In this article we will discuss about the top five techniques used for cloning of specific genes and their identification. The techniques are: 1. Direct Selection of Recombinant Clones 2. Colony Hybridization 3. Complementary DNA (c-DNA) 4. Isolation of a Specific Gene: Southern Blotting 5. Polymerase Chain Reaction (PCR).
The restriction digest always contains an assortment of donor DNA fragments depending on the number of restriction sites of the enzyme used. A specific gene that is desired to be cloned may be present on a single fragment, or more often on several fragments. When the assortments of fragments are cloned into the vector DNA, only a few of the recombinants contain the desired gene or part of the gene.
Detection of such recombinants presents a problem. Several techniques have been developed to tackle this problem. One solution is to identify the desired recombinants directly with the help of the marker genes, or indirectly by the technique of colony-hybridization using a radioactive probe of the specific DNA or RNA. Another approach is to use a pre-selected DNA segment containing the desired gene for cloning.
This can be prepared from an m-RNA by reverse transcription (RT). The DNA so produced can be multiplied by the polymerase chain reaction (PCR). The combination of the two is called RT-PCR technique. A desired gene can also be isolated by the Southern blotting technique.
The principles of these techniques are discussed below:
(a) Direct Selection of Recombinant Clones:
A recombinant clone can be selected with the help of marker genes present in the vector as well as in the donor DNA. An example of direct selection can be cloning of threonine synthesis gene (thr+) in a thr+-amps host through a vector containing an ampicillin-resistance gene (ampr). In this case, the objective would be to select a clone containing the thr+ gene.
Transformation with recombinant DNA will form some host cells which have the thr+-gene. The non-trans-formant host cells fail to grow in a medium which contains ampicillin but no threonine. Also, those host cells which take up the vector DNA other than thr+ gene fail to grow in such a medium, because threonine is absent, though such host cells possess the amp’ gene carried by the vector.
Only those trans-formants are able to form colonies on such a selective medium which have taken up the thr+ gene from the donor DNA fragment and amp’ gene of the vector. The presence of these genes in a recombinant host enables it to synthesise threonine and to destroy ampicillin. As a result, such a trans-formant can form a clone of cells.
This is diagrammatically represented in Fig. 9.136:
(b) Colony Hybridization:
By this procedure, it is possible to detect the colonies of recombinants which have taken up a desired DNA segment containing a gene of choice. For detection of such colonies a sample of the DNA containing the particular gene which has been labelled with radioactivity must be available. Alternatively, a sample of radioactive m-RNA of that gene may also be used.
After the host cells are transformed by the recombinant vector DNA, they are plated on agar to allow formation of individual discrete colonies. Among these colonies, the majorities are produced by trans-formants without the desired gene, and only very few might contain it. An impression of the colonies is next transferred to a nitrocellulose filter.
They are then treated with alkali (sodium hydroxide) which causes lysis of the cells. At the same time, the DNA molecules liberated from the cells are denatured in situ to produce single-strands. At the next step, the radioactive DNA probe, denatured to produce single-strands or the radioactive m-RNA is added to the filter.
The single-stranded probe DNA or RNA is allowed to hybridize with the single stranded DNA of the colonies. After removing the unbound radioactive nucleic acid by washing, the filter is kept in contact with an X-ray film for a few weeks to detect the presence of colonies which have taken up the desired gene (auto-radiographic technique).
The procedure is schematically shown in Fig. 9.137:
(c) Complementary DNA (c-DNA):
The molecular biological techniques make it possible to prepare a DNA which contains a desired gene. Such DNA can be used for cloning into a suitable vector and be inserted into a host, so that selection of the recombinant from a large number of clones which do not contain the desired gene can be avoided. One such method is to prepare a complementary DNA or c-DNA. It is generally applicable for isolation of a gene of the eukaryotic organisms.
Certain specialized cells of eukaryotic organisms produce a particular protein in abundance e.g. the egg-albumin in the oviduct of hens, or insulin in the β-cells of the pancreas. Such cells can act as a source of the specific m-RNA which codes for the protein because the particular species of m-RNA produced in these cells forms the major portion of the total m-RNA can be isolated in pure form.
From such isolated pure m-RNA, DNA can be prepared using the enzyme reverse transcriptase which can use m-RNA as a template for synthesis of a complementary DNA (c-DNA) forming a heteroduplex, i.e. an m-RNA strand duplexed with a single-stranded DNA.
It may be recalled that the eukaryotic m-RNA molecules have a poly-A tail at the 3′-end. The poly-A tail serves as a priming site for initiation of c-DNA synthesis by reverse transcriptase, provided a short sequence of poly de-oxy-thymidine is annealed to the poly-A tail. The enzyme reverse transcriptase then adds nucleotides to poly-dT sequence in the 5′ —> 3′ direction by copying the m-RNA template.
A characteristic feature of reverse transcription is that the synthesis of c-DNA continues even after reaching the 5′-end of m-RNA for some distance producing a hairpin bend where the c-DNA itself acts as template. This extension beyond the 5′-end of m-RNA contains 10 to 20 bases. The m-RNA molecule is then removed by treatment with alkali and the single- stranded DNA is converted to a double-stranded DNA with DNA-polymerase I of E. coli.
The hairpin bend serves as a primer for DNA synthesis. Finally, the hairpin is cleaved by treatment with SI nuclease which specifically attacks single-stranded DNA. As the final form of m-RNA in eukaryotes is without any introns, the DNA produced in this way is also devoid of any introns.
However, the quantity of DNA produced is infinitesimally small and to get an usable quantity for cloning purpose, it has to be multiplied. This is achieved by the polymerase chain reaction (PCR). Thus, a sample of DNA containing a desired gene can be prepared and used for cloning into a suitable vector. The preparation of a double-stranded DNA from an m-RNA is schematically presented in Fig. 9.138.
(d) Isolation of a Specific gene: Southern Blotting:
This method is used for identification of a specific gene in one or a few DNA fragment(s) from amongst the numerous fragments produced by restriction enzyme acting on a genome. The number of fragments produced by a restriction enzyme depends on the length of the recognition sequence of the particular enzyme.
The number of fragments decreases with the length of the recognition sequence, because the probability of the presence of longer sequences on a given DNA molecule is less than that of the presence of shorter sequences.
The principle of identifying a specific gene in a DNA fragment is more or less the same as that of colony hybridization. The technique was developed by E. Southern and has come to be known as Southern blotting. The presence of a particular gene in a fragment is detected with the help of a radioactive probe containing the DNA of that gene. The probe is prepared from a cloned DNA segment of the gene which is desired to be isolated.
The first step is to cleave the genome with a suitable restriction enzyme into large number of fragments of which only one or a few will include the gene desired to be isolated. Next, the restriction digest is subjected to electrophoresis on agarose gel causing the fragments to move to different distances depending on their size i.e. their molecular weights.
Fragments of similar size thereby form separate bands in the gel. Although these bands are not visible as such, they can be made visible under ultraviolet light when treated with a fluorescent dye like ethidium bromide. The smaller DNA fragments move faster in an electric field and form bands further away from the origin than larger fragments.
After the DNA fragments have been separated by electrophoresis, they are denatured in situ by treatment with NaOH, so that the double stranded DNA molecules form single strands. This is necessary for hybridization with the radioactive probe DNA which is similarly treated to produce single strands.
The next step involves transfer of the denatured DNA bands from agarose gel to nitrocellulose filter where they are immobilized, because single-stranded DNA binds tightly to nitrocellulose and is not removed by washing. A firm contact between the gel and the filter is essential for ensuring the transfer of bands intact without diffusion.
In the following step, the filter with the immobilized DNA bands is flooded with the probe containing 32P-labelled single-stranded DNA, thereby allowing the radioactive DNA to hybridize with the homologous immobilized DNA on the filter. For this purpose, the filter is put into an airtight plastic bag for 16-20 hrs. at a suitable temperature to allow optimal in situ hybridization.
The filter is then washed to remove unbound radioactive probe, dried and auto-radiographed by placing it in contact with an X-ray film for a sufficiently long time (few days to several weeks). On developing the X-ray plate, the band or bands of DNA which hybridized with the probe can be identified.
The corresponding band or bands from an untreated parallel agarose gel electrophorogram can be taken out and DNA can be eluted from the band or bands. This DNA contains the gene from which the probe was prepared. It can be used for cloning in a suitable vector as such, or, if required, it can be multiplied by the PCR technique.
Outline of the procedure of Southern blotting in shown in Fig. 9.139:
The blotting technique developed for isolation and identification of a specific DNA fragment can also be applied for RNA with suitable modifications. The process of blotting RNA bands on agarose gel electrophorogam has been humorously called Northern blotting. Similarly, transfer of electrophoretically separated protein bands to filters has been named Western blotting. Obviously, these designations of blotting techniques have nothing to do with the directions south, north or west.
(e) Polymerase Chain Reaction (PCR):
Polymerase chain reaction is purely a biochemical procedure by means of which a minute sample of DNA can be enormously amplified within a short time without resorting to gene cloning. This powerful technique was developed by Kary Mullis in 1984 .for which he was awarded the Nobel Prize. A DNA fragment containing a selected gene produced by the c-DNA technique or Southern blotting technique can be multiplied to millions of copies by the polymerase chain reaction in a PCR machine within a few hours.
The PCR technique utilizes the same ingredients as are required for normal DNA replication viz. the precursors which are deoxyribonucleoside triphosphates — dATP, dCTP, dTTP and dGTP, the DNA polymerase, a template of single-stranded DNA and the primers which are extended by addition of deoxyribonucleotides. In normal DNA replication, the double-stranded molecule is gradually unwound forming a replication fork and the new strands are synthesized in the leading and the lagging strands using the mother strands as template.
In the PCR technique, on the other hand, another property of DNA is made use of. A characteristic feature of DNA is that on bringing it to a certain high temperature — known as the melting temperature (Tm) — the double strands fall apart into single strands by dissolution of the H-bonds. When the temperature is lowered, the two strands again anneal to restore the double-helix structure. The PCR technique utilizes this properly of DNA for replication.
In the PCR technique, small fragments of DNA having the size of an average gene can be multiplied, but a whole genome which contains thousands of genes cannot be treated. The technique essentially consists of mixing the ingredients and heating the mixture alternately to the melting temperature of the DNA and cooling to a temperature at which new DNA strands can be generated. Each strand of the sample DNA serves as a template for new strand synthesis.
The process requires primers which are short sequences of deoxyribonucleotides complimentary to the respective 3′ and 5′ ends of the sample DNA and can form base-pairs with the single strands of the sample DNA. Through completion of each cycle which takes about two minutes, the number of DNA molecules doubles.
The PCR machine is a thermal cycler in which the temperature, time, number of cycles required for amplification of the sample DNA can be preset for automatic operation. As normal DNA polymerase is thermolabile, a thermo-stable DNA polymerase obtained from a thermophilic organism is used.
This avoids the addition of the enzyme in each cyclic operation. Generally, the DNA polymerase of Thermus aquaticus is used for DNA synthesis in PCR. This enzyme, Taq-polymerase, has an optimum temperature of 72°C and the enzyme remains stable at 94°C. Other DNA-polymerases e.g. that of Pyrococcus furiosus which has an optimal temperature of growth of 100°C can also be used.
Another reason for using thermo-stable DNA polymerases is that DNA synthesized at high temperature is relatively more error-free than DNA synthesized at ordinary mesophilic temperature, like 25° to 30°C.
The cyclic events occurring in a thermal cycler of PCR are described below and they are diagrammatically shown in Fig. 9.140.
(i) The mixture containing the DNA to be amplified, the thermo-stable DNA-polymerase, the deoxyribonucleoside triphosphate precursors and the primers is taken in a tube and placed in the thermal cycler which has been preset for the different temperatures viz. 94°C, 72°C and 60°C, time of treatment at each temperature and the number of cycles to be repeated for obtaining the adequate amplification.
(ii) The operation is started by raising the temperature to 94°C for 1 minute. At this temperature, the double-stranded sample DNA denatures to form single strands, so that each strand can act as a template.
(iii) The temperature is lowered to 60°C for 1 minute during which the added primers anneal with each strand.
(iv) The temperature is next raised to 72°C for 1 minute to allow synthesis of new strands by extension of the primers. Two DNA molecules are produced. Thereby, one cycle is completed.
(v) The temperature is further raised once more to 94°C for 1 minute to denature the newly synthesized double-stranded DNA molecules into single strands which then act as templates for another round of DNA replication.
The above steps are repeated in the automated thermal cycler until DNA sample initially fed is amplified to the desired level. It should be noted that by completion of each cycle, the initial quantity of DNA is doubled. Thus in PCR, DNA increases exponentially. Within a few hours, billions of copies of the sample DNA can be obtained. Thus, a single molecule of DNA produced by reverse transcription of an m-RNA can be multiplied enormously to be used in a cloning programme.