Different schemes of PCR

Content
Genetic Engineering and Biotechnology 1.  Recombinant DNA and PCR (Cloning and Amplification of DNA)
Restriction enzymes in cloning
Techniques used in recombinant DNA 
Cloning vectors for recombinant DNA
Plasmids as vectors
Bacteriophages as vectors
Plant and animal viruses as vectors
Transposons as vectors
Artificial chromosome vectors for cloning large DNA segments
Construction of chimeric DNA
Palindromes and staggered cleavage
Adding poly dA at the 3' ends of the vector and poly dT at the 3' ends of DNA clone
Blunt end ligation by T4 DNA ligase
Cloning in bacteria and eukaryotes
Cloning in bacteria
Cloning in eukaryotes
Molecular probes 
Labelling of probes
Applications of molecular probes
Construction and screening of genomic and cDNA libraries
Gene amplification : PCR and its applications
cDNA library from mRNA
Colony (or plaque) hybridization for screening of libraries
Gene Amplification : PCR and Its Applications
The basic polymerase chain reaction (PCR)
Different schemes of PCR


Different schemes of PCR
In the previous section, we discussed the basic outline of PCR technique, outlining how billions of copies of a DNA sequence can be obtained within a few hours, without the use of a vector and a host as done in gene cloning. However, there are several variations to this basic PCR technique, which allow varied applications of this technique.

Inverse PCR. In this technique, amplification of those DNA sequences takes place, which are away from the primers and not those which are flanked by the primers. For instance if the border sequences of a DNA segment are not known and those of a vector are known, then the sequence to be amplified may be cloned in the vector and border sequences of vector may be used as primers in such a way that the polymerization proceed in inverse direction i.e. towards the inserted segment, and not away from it towards the DNA sequence of vector from which primers have been derived (Fig. 39.21a). Similarly, if the gene sequence is known, one can use its border sequences as primers to amplify the sequences flanking this gene e.g. the regulatory sequences (Fig. 39.21b).
 
Inverse PCR : (a) use of vector border sequences as primers for amplification of inserted sequence, (b) use of border sequences of a gene to amplify its flanking regulatory sequences. (Modified and redrawn from Dharmalingam, K.-Biospectra, Nov. - Dec, 1990 page 3-8).
Fig. 39.21. Inverse PCR : (a) use of vector border sequences as primers for amplification of inserted sequence, (b) use of border sequences of a gene to amplify its flanking regulatory sequences. (Modified and redrawn from Dharmalingam, K.-Biospectra, Nov. - Dec, 1990 page 3-8).


Anchored PCR. In the basic PCR technique and the inverse PCR, one has to use two primers representing the sequences lying at both ends of sequence to be amplified. But sometimes, we may have knowledge about the sequence at only one of the two ends of the DNA sequence to be amplified. In such cases anchored PCR may be used, which will utilize only one primer instead of two primers. In this technique, due to the use of one primer, only one strand will be copied first, after which a poly G will be attached at the end of the newly synthesized strand. This newly synthesized strand with poly G tail at its 3' end will then become template for the daughter strand synthesis utilizing an anchor primer with which a poly C sequence is linked to complement with poly G of the template. In the next cycle, both the original primer and anchored primer will be used for gene amplification (Fig. 39.22).

PCR for site directed mutagenesis. This technique is used for introducing mutations at the desired place in a DNA sequence by altering the sequences of primers (Fig. 39.23). Since mutations are introduced only through primers, mutations are limited to the ends of the gene sequence. A variation of this technique allows mutations to any place of interest in the gene-the method is described as overlap extension which works as follows : (i) In two separate PCR reactions, a particular gene is amplified into two separate segments. In both reactions there is one primer at the end of the gene and the other internal to the sequence. The internal primers in two reactions are complementary to one another, so that the amplified products will have their ends internal to the original sequence. These internal ends of products in two reactions will overlap. The sequences of internal primers can be suitably modified to introduce alterations in the overlap region. (ii) The two PCR products are denatured and annealed, so that the internal ends in the overlap region will work as primers for each other.

Extension of these primers results in the formation of a complex gene, with the mutation incorporated into it (Fig. 39.24).


Directed mutagenesis (at an internal site) through PCR, using 'overlap extension' technique. (Modified and redrawn-from Dharmalingam, K.-Bio-spectra, Nov.-Dec., 1990 page 3-8).
Fig. 39.24. Directed mutagenesis (at an internal site) through PCR, using 'overlap extension' technique. (Modified and redrawn-from Dharmalingam, K.-Bio-spectra, Nov.-Dec., 1990 page 3-8).
 
Anchored PCR, using only one primer at the initial stage of amplification. (Modified and redrawn from Dharmalingam, K.-Biospectra, Nov, -Dec, 1990 page 3-8).
Fig. 39.22. Anchored PCR, using only one primer at the initial stage of amplification. (Modified and redrawn from Dharmalingam, K.-Biospectra, Nov, -Dec, 1990 page 3-8).

Directed mutagenesis (at the end of a gene sequence) through PCR, using a primer with altered base seqence. (Modified and redrawn-from Dharma lingam, K.-Biospectra, Nov.— Dec. 1990 page 3-8).
Fig. 39.23. Directed mutagenesis (at the end of a gene sequence) through PCR, using a primer with altered base seqence. (Modified and redrawn-from Dharma lingam, K.-Biospectra, Nov.— Dec. 1990 page 3-8).

RAPDs using random primers. PCR has also been used for developing molecular markers termed 'Randonly Amplified Polymorphic DNAs' (RAPDs, pronounced as 'rapids'). In this technique instead of using a pair of primers, a single short oligonucleotide primer is jsed, which binds to many differnt loci and allows amplification of random sequences from a complex DNA template (e.g. whole genomic DNA from a plant or animal). This has several advantages over RFLPs earlier used as molecular markers (for more details, consult next main topic of genetics).