 volumes of ethanol. Place at -70oC for 30 min.
  7. Centrifuge at 12 000 g for 15 min. Resuspend the DNA pellet in TE buffer.

 

 

 

Polymerase chain reaction technology: PCR developed as an alternative to cloning using plasmids or other invivo vectors. It was first described in 1987. It uses machines provided with polymerase enzymes and when given nucleotide bases, it is able to amplify the gene in large numbers.

 

Nucleic acid hybridisation: The concept of hybridisation is a fundamental one in molecular biology of genes. DNA molecule is supposed to be double stranded. Hence a single chain of DNA must attach itself to a complementary chain and this process is then called hybridisation. Hybdridisation can occur between single chains of DNA and a single chain of DNA with a single chain of RNA.

 

6. Protein translation

 

Molecular synthesis of protein molecules and other peptides can now be performed in the laboratory; thanks to the recombinant DNA methods. Once several copies of the gene to be used or which expresses the peptide has been cloned, the next stage is to find the appropriate vector that can be used to translate this into protein. The following peptide hormones were the initial ones to be synthesized using the recombinant DNA methods.

 

Proteins can now be synthesized in vitro using vectors like Escherichia coli, bacteriophage and even eukaryotic cell, such as yeast cells, monkey kidney cells etc.

The strategy for molecular synthesis will begin from the isolation of a gene ready for synthesis or expression of its peptide product. This gene is then cloned. The cloned gene is inserted into a vector which will then express its final product. The expression of the gene is dependent on several factors.

 

Gene structure for translation

A gene can be divided into three parts

Structural gene  is the sequence which contains nucleotide sequences that will code for  the appropriate amino acids which will constitute the peptide molecule. That is to say, only structural gene will be transcribed.

Operator gene: This operates the structural gene and is close to it

Promoter gene. This is close to the operator gene. A gene cannot be expressed at all without the promoter gene. For the promoter to function, the operator must be activated by a chemical compound. Certain prokaryotic promoters are well known . For example the lac operon. The operator of lac operon is activated by lactose.

 

 

Structure of protein gene

 

Table:   Biosynthesis of some human peptides. After Emery AEH, Recombinant DNA technology. London: John Wiley, p 166.

 

Cloned gene

Peptide

Number of amino acids

Synthetic

cDNA

Reference

Somatostatin

14

+

 

Itakura et al., 1977

Insulin

      Peptide A

21

+

 

Goeddel et al, 1979a

Insulin

       Peptide B

30

+

 

Growth hormone

191

+

+

Goeddel et al., 1979b

(1-24)

(24-191)

F-interferon

166

 

+

Taniguchi et al., 1980

L-interferon

165

 

+

Nagata et al., 1980., Goedel et al., 1980

 

Synthesis of somatostatin by rDNA: This is a simple peptide with only 14 amino acids. In order to synthesize it lac operon is attached to somatostatin structural gene and the whole gene complex is then inserted into E coli and E coli is exposed to lactose which activates the lac operon. Lac operon contains the following subunits

These are then attached to somatostatin gene sequence. Hence after translation β -galactosidase will be attached to somatostatin molecule forming a complex molecular structure. Cyanogen bromide is a chemical agent that cleaves peptides. It  will then break the bond between β-galactosidase and somatostatin, thereby freeing somatostatin molecule.

 

Synthesis of insulin by rDNA: Human insulin is essential in treating human diabetes mellitus. In the past porcine insulin is used for treatment being the one nearest to human insulin in structure. But it as the unfortunate side effect of developing antibodies and therefore tolerance. It is best to use human insulin and until recently that was impossible. With the use of recombinant DNA human insulin can now be synthesized in the laboratory using eukaryotic or prokaryotic translational vectors.

 

Insulin is made up of 2 chains held together by disulphide bonds (A & B chains)

The genes for A and B chains are obtained and cloned separately and the plasmid pBR-322 is now attached to lac operon together with insulin gene A or B. After expression cyanogen bromide is used to break the bond between peptide and β- galactosidase, thereby freeing each peptide. The two peptides are now available for coupling. They are joined together by sulphonation and air oxidation and then marketed as human insulin which does not produce antibodies. Its most common trade mark is Humulin.

 

Simplified diagrammatic representation of the bacterial synthesis of human insulin. After Emery A, 1984, An Introduction to Recombinant DNA technology. London: John Wiley, p 165.

 

Synthesis of human growth hormone by rDNA

Human growth hormone is the antagonist of somatostatin. It has 191 amino acids. It is synthesized in 2 fragments which are later bonded together. These fragments are broken at amino acid 23 and 24

For the synthesis of HGH, mRNA is extracted from pituitary gland and then converted into cDNA

The resulting cDNA is exposed to a special endonuclease enzyme HAeIII which is able to attack the cDNA and break it into two (1-23; 24-191)

 

The two fragment genes are now cloned separately and digested also with HAeIII which then open them up for fusion to occur with plasmid vector. They can now be reproduced in large quantities.

 

 

 

 

Figure: Simplified diagrammatic representation of the steps involved in generation recombinant plasmid for the bacterial synthesis of human growth hormone. After Emery AEH, An Introduction to Recombinant DNA. Chichester: John Wiley, p 166.

 

Synthesis of interferons by rDNA

These are peptide molecules produced by human cells after infection with viral agent. Hence interferons can be used to treat viral diseases but they are manufactured in very small quantities in the human body. But modern recombinant DNA can now produce these very vital agents in large quantities for therapy.

Today three types of interferons are recognized and they are

 

Synthesis of vaccines by rDNA

Vaccines can be produced using recombinant DNA technology. A portion of an infective agent can be obtained by synthesing its gene. The synthetic gene is then available to produce the antigen which then stimulates the antibody production. Sometimes the antigen may work as receptor to mop up infectious agents. In this case the antigen may be obtained directly form the human tissue receptor rather than infectious agent. Example is CD4+ receptors found in T lymphocytes to which HIV particles attach. Method have been adopted in which CD4+ receptors are produced in large amounts through recombinant DNA techniques  and injected into HIV infected patients, so that free CD4+ receptors are attached to the viral particles and then mop them up. The method presents how useful recombinant DNA can be in providing strategies for drug design and manufacture, but unfortunately, it has not been successful in HIV therapy.

 

Post translational modification

Sine procaryotic cells may have been used to procure translation in vitro systems, it is sometimes desirable to modify what has been translated for use in eukaryotic systems.

To make the peptide products active biologically the following methods may be used

The above methods are reversible. Other methods exists which are irreversible and they include attachment of coenzyme such as biotine

Aside from the use of prokaryotic cells for in vitro translational methods, the following eukaryotes can also be used.

 

7. Diagnostic methods in gene technology

Molecular blots

These are techniques designed to predict the availability of genes in cells or tissues (Southern) or their transcripts (Northern). Transcripts of genes are mRNA for the particular gene. Also protein products are identified in molecular blots (Western). This is based on the principle that all genes are present in all cells of the body but only active or are derepressed in the cell or tissue of expression. Thus the insulin gene can be found in all cells of the body but it is only expressed in cells that normally elaborate insulin and they are the β  cells of the islets of Langerhans. Therefore in these β cells you can find mRNA for insulin and of course the DNA. But the DNA for insulin (insulin gene) is found equally in all cells of the body rendering all cells capable of being used to identify the DNA. This principle is used extensively in antenatal diagnosis. For example, to make a diagnosis of the hemoglobinopathies such as sickle cell disease, it is not necessary to obtain  red blood cells. Any cells of the body would do and hence amniotic fluid cells can be used for such function. On the other hand, if we wish to determine which cell expresses a particular gene, that is to say which cell elaborates the gene product, then we have to use mRNA technique to identify transcripts of the gene in the nucleus of the cell to be sure the gene is active there. Hence we expect to find mRNA for insulin only in the B cells of eh islets of Langerhans.

 

 

Southern blot technique[1]

The technique now known as Southern blot was first described by a man known as E.M. Southern in 1975 in Edinburgh. It is useful method of identifying gene or gene sequence in any tissue of the body of an individual without necessarily obtaining the cell in which the gene is active). As mentioned earlier, this is based on the  principle that all genes are present in all cells of the body repressed, but only active or are derepressed in the cell or tissue of expression. For diagnosis, we need a gene probe, i.e. a sequence of nucleotides identifical to the one being sought after for diagnosis but which does not contain repetitive DNAs that might confuse the blotting process. Sites containing repititive genes are available in genome and may have repeats up to 30000 times. Hence a gene probe to be used in molecular blot should not contain repetitive DNA or interpretation of hybridisation will be difficult. This gene probe must also contain only exons and not introns. Since cDNA normally has the above properties, it is highly favoured in Southern blot techniques It is normally labelled with radioactive material. A good example of such radioactive materials could be 32P.

 

 

Preparation of probes using the large (Klenow) fragment of E coli DNA polymerase I.

→Labelled strand; ds and ss DNA, double – and single-stranded DNA, respectively. In Arrand JE, Preparation of Nucleic acid probes. Hames B D and Higgins S J eds. Nucleic acid hybridisation; a practical approach. Oxford: IRL press, 1985, p 37.

 

In this technique, DNA is extracted from tissue and subjected to restriction enzyme which break the genome into several fragments. One of the fragments will contain the gene to be identified. Already a gene probe has been prepared and labelled.  The fragments are then subjected to agarose gel electrophoresis and blotted on nitrocelluose filter paper which has been impregnanted with the labelled gene probe. If the gene sequence is available on tissue it will be hybdridised and the point of hybridisation is then picked up by autoradiography.[2]

Southern blot technique after Emery (1984)

 

Clinical conditions diagnosable by Southern blot

Several techniques are associated with prenatal diagnosis of genetic conditions in addition to Southern blot. Some are a follows,

 

It  is  now possible to apply the technique of Southern for the diagnosis of several medical conditions. There are 2 categories in which DNA techniques can be valuable for diagnosis

  1. Genetic materials that are unifactorial e.g. sickle cell disease, albinism etc
  2. The common multifactorial inheritance conditions

 

Most of the unifactorial conditions can be diagnosed with the aid of the restriction fragment length polymorphism (RFLP). DNA has variations in restriction sites which leads to polymorphism and the restriction fragments produced by specific restriction enzymes can be used as linkage markers for detecting genetic condition such as sickle cell disease.

 

 

DNA polymorphism. After Emery (1984).

 

Sickle cell disease

In this condition, southern blot diagnosis exploits the restriction fragment. The β  haemoglobin gene probe must hybridise with restriction fragment of 7.6kb in Caucasians when the restriction enzyme HpI is used. In blacks it is found in 7.0kb for normal β   globin gene. However the sickle cell gene is found in fragment  having 13kb.

 

 

Diagrammatic representation of the steps involved in the detection of a hypothetical β-globin gene. From P.F.R. Little. Antenatal diagnosis of haemoglobinopathies. In R Williamson (ed). Genetic Engineering Academic Press, 1981, vol 1, 61-102 and Walker J.M. and Gaastra W, eds. Techniques in Molecular Biology, London: Croom Helm, 276.

 

Thalassemia

α globin can be used for its diagnosis

 

Phenylketonuria

The restriction enzymes used include Mspl, sphl and HindIII

 

Myotonic dystrophy

In this condition the gene locus is found in chromosome 19 and is linked to the following loci

It is possible to use the linkage in the diagnosis with the appropriate restriction enzymes

 

Congenital adrenal hyperplasia

The absence of the enzyme 21-hydroxylase leads to the formation of ambiguous external genitalia. The gene for the enzyme leads to major histocompatibility complex (MHC) which is located on chromosome 6. A technique has been developed for its diagnosis

 

Others

Other conditions in which DNA technology has been used for diagnosis include

 

Multifactorial conditions

These are  conditions caused by multiple genes (nature) and also environmental factors (nurture) in which recombinant DNA diagnosis can now be made are as follows

 

Y chromosome diagnosis

Probes have been developed which contains DNA sequences that are specific for the Y chromosome. This can be used to identify the male fetus.

 

Antenatal diagnosis

Cells used for antenatal diagnosis are usually obtained from the amniotic fluid and they contain cells of fetal skin . This will suffice, but if not adequate chorion biopsy should be tried.

 

Infectious disease identification/diagnosis

The techniques for identification of infectious conditions using Southern blot is increasingly made sophisticated.  Probes are made from parts of any infectious agent and they will hybridise with infectious agents DNA when present. This has led to the detection of toxins production using special toxin probes, gene characteristics and even antibiotic resistance in infectious agents.

 

Techniques in molecular biology associated with DNA diagnosis

Preimplantation genetic diagnosis[3] In this method, small numbers of cells are obtained from in vitro fertilised eggs before implantation, sometimes at the blastocyst stage. This affords an opportunity for early detection of abnormalities that the abnormal embryos are not transferred but wasted. The technique used for identification of genes include PCR DNA based amplification and fluorescent in situ hybdridisation (FISH). The two techniques require only a few cells. The technique is now quite versatile and can detect sex of the embryo so that diseases which are linked to sex chromosomes can be detected or discriminated against (e.g. hemophilia which only affects men and make women carriers or genes linked to Y chromosome). It can also be used alone or in conjunction with sperm sifting methods to provide gender selection of family balancing. Check information from www.givf.com for more.

 

Fluorescent activated cell technique. In this technique genomic DNA containing several chromosomes are subjected to special fluorescent dyes. They are then picked up by fluoroscopy. It is then possible to obtain a particular chromosome that is needed for DNA analysis. The technique can therefore be used for the production of probe for Southern blot techniques.

 

Oligonucleotide probes: In southern blot technique it is sometimes unnecessary to use the whole gene as probe since there may be several repititions. It is therefore possible to synthesize a small portion of the gene containing a sequence of nucleotides which may be used as probe. This is then called oligonucleotide probes or oligoprobes.

 

Nick translation: This is a method for the labeling of DNA for use in molecular blots. It utilises DNase I to create single stranded nicks in double stranded DNA. It then uses 5’3’ E coli exonulease to break the DNA at the nicks, followed by E coli 5’3’polymerase to incorporate new strand made by incorporation of labeled deoxyribonucleotides thereby producing the nick at the ends of the new strands moving along the 5’ to 3’ direction. Denaturing the double stranded DNA produces short part labeled single stranded DNA which can be used as gene probes.

 

Northern blot[4]

As mentioned earlier, Northern blot is a technique designed to identify mRNA in tissues and not DNA. Hence it is able to pick transcripts of genes when such are present and therefore able to determine where genes are expressed or derepressed. If Northern blot is positive, it means that the gene product is produced in the cell or tissue under study. It is therefore a valuable tool in research where new peptide products are being sought for and found daily. When combined with a special microscopic technique known as in situ hybdridisation, it then becomes a formidable tool for predicting areas of peptide secretions in minute tissues and cells. See more[5]

 

mRNA is extracted from the nucleus and subjected to restriction enzyme to produce several restriction fragments. One of the fragments contains the transcript to be identified. They are subjected to agarose gel electrophoresis. The resulting gel is blotted on to DBM (diazobenzyloxymethyl) paper rather then nitrocellulose filter since nitrocellulose filter does not have affinity for RNA. The DMB paper is already impregnated with gene probe which is radiolabeled. The area of resulting hybridization is picked up by autoradiography.

 

Western blot

It is now possible to use a method based on nucleic acid blots (Southern and Northern) to detect protein with specific amino acid sequences in tissues or cells. This protein blotting method is commonly called Western blot. While there is not yet any Eastern blot, it should be noted that although there is a man called Southern, there is no one called Northern or Western. There are three types of Western blot techniques and are as follows

 

 

Capillary blotting method is very slow. It may take a few days to complete. It relies on the ability of the peptides or protein to diffuse into nitrocellulose filter from its SDS- polyacrylamide gel electrophoretic medium by contact.  First the nitrocellulose filter is impregnated with protein probe which radiolabelled. In electroblotting method, electrical current is used to aid the transfer of substance across the filter and is therefore of very short duration. It is currently the most favoured technique in protein blotting.

 

 

The major steps in a typical protein blotting experiment. From Keth, In Walker JM and Gaastra Wim eds. Techniques in Molecular Biology London: Croom Helm, (1983), p54

 

8. GENE THERAPY[6]

Originally gene therapy was a theoretical proposition which was embodied in the concept of genetic engineering. The concept of genetic engineering meant that it was possible to engineer an abnormal gene in order to effect a complete genetic cue. The haulmark of this concept was of course the development of the strategies for incorporation a foreign gene into the genome of the cell. The very first gene injection to be carried out in the human for treatment was made in 1994 using the vascular endothelial growth factor, although the method had been used with considerable success in agricultural sector to produced transgenic plants and animals.

 

Types of gene therapy[7]


 

Gene therapy started initially as gene injection. This involved several strategies for incorporating genes into somatic cells or into early embryos. When an organism incorporates an alien gene it is then said to be transgenic. Genes can be used to treat congenital abnormalities which are caused by mutation through the replacement by cDNA or polynucleotide sequences.

 

Another strategy is to induce a suppressor mutation. This is done with the use of tRNA which then suppresses the mutation existing in the gene to be expressed. The change which occurs in the tRNA helps to code for the normal amino acid rather than the abnormal one and hence cures the condition permanently (see Wang et al 1983)

 

Strategies for incorporating genes into cells

  1. The use of microcells. This is the incorporation of DNA molecules into the chromosomes, which are the microcells. The microcells act as vectors for the replication of genes. Microcells are chromosomes which have picked out at metaphase  of dividing cells.
  2. Role of calcium precipitation. Cells to be replaced with genes are taken from the body and placed in a test tube to which calcium salt has been added. Calcium breaks down the cell wall  and allows the gene to be incorporated into the cell. The cells are then grown in culture to increass their number. They will replicate the gene that has been incorporated and they are then replaced into the body. This strategy is vital for the treatment strategy of the hemoglobinopathies, since red cells which are immature can be injected with normal genes and replaced into the body. The strategy is also vital in replacing diseased endocrine cells.
  3. Direct intranuclear injection. Using micropipette methods, a gene can be directly injected into the nucleus of a cell for its incorporation thereby bypassing cell membrane and the need to provide calcium for the purpose of transport across the cell membrane. This strategy is often used for injecting early embryonal cell at the zygotic stage. At the stage of morula the method may change to the incorporation of genes using calcium for transport across cells since this must involve many cells at the same time.
  4. Use of viral carriage:  Certain retroviruses and others such as SV-40 (simian virus -40) can be used for carrying genes into tissues. The virus is injected into the cell and the cell is grown in culture. Once the virus is incorporated into the genome of the cell it releases its contained gene which may form a provirus in that genome. With the culture of the cell, several copies of the cell containing the virus are made and then reinjected into the body to influence either somatic or germ cell.

 

Other modern methods include

 

Problems of gene injection

  1. If somatic cells are to be used for gene therapy then injected transgenic cells are to grow faster than the original cells in the body or else they will be outnumbered by the original cells. For example, supposing we use immure red cells such as erythroblast for gene injection. We then grown these cells in culture after injecting them with genes. They are now ready to be reinjected into the bone marrow. If the original erythroblasts produced by the body are not outnumbered by the transgenic cells, the strategy for treatment will fail. Also if the injected cells lack enough growth potential, strategy is also bound to fail.
  2. It is necessary sometimes to use early embryonal cells for gene injection especially when the effect of gene incorporation is meant to be far reaching and to affect many cells if not all cells of the body. For example, the incorporation of growth hormone gene must affect all the cells in the body. To use early embryonal cells then there must be a need to have an in vitro fertilisation program on and also the need for an embryologist. Ordinarily early embryos are difficult to obtain and also to manipulate. If the effect of the gene is to be localised or if the gene must express in only a few tissues and not all, there is a need to provide the tissue with tissue specific enhancers which allow the incorporation of the gene into the genome and also allow its expression only in the specific tissue to be targeted.
  3. It is not only the gene in question that would be needed if embryonal cells are to be injected. Other sequences must accompany the gene in order to make the incorporation effective and they include tissue specific enhancers (as mentioned above), specific flanking sequences and promoter sequences.
    1. Promoter sequences are known to activate the gene in its prescribed loci of function. If absent gene will not be expressed
    2. Tissue specific enhancers: Certain sequences are known to enhance tissue specificity
    3. Flanking sequences: These are not known to provide any functions but are nevertheless needed in certain cases if a good incorporation of gene is to be expected in tissues

 

The important reason why a tissue specific enhancer is to be incorporated into the sequences to be transferred comes from the earlier observations that a globin gene injected into an early embryonal cell without tissue enhancers may be lead to activation of the gene in muscle, testis etc. This activation would naturally cause the cells in these area to produce hemoglobin with disastrous consequences.

  1. The effect of the control of the foreign gene is presently being mapped out. Just like other areas in recombinant DNA technology, the fears of the dangers of incorporated genes have almost completely gone and injection of genes are being made daily in the human clinical practice.

 

Gene therapy strategies

Susan B. Kesmodel and Francis R. Spitz (see Molecular medicine 5; 3 and http://www.expertreviews.org/

Gene therapy strategies

Strategy

Mechanism

Refs

Mutation compensation

Restoration of tumour suppressor gene function

24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 110, 112, 116, 117

Inactivation of oncogenes

12, 37, 38, 39, 40, 41, 121, 122

Molecular chemotherapy (‘suicide’ gene therapy)

HSV-tk/GCV

42, 45, 46, 47, 48, 49, 50, 51, 107, 108, 109

Escherichia coli CD/5-FC

42, 43, 45, 49, 52, 53, 54, 111

Immunopotentiation

Delivery of IL-2

75, 107

Delivery of IL-12

62, 65, 66, 67, 74, 108

Delivery of GM-CSF

63, 64, 68, 74, 108, 109

Delivery of IFN-g

76, 115, 124

Tumour vaccines derived from TAAs (MART-1, gp100)

70, 71, 72, 73

Upregulation of MHC/costimulatory molecules

10, 55, 75, 76, 123, 124, 125

Inhibition of immunosuppressive molecules

123, 126

Viral-mediated oncolysis

Viruses with functional gene sequence deletions: ONYX-015 14, 77, 79, 80, 81, 82, 83, 84, 113, 114

Viruses with functional gene sequence deletions: G207

14, 77, 85, 86, 87

Viruses with tumour-/tissue-specific promoter/enhancer sequences (PSA88, DF3/MUC-189, AFP90)

14, 88, 89, 90

Antiangiogenic therapy

Downregulation of VEGF activity

97, 98, 99, 100

Delivery of endogenous inhibitors of angiogenesis

100, 102, 103, 105, 106

Abbreviations: CD/5-FC, cytosine deaminase/5-fluorocytosine; GM-CSF, granulocyte–macrophage colony-stimulating factor; HSV-tk/GCV, herpes simplex virus thymidine kinase/gancyclovir; IL, interleukin; IFN-g, interferon g; MHC, major histocompatibility complex; TAAs, tumour-associated antigens; VEGF, vascular endothelial growth factor. See references[8]

 

 

 

 

 

 

 

 

 

 

 

 

Strategies for gene injection. After Emery 1984.

Modern strategies of gene injection with special reference to oncology[9]

Gene injection is now used almost routinely to treat some cancers and the following strategies are being adopted gradually

 

9. Cloning-to-clinic[10]

This simply means cloning a gene and using its peptide product in the clinic for treatment. Cloning-to-clinic is therefore both basic to the science of pharmacy (and indeed pharmacology) as it is to clinical medicine and therapeutics.  It is very much of interest in the design of new drugs for example. If a receptor of a drug can be identified, its gene cloned and then large quantities of the gene product obtained by in vitro translation, it is possible to construct a molecule that is antagonistic to it. Once these molecule is obtained it can then be used to treat the condition in which the receptors need to be blocked.

 

Pharmaceutical protein

Market size (kg)

Market value ($ x 106)

Minimum sheep expression required (g/litre)

α1-antitrypsin

7500

750

2.0               50,000

Plasminogen activator

75

750

0.1               10,000

Factor IX

2

60

0.01              3,000

Erythropoietic

0.2

300

0.001             3,000

Factor VIII

0.975

375

0.0001          10,000

Data from James 1993 (see Moor  1997)

 

 

 

Example of this method is in the condition acquired immune deficiency syndrome therapy.  It has been possible to clone AIDS virus target molecules (e.g. CD4+ receptors in T lymphocytes. It has also been possible to provide recombinant products which are antagonistic to these receptors. It is then possible to try out whether the antagonistic molecules can bind to the CD4+ receptors so that when the viral particles enter the body they will be displaced by the anatagonists and would therefore not find any place to bind. Other molecules which have been studied include hemoglobin, dihydrofolic reductase, lysozyme, phospholipase A, thyroid hormones binding prealbumin, renin receptor, insulin receptor, atrial natriuretic factor receptor.

 

Peptide products for cloning to clinic

These are directly synthesized from their genes and are used in three categories

 

First generation gene products

The following compounds are synthesized using recombinant DNA techniques with generation of clones of genes from vectors and then translation in vector cells, including post-translational modifications. They are human insulin, growth hormone, tissue plasminogen activator, interleukins, hepatitis B vaccine, erythropoietin, colony stimulating factors, superoxide dismutase, growth factors, clotting factors, and pulmonary surfactant.

 

Second generation gene products

These are improved compound obtained from first generation products  Examples in which this has proven useful include dihydrofolate reductase, phopholipase A2, hemoglobin, lysozyme and thyroid hormone binding prealbumin. Others are renin, insulin and atrial natriuretic factor receptors. It is also possible to develop antagonists against proteins or peptides obtained as first generation products and synthesize enzymes which may be useful in production of many first generation gene products (Baxter 1988).

 

Delivery methods

Recombinant DNA products are proteins. They would have to be introduced into the body mainly by injections, since protein products do not cross the cell membrane barrier easily unless side by carrier proteins. But recombinant DNA techniques are also being used to design effective delivery systems. Examples are

 

 

Production of bioactive peptides for therapeutic purposes from transgenic animals[11]

The development of two interesting techniques has provided the means of modifying the genes of animals in a manner in which the offsprings can be used for the production of biomedically active proteins.

·        The first is the procurement of reliable source of mammalian eggs for genetic manipulation

·        The second is the cloning of relevant genes and  the preparation of gene constructs for the insertion into the genome of developing egg

Three areas have been covered in recent times in the production of peptides

  1. The provision of rare protein which are needed for patients with specific defect. Two main proteins can be used as examples of use of transgenic animal.

·        Blood clotting factor IX

·        α1 antitrypsin.

These two proteins can now be produced using genes which are placed in early embryos of sheep and targeted to the mammary glands of the developing sheep. Hence the peptide products are produced in milk and can be easily harvested from there. It does not seem to have any side effect on the developing sheep.

 

 

Strategy for embryo injection into sheep for procuring transgenic sheep, which will produce proteins in milk. After Moor (1987) and James (1993.

 

  1. Provision of human monoclonal antibodies which can be used via immunotherapy for the treatment of cancers and other diseases. It is possible to provide animals that can produce human antibodies.
  2. The possibility of providing animals that can produce transplant organs for transplantation in patients who require them.

 

 

10. Electron microscopy  and molecular genetics

Since 1959 Kleinschmidt and Zahn have described the visualization of double stranded DNA filaments. It is now possible using electron microscope to characterize single stranded DNA and RNA and also hybrid molecules.

 

EM is therefore now a tool of increasing interest in molecular biology and can be used for any of the following

  1. Characterisation of DNA structure whether linear, circular degree of supercoiling and length of a DNA fragment
  2. Analysis of complex structures such as replicating intermediates.
  3. The detection and analysis of complementary sequences (including analysis of homology and non homology) either between two populations (heteroduplexes) or between and RNA and a DNA molecule (hybrids) by D loop mapping or R loop and R hybrid mapping respectively.
  4. The visualization by EM of hybrids between DNA and RNA provides reliable information for the characterization of partial and total homology between the two populations of nucleic acid molecules. For DNA-DNA hybridization regions of homology will be double stranded and regions lacking homology will be single stranded segments (thinner filaments) or D-loops. For DNA-RNA hybdridisation essentially two methods have been developed to map sequences along a DNA molecule which are complementary to particular DNA. The first method is the R loop technique in which RNA molecule is hybridised to its complementary sequence in duplex DNA. The second method is the R-hybrid technique where RNA molecule hybridizes to denatured single stranded DNA molecules.
  5. The study of protein nucleic acid interactions

 

The following procedures are followed in EM analysis of nucleic acid hybrids

  1. Denaturation of the nucleic acids followed by renaturation under the desired conditions.
  2. Spreading of the hybrids in the presence of proteins and adsorption on carbon films prepared on electron microscope grids.
  3. Visualization and length measurement of the hybrid molecules either directly on a video monitor connected to the EM or on prints contained from EM photographs

 

The shortest DNA-DNA or DNA-RNA hybrids recorded is 30±10bp region of exon 11 of the conalbumin gene.

 

EM staining procedure (Oudet and Schatz 1985).

i.                     Prepare a stock solution of 0.5% uranyl acetate in 50mM HCl. After dissolution, filter the solution through a disposable filtert (0.22μm; Millipore) and store it at room temperature in a dark bottle covered by aluminium foil to prevent exposure to light. It is stable for 2-3 months.

ii.                   Just before use, prepare the staining solution by mixing 0.5ml of the uranyl acetate stock solution with 49.5 ml of 90% ethanol in water. This solution must be used within 60min of preparation and at room temperature.

iii.                  Replace the drop of water present on the surface of the grid with the staining solution by touching the surface of the staining solution for a few seconds.

iv.                 Dip the grid into a small beaker containing the same staining of the staining solution for a few seconds.

v.                   Dip the grid into a small beaker containing the same staining solution for a at least 1 min.

vi.                 Next, rinse the grid for 1 min in 90% ethanol, then apply it to a sheet of filter paper to remove the excess solution. Allow it to air dry.

 

 

11. In situ hybridization histochemistry

The full name of this technique implies it is a bona fide histochemical technique albeit applied to genes. It therefore localizes DNA or RNA strands in cytological materials. It is the only way we have to prove conclusively that a particular peptide product is produced in a particular cell, since classical histochemical technique may pick up chemical substances which have diffused from other cells or tissues to the area of localization, although form the experience of classical histochemists/cytochemists, this seems highly unlikely.

 

Hybridisation of nucleic acid probe to nucleic acids within cytological preparations permit high degree of spatial localization of sequences complementary to the probe. For example  in situ hybdrisation can be used to map the sites of a particular sequence.

 

Table Human genes which have been assigned by cell hybrid analysis and /or in situ hybridization. After Emery (1984), p 62.

Genes

Chromosomal location

Immunoglobulin

       α chain

2p12

Collagen, type 1 α 2

7q21

α- interferon

9p21

β-Interferon

9p21

β- Globin

11p15

Insulin

11p15

γ-Interferon

12q24

Immunoglobulin

      Heavy chains

14q32

α Globin

16p12

Collagen, type 1 α 1

17q21

Growth hormone

17q22

Immunoglobulin

      λ  light chain

22q11

 

 Hybridisation also to interphase nucleus can be used to study the functional organisation of particular sequences. It is possible to use the technique to detect DNAs  that are present in only  a very small subset of cells. Such DNAs might never be detected because of dilution of other DNAs of no interest.

 

There are two types of in situ hybridisation

 

Equipments and tools for in situ hybdridisation

Other things needed include

 

CYTOLOGICAL PREPARATIONS (Purdue, 1984)

 

Pre-treatment of Slides before Hybridisation

1.       Incubate the slides in 2 x SSC[12]   at 70oC for 30 minutes.

2.       Transfer the slides to 70% ethanol at room temperature for 10 min. Wash again in 70% ethanol for 10 min and then in 95% ethanol for 5 min. Air dry.

3.       Arrange the slides in a moist chamber containing 2 x SSC. Place 200 ml of RNase[13] (l00 mg/ml in 2 x SSC) over the preparation on each slide and cover each with a coverslip (2.2 cm x 4.0 cm).

4.       Incubate the slides at room temperature for 2 h. Then remove the coverslips gently by dipping the slides into a beaker of 2 x SSC to float off the coverslips.

5.       Wash the slides in 2 x SSC (3 x 5 min), 70% ethanol (2 x 10 min) and 95% ethanol ( 5 min.) Air dry.

6.       Suspend the slides in 0.1M triethanolamine-HCl, pH 8.0. Stir the solution vigorously and add acetic anhydride to 5 ml per litre.

7.       When the acetic anhydride is thoroughly dispersed, stop the stirring and leave for 10 min.

8.       Wash the slides in 2 x SSC (5 min), 70% ethanol (2 x 10 min) and 95% ethanol (5 min). Air dry.

9.       Place the slides in 70mM NaOH for 3 min. Then wash the slides in three changes of 70% ethanol (10 min each) and two changes of 95% ethanol (5 min each).

10.    Air dry the slides.

 

Hybridisation

Hybridisation is performed either in aqueous salt solution such as TNS buffer (0.15M NaCl), 10mM Tris-HCl, pH 6.8) at high temperature or in the presence of formamide[14] at lower temperature.

 

Hybridisation in aqueous salt solution at high temperature.

This is carried out typically in 0.3 M NaCl, 20mM Tris-HCl, pH 6.8, at 67oC. The probe is generally used at 1-10 ng per slide.  If a DNA probe is being used, sheared, denatured E. coli  DNA is also added at 40 mg per slide to reduce non-specific binding of the probe. If an RNA probe is being used, E. coli rRNA is added instead.

(i)      If the probe is double-stranded DNA or double-stranded complementary RNA (cRNA), it should be denatured immediately before use as follows.  Mix the probe with the carrier E.coli DNA or E. coli rRNA in water to 90% of the final volume required to give the correct concentrations. Incubate the mixture at 85oC for 3-15 min and then chill quickly. Bring to 0.3M NaCl, 20 mM Tris-HCl, pH 6.8, by adding 1/10th volume of 10 x concentrated stock buffer. Single-stranded probes such as M 13 clones or SP6 transcripts do not need to be denatured and so can be dissolved directly in 0.3 M NaCl, 20 mM Tris-HCl, pH 6.8, together with the carrier DNA or rRNA.

(ii)     Place 15-20 ml of the hybridisation mixture over each cytological preparation and cover it with a coverslip (18 mm2).

(iii)   Place the slides over a reservoir of hybridisation buffer in a tightly-sealed moist chamber[15] in an oven at 67oC. Note that the amount of liquid in the chamber must be sufficient to prevent evaporation from under the coverslip, otherwise the autoradiographs will show a high background. Typical hybridisation times are 12-14 h at 67oC.

 

Hybridisation in formamide solution

This is carried out typically in 40% formamide in 4 x SCC at 40oC. The mounts of probe and E coli DNA used per slide are as for hybridisation in aqueous salt solution (see above).

(i)      If the probe is double-stranded DNA or double-stranded cRNA, it should be denatured immediately before use. To do this, dissolve the probe plus carrier E coli DNA or rRNA in water to 40% of the final volume desired. Heat the mixture at 85oC for 3-15 min and then chill quickly. Add 20% of the final volume of 20 x SSC and 40% of the final volume of formamide. Single-stranded probes such as M13 clones or SP6 transcripts do not need to be denatured and so can be dissolved directly in 40% formamide, 4 x SSC plus carrier E coli DNA or rRNA.

(ii)     Place 5ml of the hybridisation mix over each preparation and over with a coverslip (18 mm2).

(iii)     Seal the coverslip with a thick coat of rubber cement. The seal saves the expense of filling a moist chamber with formamide buffer to prevent evaporation from under the coverslip.

(iv)   Incubate the sealed slides in a closed container on moist paper towels to prevent drying of the rubber cement. Typical incubations are for 12-14 h at 40oC.

 

Treatment of Slides after Hybridisation.

12.    Following hybdridisation, remove the slides from the moist chambers. Immediately wash off the coverslip and hybridisation mixture by dipping the slide into a beaker of 2 x SSC. (If the coverslip is sealed, peel off the rubber cement first.) Place the slides in a rack in a staining dish containing 2 x SSC and leave for 15 min.

13.    Remove non-specifically bound nucleic acid probe as follows:

RNA probes

(i)      Place the slides in RNase (20 mg/ml in 2 x SSC) at 37oC for 1 h.

(ii)     Rinse the slides twice in 2 x SSC for 10 min each time

(iii)    Dehydrate the preparation in 70% ethanol (2 x 10 min) and 95% ethanol (5 min). Air dry.

 

14.    The preparations are now ready for autoradiography

 

 

Hybridisation to DNA in Mammalian Metaphase Chromosomes

 

Removal of Endogenous RNA

1.       Treat the preparations with RNase (100 mg/ml in 2 x SSC) at 37oC for 1 h.

2.       Rinse in 2 x SSC (3 x 5 min).

3.       Dehydrate in 70% ethanol for 2 x 10 min then 95% ethanol for 5 min. Air dry.

 

Denaturation of Chromosomal DNA

4.       Immerse the slides in 70% (v/v) formamide in 2 x SSC at 70 oC for 2 min.

5.       Dehydrate in ethanol as in step 3.

 

Hybridisation

6.       Apply 20 ml of labelled probe at 0.05-0.2 mg/ml in 50% (v/v) formamide in 0.3 M NaCl, 30 mM trisodium citrate, 10% dextran sulphate, 40mM sodium phosphate buffer, pH 6.0, containing a 1000-fold excess of sheared, denatures non-competing DNA.

7.       Cover the hybridisation solution with a coverslip (18mm2) and seal quickly with rubber cement.

8.       Incubate at 37oC for 11 h.

 

Removal of Non-specifically Bound Probe

9.       Rise the slides in 50% formamide in 2 x SSC at 39oC

10.    Rinse the slides in 2 x SSC at 39oC

11.    Dehydrate the preparations in ethanol as in step 3. Air dry.

12.    The preparations are now ready for autoradiography (MSc programme).

 

 

Hybridisation to RNA in cytological Preparations

 

The best cytological technique for localizing RNA in cells according to Pardue (1983) must

 

Pretreatment of slides

  1. The cytological preparation may first be digested with protease to improve access of eh probe to cellular RNA
  2. Acetylate the prepartion to reduce non-specific binding of the probe. Alternatively, wash the preparation with physiological buffer (the composition will depend on the tissue under study), transfer to distilled water and use immediately for hybridisation. In this case, take care that no water is left on the slide to cause dilution of the probe.

Hybridisation

  1.   Hybridisation can be carried out in 0% formamide, 4 x SSC, 0.1M sodium phosphate buffer, pH 8.0, at 50oC. Somewhat more stringent conditions are obtained in 50% formamide, 5mM EDTA, 0.3 M NaCl, 20mM Tris-HCl, p 8.0 at 50oC. The  probe is generally used at 1-10 ng per slide. For DNA probes, sheared denatures E coli is added at 40ug over slide to reduce non-specific binding of the probe. For RNA probes, E coli rRNA is used instead.

                                                               i.      Prepare the hybdridisation mixture containing the labeled probe and E coli DNA or rRNA (see above) at the required concentrations. Denhardt’s solution [0.02% each of bovine serum albumin (BRL), Ficoll (Sigma), and polyvinylpyrollidone (Sigma)] can also be added  to reduce the background. As with DNA hybrids, dextran sulphate can be added to accelerate hybridization.

                                                             ii.      Place 5 μl of the hybdridisation mixture over each preparation and cover with a coverslip (18mm2). Seal the coverslip with a thick coat of rubber cement. In some cases, after methacrylate embedding, dry peparations do not wet well with the formamide solution. Therefore when using the methacrylate procedure, leave the slides in water until just before the probe is applied. Then wipe each slide and flip it hard with a finger to remove remaining drops of water. Apply the probe before the preparation dries completely. Cover each preparation with a coverslip and seal either rubber cement as before.

                                                            iii.      Incubate the slides in a closed chamber on moist paper towels at the chosen temperature (typically 50oC). Typical incubations are for 12-14 h.

Treatment of Slides after hybridization

  1. Following hybdrisation, remove the slides from the moist chambers, peel off the rubber cement and rinse off the coverslip and hybridization mixture by dipping each slide into a beaker of 2 x SSC.
  2. Remove non-specifically bound probe as follows:
    1. RNA probes

                                                               i.      Place the slides in RNase (20μg/ml) in 0.5M NAcl, 10mM Tris-HCl, pH 8.0, at 37oC for 30 min

                                                             ii.      Wash the slides in this buffer at 37oC for 30min, in 0.1 x SSC at room temperature for 10 min.

                                                            iii.      Dehydrate the preparations in 70% ethanol (2 x 10 min) and 95% ethanol (5 min). Air dry.

 

    1. DNA probes

                                                               i.      Remove the non-hybridised probe by repeated washings in the hybridization buffer at a temperature a few degrees below the temperature of hybdridisation

                                                             ii.      Wash the slides at room temperature in 0.1 x SSC

                                                            iii.      Dehydrate the preparations in 70% ethanol (2 x 10 min) and 95% ethanol (5 min). Air dry.

 

  1. The preparations are now ready for autoradiography.

 

 

12. Immunotherapy and monoclonal antibody production

 

Table: Effects of antibody–antigen interaction. After SikoraK and Smedley HM . Monoclonal antibodies. Oxford: Blackwell, p.26.

Interaction

Effects

Immune complex formation

Removal of antigen, serum sickness, disease states

Complement activation

Lysis of bacteria or cells

Macrophage activation

Cell lysis

Killer cell activation

Cell lysis

Immunoregulation

Control of immune system

 

 

Immunotherapy is the use of immune system to treat disease. This could be by providing antibodies which may be either monoclonal or polyclonal. A monoclonal antibody is one which is produced by only one clone of lymphocytes. It was developed by Kohler and Milstein in 1975.[16] When a single B lymphocyte is stimulated by an antigen, to produce antibody, the resulting antibody is monoclonal but when it involves more than one B lymphocyte it builds up polyclonal. Usually in antibody production through immunisation, antigen is injected into the animal body but various parts of the antigen have the capacity to stimulate antibody production leading to the development of polyclonal antibodies.

 

To obtain monoclonal antibody only one single lymphocyte cell (B) will be isolated and fused with a myeloma cell.

 

 

 

The resulting hybdridoma grows and divides, keeping its ability to produce the monoclonal antibody which the original lymphocyte had the ability to produce.

 

Steps in making a monoclonal antibody. After Sikora K and Smedley HM . In Monoclonal antibodies. Oxford: Blackwell, p.26.
Monoclonal antibodies have been used for recognizing subset of lymphocytes

 

Table: MCA’s recognising lymphocyte differentiation antigens. Sikora and Smedley, 1984, p. 73

Subset

MCA’s

Normal T cells

Pan T

T-cell leukaemia

OKT 3

Mycosis fungoides

L17 F12

Helper/inducer T cells

OKT 4

Sezary syndrome

Sk 4

Thymic lymphocytes

OKT 6

Thymoma

Suppressor/cytotoxic cells

OKT 8

Some T-cell neoplasms

B lymphocytes and neoplasms

anti-Ig MCA’s

 

Immunization

Freund’s adjuvant is used which is a mixture of dead tuberculosis organisms in a fatty base. It is injected into the animal, preferably rabbit on a weekly basis 3 times either subcutaneously or intraperitoneally. The final dose is given 3 days before the death of the animal. The spleen of the animal is then obtained after sacrifice. The spleen is placed in tissue culture fluid. Everything after this must be completely sterile to avoid bacterial contamination. The spleen contains both red blood cells and lymphocytes. Ficoll is dense fluid in which centrifugation occurs to separate the red cells from the lymphocytes which remains at the top of the Ficoll. Harvest lymphocytes using a Pasteur pipette and washed by centrifuge. The cells are mixed with myeloma cells to form hybdridomas.

 

The use of Ficoll to separate viable lymphocytes from a spleen cell suspension containing red cell and platelets. After Sikora and Smedley, p 15.

 

Immunotherapy and oncology

Certain surface antigens on tumours can be targeted for the production of antibodies, eg monoclonals to fight cancer. The following tumors can be treated with MCA

 

There are problems associated with use of MCA for treatment of cancer. They include

 

  1. Immune complex formation of MCA
  2. Poor vascularity of tumor
  3. Patchy antigen distribution in tumor
  4. Internalization of antigen on exposure to MCA
  5. Antigen shedding by tumor- ‘smokescreen effect’
  6. Evolution of antigen negative clones

(Sikora and Smedley, p. 106.

 

12. Oncogenes

Certain retroviruses contain genes which confer on them the ability to produce neoplasms in infected animals. These were labeled oncogenes.. Human transforming DNA sequences were also found to be homologous to various viral oncogenes. The human transforming DNA sequences were then labeled cellular oncogenes (c-onc) while the viral sequences were called viral oncogenes (v-onc). Cellular oncogenes have been detected in many malignant tumors such as 

 

 

Certain oncogenes are known to be activated in fetal tissue and thus appear that certain oncogenic sequeces could not only induce cancer but also assist in developmental processes in embryogenesis.

 

 

Some examples of viral oncogene (v-onc) and the chromosomal location of homologous human cellular oncogenes (c-onc). After Emery A, p 114.

Origin

v-onc

c-onc location

Cat sarcoma

 

 

   Synder-Theilin strain

fes

15

   McDonough strain

Fms

5

Chicken sarcoma

 

 

   Rous strain

src

20

Chicken erythroblastosis

erb-A

17

 

erb-B

7

   Myeloblastosis

myb

6

    Myelocytomatosis

myc

8

Mouse sarcoma

 

 

    Moloney strain

mos

8

Mouse leukaemia

 

 

    Abelson strain

abl

9

Rodent sarcoma

 

 

    Harvey strain

H-ras-1

11

 

H-ras-2

X

    Kirsten strain

H-ras-1

6

 

H-ras-2

12

 

N-ras

1

Simian sarcoma

sis

22

 

 

 

 

13.  Genomics

Genomics is the study of the genes which make up the genome. A genome is the totally of all DNA in an individual’s body. In human biology the genome was almost completely unraveled in the year 2003 after the completion of the so called Human Genome project.[17] The project aims mainly at identifying sequences of all human genes with a view to storing their data to be made available at fee gene sequences databases worldwide. With such gene sequences easily available the genes could be easily manufactured cloned and passed through rDNA technique which allows the synthesis of the protein products and the identification of the actions and properties of these proteins or peptides.  More importantly, such genes could be constructed for gene therapy, a method of therapeutics of the future.[18]

It is said that the genome of all human beings is the same in 99.9% of cases and only differ in the remaining 0.1%. This therefore suggests that race plays very little part in determining the variations in humans at the genetic level. Some would however wish to exploit this 0.1% in what may be called genism, but that is not true science.

 

14 Proteinomics

Proteinomics takes into consideration the synthesis and identification of protein products of genomic genes for their possible exploitation in clinical therapeutics and pharmacy.[19] The exploitation may also go far beyond therapeutics and may dabble into areas of relevant research into the functions of biological proteins and the analysis of their properties. But by far the most important areas of the utilization of proteinomic information would be in therapeutics and clinical pharmacy. While genomics studies the genome which is the totality of all DNA in an organism, protenomics studies the proteome,[20] which is the totality of all proteins in the cell.[21]

15 Protein kinases[22]and rDNA pharmaceutical products

An example of rDNA products in biology would be protein kinases which exist in 60 different rDNA products

 

15. Strategies for rDNA pharmaceutical products[23]

Modern rDNA pharmaceuticals recognize the production of these premier rDNA drugs

·        Decrease symptoms of hepatitis

·        Decrease spread of herpes zoster

·        Shrink certain tumors

o       α-IFN (α-interferon) for the treatment of certain leuakemias, Kaposi’s sarcoma, malignant melanoma, multiple myeloma and some kidney cancers.

o       β-IFN-1b- multiple sclerosis

 

 

 

 

Erythropoeitin

Others

n      MoAb against cell-surface tumor Ags can be used for diagnosis and immunotherapy

n      MoAb can be conjugated to toxins or radioactive isotypes to kill tumor cells = Immunotoxins

DNA vaccines for the following conditions

n      Malaria

n      AIDS

n      Herpes

n      Tuberculosis

n      Rotavirus (childhood diarrhea)


[1]The Southern Blot is one way to analyze the genetic patterns which appear in a person's DNA. Performing a Southern Blot involves:

1. Isolating the DNA in question from the rest of the cellular material in the nucleus. This can be done either chemically, by using a detergent to wash the extra material from the DNA,or mechanically, by applying a large amount of pressure in order to "squeeze out" the DNA.

2. Cutting the DNA into several pieces of different sizes. This is done using one or more restriction enzymes.

3. Sorting the DNA pieces by size. The process by which the size separation, "size fractionation," is done is called gel electrophoresis. The DNA is poured into a gel, such as agarose, and an electrical charge is applied to the gel, with the positive charge at the bottom and the negative charge at the top. Because DNA has a slightly negative charge, the pieces of DNA will be attracted towards the bottom of the gel; the smaller pieces, however, will be able to move more quickly and thus further towards the bottom than the larger pieces. The different-sized pieces of DNA will therefore be separated by size, with the smaller pieces towards the bottom and the larger pieces towards the top.

4. Denaturing the DNA, so that all of the DNA is rendered single-stranded. This can be done either by heating or chemically treating the DNA in the gel.

5. Blotting the DNA. The gel with the size-fractionated DNA is applied to a sheet of nitrocellulose paper, and then baked to permanently attach the DNA to the sheet. The Southern Blot is now ready to be analyzed.

In order to analyze a Southern Blot, a radioactive genetic probe is used in a hybridization reaction with the DNA in question (see next topics for more information). If an X-ray is taken of the Southern Blot after a radioactive probe has been allowed to bond with the denatured DNA on the paper, only the areas where the radioactive probe binds [red] will show up on the film. This allows researchers to identify, in a particular person's DNA, the occurrence and frequency of the particular genetic pattern contained in the probe.

 

 

[2] Schedule:

Day 1:

Digest DNA

Run an agarose gel overnight

Day 2:

Blot gel

Label the probe

Day 3:

Dry membrane (can be left at this point)

Purify probe

Pre-hyb

Hyb overnight

Day 4:

Post hyb

Expose film overnight

Day 5:

Develop film

Strip membrane

 

Blotting the Gel:

Use powder-free gloves.

1.        Stain the gel in 10 mg/l Ethidium bromide. Take a picture to see how well the enzymes cut, include a ruler.

2.        You can, if you so desire, cut the wells off of the gel. Trim one corner of the gel as a reference.

3.        Acid wash the gel for 8 minutes in 10.4 ml conc HCl + 489.6 ml dH2O.

4.        Rinse once with dH2O.

5.        Denature 2 times (15 minutes each) in 58.6 g NaCl + 20 g NaOH / liter

6.        Rinse twice with dH2O.

7.        Neutralize 2 times (15 minutes each) in 30.3 g Tris + 43.9 g NaCl + 16.75 ml HCl / liter.

8.        Rinse twice with dH2O.

9.        Blot overnight onto Magna NT with 10X SSPE. Write name on membrane with a ballpoint pen. Wet membrane in 10X SSPE and set up as shown:

  1.  

 


Labeling the Probe

1.        Bring 50ng of the DNA to be labeled to a volume of 17 µl with H2O.

2.        Boil for about 3 minutes to denature the DNA.

3.        Chill on ice.

4.        Add the following to the DNA sample:

5 µl

Oligo Labeling Buffer (OLB)

1 µl

10 mg/ml BSA (nuclease-free)

0.5 µl

dA/dT/dG mix (1mM each)

2 units

Klenow fragment of DNA Polymerase I

5.        In the Hot Lab, add

2.5 µl

alpha-(32P)-dCTP (25 µCi) (3000 Ci/mmole)

6.        Incubate at 37°C for at least 3 hours or at room temperature overnight.


Drying the Membrane

1.        Stain the gel and photograph. Did all the DNA transfer?

2.        Wash the membrane in 5X SSPE.

3.        Put the membrane on a piece of Whatman paper wetted in 10X SSPE, which is on a piece of dry Whatman paper. Crosslink @ 1200.

4.        Let the membrane sit on the bench for about 5 minutes.

5.        Bake for 2 hours at 80°C under vacuum.

6.        Store at room temperature.


Purifying the Probe

1.        To stop the reaction add:

1 µl

0.25M EDTA

1 µl

10% SDS

2.        Spot 1 µl of the stopped reaction onto each of two small pieces of DE81 filter.

3.        Wash one of the pieces of DE81, using the vacuum flask filter thing, with:

50 ml

NH4 Formate (0.3M pH=7.8)

50 ml

NH4 Carbonate (0.25M)

25 ml

95% EtOH

 

Purifying the Probe

1.        To stop the reaction add:

1 µl

0.25M EDTA

1 µl

10% SDS

2.        Spot 1 µl of the stopped reaction onto each of two small pieces of DE81 filter.

3.        Wash one of the pieces of DE81, using the vacuum flask filter thing, with:

50 ml

NH4 Formate (0.3M pH=7.8)

50 ml

NH4 Carbonate (0.25M)

25 ml

95% EtOH

4.        Measure the activity of both pieces of filter in the scintillation counter to determine the percent incorporation

  1. Measure the activity of both pieces of filter in the scintillation counter to determine the percent incorporation (70% is good). 
  2. Plug the bottom of a 1 ml disposable syringe with a little bit of glass wool and put the syringe in 15 ml conical tube.
  3. Fill the syringe with Sephadex G-50 in STE Buffer (TE + 100 mM NaCl + 0.02% NaN3).
  4. Spin the tube/syringe in the clinical centrifuge at top speed for 2 minutes. Add more Sephadex and spin again. Repeat until the packed Sephadex volume is approximately 0.9 ml.
  5. Cut the lid off of a 1.5 ml Epindorf tube. Place the decapped tube in another 15 ml conical tube. Put the syringe in the new tube so that the tip of the syringe is in the Epindorf tube.
  6. Add enough STE to the stopped probe reaction to bring the volume to 100µl.
  7. Transfer the 100µl probe reaction to the top of the Sephadex-packed syringe. Cover the top of the spin column with Parafilm.
  8. Spin the column in the clinical centrifuge for 2 minutes at top speed.
  9. Wash the reaction tube with 100µl STE. Add it to the column and spin for another 2 minutes.
  10. Remove the labeled probe from the Epindorf tube with a Pasteur pipette and dispose of the waste in the appropriate containers.
  11. Spot 1µl of the probe solution a a piece of DE81 filter. Put the filter in a scintillation vial and add 2 squirts of scintillation fluid. Measure the activity of the probe using the scintillation counter. A final concentration of 106 to 2 x 106 cpm per ml of hybridization solution is optimal.
  12. Before adding the labeled probe to the hybridization solution, boil it for 3 minutes and chill on ice.


 


Prehybridization and Hybridization

  1. Make 25 ml of prehyb solution and add it to a heat sealable bag with the membrane. Seal the bag.

Reagent

[Stock]

Volume

[Final]

Formamide

-

12.5 ml

50%

SSPE

20X

6.25 ml

5X

Denhardt's

50X

5.0 ml

10X

SDS

20%

1.25 ml

1%

Salmon Sperm DNA

10 mg/ml

0.75 ml

300 µg/ml

  1. Seal the bag in a box of water and shake at 42°C in the dry shaker for 4 hours.
  2. Cut the bag at one corner and drain out the prehyb solution.
  3. Make another 25 ml of prehyb solution and add it to the bag through the cut corner. Add the heat denatured probe to the bag (prehyb solution + probe = hyb solution). Seal the bag.
  4. Incubate with shaking at 42°C overnight.

Posthybridization

  1. Cut the bag and drain the hybridization solution into a 50 ml plastic tube. Label the tube and store at -20°C. It can be reused several times.
  2. Wash the membrane once with 500 ml 2X SSPE, 1% SDS for 25 minutes at room temperature.

Reagent

[Stock]

Volume

[Final]

SDS

20%

25 ml

1%

SSPE

20X

50 ml

2X

H2O

-

425 ml

-

  1. Wash membrane 3 times with pre-heated 0.2X SSPE, 0.2% SDS for 20 minutes at 65°C to 68°C.

Reagent

[Stock]

Volume

[Final]

SDS

20%

15 ml

0.2%

SSPE

20X

15 ml

0.2X

H2O

-

1470 ml

-

  1. Expose X-Ray film.

Stripping the Membrane

  1. Rinse the membrane in sterile, distilled H2O.
  2. Incubate the membrane in 0.2N NaOH, 0.1% SDS at 42°C with shaking for 30 minutes.

Reagent

[Stock]

Amount

[Final]

SDS

20%

2.5 ml

0.1%

NaOH

-

4 g

0.2N

H2O

-

to 500 ml

-

  1. Wash in 2X SSPE
  2. Wrap in Saran Wrap, squeezing out extra moisture with a pipette and store at -20°C. To re-probe, begin at the pre-hybridization stage.

http://www.cbs.umn.edu/~amundsen/chlamy/methods/south.html

[3] Preimplantation genetic diagnosis (PGD) offers an alternative to more traditional methods of prenatal genetic testing (chorionic villous sampling or amniocentesis), and allows genetic analysis to be performed on early embryos prior to implantation and pregnancy. This provides couples at risk for certain genetic diseases the opportunity to know that any pregnancy they achieve should be unaffected. Technical advances in molecular genetics and cytogenetics now enable GIVF physicians and scientists to be able to diagnose some inherited genetic or chromosomal disorders from a single cell of an early embryo. The information gained by PGD is used to select for replacement in the uterus only those embryos considered unlikely to be affected by the specific genetic disorder for which testing is performed.

Couples who have PGD will undergo an in vitro fertilization (IVF) cycle for the purpose of creating embryos from the woman's eggs and man's sperm which will have genetic testing prior to replacement into the woman's uterus. The genetic material of the embryos (which is derived from both parents) is not altered in any way during a PGD cycle, and early embryological development is similar to natural conception, except that it occurs in the laboratory (In Vitro, literally "in glass").

Embryos that show normal development are biopsied with micromanipulation techniques involving the use of very fine glass needles and tools under microscopic observation and control to obtain sufficient cells (blastomeres) for analysis. Based on animal experiments as well as 10 years of experience with PGD in humans, including over 200 normal births, it is felt that removal of small numbers of cells is unlikely to affect the continued development of a healthy embryo and fetus.

The cells removed from each individual embryo are analyzed by genetic testing using either PCR-based DNA amplification, or fluorescent in situ hybridization (FISH). Those embryos considered to be unaffected on the basis of this testing will then be available to be transferred into the woman's uterus or cryopreserved for future use.

The success of a PGD/IVF cycle is influenced by a number of factors. In general, the number of eggs and quality of the eggs decreases with advancing maternal age. In addition, the individual response of women to stimulation with medications which cause the ovaries to produce multiple eggs cannot always be predicted in advance. Most male-factor (sperm quality) problems can now be overcome with the use of intracytoplasmic sperm injection (ICSI). Sperm for fertilization can also be obtained from men who have undergone vasectomy or have other causes of obstructive azoospermia by the use of non-surgical sperm aspiration (NSA). The percent of fertilized eggs that will become embryos will vary depending on egg and sperm quality among other factors.

Preimplantation genetic diagnosis addresses the need of a special group of selected patients, and, because of the need for complex genetic and IVF services, it will probably continue to be limited to a few specialized centers. The Genetics & IVF Institute (GIVF) physicians and scientists combine the necessary expertise in assisted reproduction and molecular genetics to maintain an active and successful PGD program. To date, GIVF has provided PGD analysis in over 300 IVF cycles. Our clinical PGD program currently includes:

PGD for prevention of X-linked diseases. GIVF offers PGD for sex-determination in embryos, transferring only female embryos that would not be at risk to be affected with X-linked diseases such as Duchenne Muscular Dystrophy and X-linked hydrocephalus. When combined with our MicroSort® technique for sperm separation, the vast majority of embryos are female, increasing the number of unaffected embryos available for uterine transfer.

PGD for gender selection/family balancing. GIVF offers PGD for gender selection for the purpose of family balancing for couples that meet qualifying criteria.  For additional information, please go to the PGD for Family Balancing page.

PGD for Spinal Muscular Atrophy Type I. GIVF has successfully performed PGD for deletions in the survival motor neuron (SMN) gene that has been identified in 98% of SMA type I cases. Several healthy and unaffected children have been born as a result of this program. The results of our PGD program for SMA have been published in the journal Neurology. For more information about SMA click on the Families of SMA Website.

PGD for Huntington Disease. GIVF offers PGD options for individuals who are known to carry the HD gene as well as for at-risk individuals who do not desire presymptomatic testing. This Non-Disclosing HD PGD program provides the most accurate testing possible, and allows at-risk couples new reproductive options. This non-disclosing PGD program for HD is the only such program in the world.

PGD for Cystic Fibrosis (deltaF508). GIVF's PGD program for Cystic Fibrosis identifies the presence of the deltaF508 mutation, which accounts for 75% of identified CF mutations. In the majority of cases in which a couple faces a 25% to 50% risk for an affected child, one or both of them would carry the deltaF508 mutation. 

PGD for Chromosome Translocation. Couples in which one partner carries a chromosomal translocation may experience recurrent pregnancy loss or the birth of a child with multiple congenital anomalies.  PGD may be used to select which embryos to transfer in order to prevent such outcomes.  GIVF has been offering PGD for chromosome translocations since March 2001.

 PGD for Chromosomal Aneuploidy Screening.   Preliminary studies suggest that screening early embryos for common chromosomal aneuploidies may be helpful in couples undergoing IVF with a history of recurrent pregnancy loss, repeated IVF failure or advancing maternal age. DNA probes for the most common chromosomal aneuploidies associated with early miscarriage and advanced maternal age are used to perform FISH screening.

 

[4] Northern blot protocol

Stratagene suggests radiolabeling 25–50 ng of sample DNA or RNA. The

specific activity of the labeled probe should be >1 × 108 cpm/µg when using

fresh (less than 1 week old) radiolabel. Stratagene offers the Prime-It® II

random primer labeling kit for generating high-specific activity probes.

Unincorporated radiolabel must be removed from the probe prior to

hybridization. Excess radionucleotides are efficiently removed from labeled

probes using Stratagene’s NucTrap® probe purification columns.

If using other commercially available labeling or purification kits, please

follow the manufacturer’s instructions. For additional labeling and

purification protocols please consult references 1 and 2.

Hybridization of Radiolabeled Probe to the MessageMap® Blot

1. Generally, 180 µl of the MiracleHyb hybridization solution per cm2 of

the MessageMap blot should be used for standard experiments. More or

less MiracleHyb hybridization solution may be used as desired, but it is

necessary to ensure that there is sufficient solution to cover the

membrane at all times during the prehybridization and hybridization.

The table below gives recommended minimum volumes of MiracleHyb

hybridization solution for common hybridization containers.

Container*

Minimum volume of MiracleHyb

hybridization solution

50-ml conical tubes 5 ml

heat-sealable bags or roller bottles 10 ml

2. For double-stranded probes, prehybridize the MessageMap blot in

MiracleHyb hybridization solution at 68°C for 15 minutes.

For oligonucleotide probes and riboprobes, calculate the melting

temperature (Tm) (see Appendix I: Hybridization and Melting

Temperatures for the mathematical formula). Prehybridize the

MessageMap blot in MiracleHyb solution for 15 minutes at 5–10°C

below the Tm.

Note It is normal for the MiracleHyb hybridization solution to

become slightly opaque during the pre-hybridization step,

especially at higher incubation temperatures ( . 50°C).

* If 50-ml screw cap conical tubes or bottles are used, ensure that the blot is positioned with

the Stratagene logo facing inward and not pressed against the walls of the vessel. If

processing more than one blot, place each blot in a separate vessel

MessageMap® Northern Blot 5

3. Aliquot into a screw-cap microcentrifuge the correct amount of

MiracleHyb probe preparation buffer (50 µl of MiracleHyb probe

preparation buffer per 5 ml of prehybridization solution used in Step 2.)

Note Always use screw-cap microcentrifuge tubes when boiling

radioactive solutions

4. Add radiolabeled probe to the MiracleHyb probe preparation buffer and

mix by pipetting. For double-stranded probes, boil the diluted probe for

2 minutes. Briefly spin in a bench top microcentrifuge. The boiling

step is not necessary for oligonucleotide or RNA probes.

For best results, use random-primed radioactive probes with the

following concentration and specific activity:

Suggested Probe Concentration

1.0 × 106 total counts/ml of hybridization solution

Specific Activity of the Probe

108 cpm/µg or greater

5. Add the probe/MiracleHyb probe preparation buffer mixture to the

prehybridization solution containing the MessageMap blot. It is

important that the probe is added to the prehybridization solution

and is not pipetted directly onto the membrane.

Hybridization Conditions

Carry out the hybridization at 68°C. (When using oligonucleotide probes or

other short probes, perform the hybridization at 5–10°C below the Tm.)

Probe may be hybridized to the blot (on roller bars or a similar device for

gentle agitation) for as few as one to two hours for high-abundance

messages or standard Southern blot analysis. For low-abundance messages

or when maximum sensitivity is desired, hybridize overnight (.16 hours).

Washing the Blot

Perform the following membrane washes (gentle agitation is required) for

double-stranded probes, oligonucleotide probes and riboprobes:

1. Wash twice for 15 minutes each at room temperature with excess

2× SSC buffer and 0.1% (w/v) SDS wash solution§.

2. Wash once for 30 minutes at 60°C with excess 0.1× SSC buffer and

0.1% (w/v) SDS wash solution§ for a high-stringency wash.

§ See Preparation of Media and Reagents.

6 MessageMap® Northern Blot

Detection

Using forceps, grasp the MessageMap blot by one corner and lift it out of

the wash solution. Hold it vertically so that the excess liquid is allowed to

drain off. Remove the last traces of excess liquid by touching the bottom

corner of the membrane to a clean paper towel or tissue. Wrap the blot in

plastic wrap. Expose the wrapped MessageMap blot to autoradiography film

with an intensifying screen at –80°C for 2 hours–overnight. For very lowabundance

messages or increased signal intensity, film exposure may be

extended up to one week without greatly increasing background.

Stripping the Blot for Reuse

1. Heat the 0.1× SSC buffer and 0.1% (w/v) SDS wash solution to

boiling.

2. In a glass dish, pour the 0.1× SSC buffer and 0.1% (w/v) SDS wash

solution over the MessageMap blot and wash the blot twice for

15 minutes each.

Proceed with the prehybridization step for the next hybridization. If the

MessageMap blot will not be used immediately for a second round of

hybridization, remove the excess liquid from the membrane by draining (see

Detection above), then store the MessageMap blot in plastic wrap,

desiccated.

β-actin cDNA Control

Use 25 ng of the β-actin cDNA control when preparing the control probe

using random primer labeling protocols. Probes suitable for this experiment

should have a specific activity of >1 × 108 cpm/µg. The hybridization

solution for the control experiment should contain 1 × 106 cpm/ml β-actin

probe. A sharp band ~2.5 kb* should be detected on the film after 1–2 hours

at –80°C with an intensifying screen.

* In some cases the β-actin control might also hybridize to α-actin, causing two bands to

appear on the blot.

www.sratagene.com

[5]  I. Electrophoresis

 II. Sample preparation

 III. Gel run

 IV. Northern transfer of RNA

 V. Hybridization

 VI. Stripping and re-hybridization

 


Buffers:

 10 x MOPS:
0.4 M Morpholinopropanesulfonic acid (free acid); 0.1 M Na-acetate-3 x H2O; 10 mM EDTA; adjust to pH 7.2 with NaOH; store dark in fridge:
[500 ml: 41.9 g MOPS, 6.8 g NaAc, 10 ml 0.5 M EDTA]

 Loading Buffer:
1 x MOPS; 18.5 % Formaldehyde; 50 % Formamide; 4 % Ficoll400; Bromophenolblue; store at -20 °C:
[1 ml: 100 µl 10 x MOPS, 500 µl Formamide, 185 µl Formaldehyde, 40 mg Ficoll400, Bromophenolblue, 215 µl H2O]

 Prehybridization-buffer:
5 x SSC; 50 % Formamide; 5 x Denhardt's-solution; 1 % SDS; 100 µg/ml heat-denatured sheared non- homologous DNA (Salmon sperm DNA or yeast tRNA)
[100 ml: 25 ml 20 x SSC, 50 ml Formamide, 5 ml 100 x Denhardt's, 1 g SDS, 1 ml 10 mg/ml DNA]

 Hybridization-buffer:
Prehybridization buffer with 5 % Dextransulfate (Na-salt, MW 500,000, 50 % stock-solution) and without non-homologous DNA

 100 x Denhardt's solution:
[for 500 ml: 10 g Ficoll 400; 10 g polyvinylpyrrolidone MW 360000; 10 g BSA fraction V; H2O]
store at -20 °C.

 20 x SSC:
3 M NaCl; 0.3 M Na-citrate
[1 l: 175.3 g NaCl, 88.2 g NaCitrate]

 Strip-solution:
5 mM Tris pH 8; 0.2 mM EDTA; 0.05 % Na-pyrophosphate; 0.1 x Denhardt's solution
[500 ml: 2.5 ml 1 M Tris, 200 µl 0.5 M EDTA, 5 ml 5 % NaPP, 1 ml 50 x Denhardt's]


 

[6] Some strategies for treating genetic disease. Emery (1983)

Therapy

Disorders

1. Replacement of deficiency protein

 

         Antihemophilic globulin

Haemophilia

2. Replacement of deficient vitamin or coenzyme

 

          B6

Cystathioninuria

          B12

Methylmalonicadidaemia

          Biotin

Propionicacidaemia

          D

Vitamin D-resistant rickets

3. Replacement of deficient products

 

         Cortisone

Adrenogenital syndrome

          Cysteine

Homocystinuria

          Thyroxine

Congenital cretinism

          Uridine

Oroticaciduria

4. Substrate restriction in diet

 

          (a) Amino acids

 

                 Phenylalanine

Phenylketonuria

                 Leucine, isoleucine and valine

Marple syrup urine disease

                 Methionine

Homocystinuria

          (b) Carbohydrates

 

                  Galactose

Galactosaemia

           (c) Lipids

 

                   Cholesterol

Hypercholesterolaemia

           (d) Total protein

Defects in urea cycle

5. Drug therapy

 

            Aminocaproic acid

Angioneurotic oedema

            Cholestryramine

Hypercholesterolaemia

            Insulin

Diabetes

            Pancreatin

Fibrocystic disease

            Penicillamine

Wilson’s disease

6. Preventive therapy

 

            Avoidance of certain drugs

G6PD deficiency, prophyria

            Rh gamma globulin

Rh incompatibility

7. Replacement of defective tissues

 

             Kidney transplantation

Polycystic kidney disease

             Corneal graft

Congenital keratoconus

             Bone marrow

Immunodeficiency

8. Removal of noxious substances

 

              Dialysis

 

              Portocaval anastomosis

Glycogenoses

9. Removal of diseased tissues

 

              Colectomy

Polyposis coli

              Splenectomy

Hereditary spherocytosis

               Neurofibromata

Neurofibromatosis

 

[7] Non viral injection for the vasculature