principle and application of polymerase chain reaction PCR

APPLICATIONS OF PCR-

• PCR has many exciting and varied applications; some of these are briefly outlined below.

1. PCR can be used to amplify a specific gene present in different individuals of a species and even in different somatic cells or gametes, say, human sperms, of an individual. These copies can be used for cloning. Alternatively, they can be sequenced to obtain information on the mutational changes in the genes of different individuals, cells or gametes. Such data can be used in disease diagnosis, population genetic studies etc.



2. PCR has been used to study DNA polymorphism in the genomes using random sequences as primers. Synthetic nucleotides of 10 base length are used as random primers to amplify polymorphic DNAs having sequences specific to the primers used. 

• Such an application of PCR generates random amplified polymorphic DNAS (RAPDS, pronounced as 'rapids'), which is detected as bands after electrophoresis. RAPD bands of different strains or species can be compared. They can be used to construct RAPD maps, similar to RFLP maps (Appendix 6.III); such maps have been constructed in maize, soybean, mouse, man, etc. 

3. PCR can be used to detect the presence of a gene transferred into an organism (transgene) by using the end sequences of the transgene for amplification of DNA from the transgenic organism. Amplification will occur only when the transgene is present in the organism; the amplified DNA is detected as a band on the electrophoretic gel.

• It may be emphasized that such detections can also be done using nucleic acid hybridization (colony, dot blot, Southern and northern hybridizations). But these approaches take much longer time (several weeks) and use radioactivity, while PCR detection takes a single day and uses no radioactivity; it uses only ethedium bromide staining for detection of the DNA band produced by amplification.

4. DNA amplification of individual sperms is used to estimate the frequency of recombination between specified genes; for this application, PCR is coupled with DNA sequencing. The Recombination frequencies can be used to construct linkage maps, which were hitherto not possible in species like man.

5. Microdissected segments of chromosomes, e.g., of salivary gland chromosomes of Drosophila, can be used for PCR amplification to determine the physical location, of genes in chromosomes.

6. PCR can be used to determine the sex of embryos. Thus the sex of in vitro fertilized cattle embryos could be determined using Y chromosome-specific primers before their implantation in the uterus.

7. PCR is used to generate single strand used for DNA sequencing, and for thermal cycle sequencing, which is extensively used in automated DNA sequencing.

8. PCR can be to used to produce cDNA copies of mRNA; this is called reverse transcription PCR (RT-PCR; Section 3.3.8). Thermostable but unusual polymerase isolated from Thermus thermophilus has reverse transcriptase activity in the presence of Mn2+, but it functions as DNA polymerase in the presence of Mg2+. This raises the possibility of linking the steps of reverse transcription and PCR into one.

9. DNA fingerprinting is now almost exclusively based on PCR.

10. PCR is being used to analyse the preserved tissues of extinct species, including ancient human remains. By studying the sequence similarities in concerned genes, phylogenetic trees can be mapped out. Mitochondrial DNA, with its high copy number, has been widely used in evolutionary studies.

11. PCR has assisted in the mapping of human genome. Primers with homology to interspersed repetitive sequences (IRS) have been helpful in determining the proper order of multiple fragments of human genomic DNA being mapped.

12. PCR is finding an increasingly important application in taxonomy, e.g., in microbial taxonomy.

13. It is being applied for prenatal diagnosis of genetic diseases, e.g., sickle cell anaemia (one of the first applications of PCR).

14. RT-PCR provides information on the activity of tumour cells and viruses.

15. PCR is particularly useful in monitoring retrorival infections. For example, PCR has enabled an early detection of HIV infections in newborns; conventional detection methods can not be used in such cases since babies have sufficient antibodies from the infected another.

16. PCR can be used for detection of infection by such pathogens, which are very difficult to culture in vitro, e.g, syphilis pathogen. PCR can be performed on formalin-fixed tissues; this makes collection, storage and handling of tissues safer and convenient.

ADVANTAGES OF PCR-

• The idea of PCR is astonishingly simple, and the technique is relatively straight-forward. But its impact on molecular biology has been as immense as the discovery of restriction enzymes during the 1970s. The various advantages offered by PCR are summarised below.

1. PCR is a highly sensitive technique; it allows the detection of even a single copy of the target sequence present in the DNA samples and generates millions of copies of this sequence.

2. The quantity of DNA required for PCR is, therefore, astonishingly low: PCR uses nanogram quantities of DNA as compared to microgram quantities used for genes cloning. 

The only caveat is that the DNA sample must contain at least one copy of the target sequence.

3. PCR procedure is very rapid, requiring only few hours as compared to days or even weeks in the case of gene cloning.

4. PCR procedure is relatively very simple, and does not require specific skills. In contrast, gene cloning is tedius and requires considerable skill in planning and execution.

5. The purity of the DNA preparation is not critical in case of PCR, while it has to be of high purity in the case of cloning. But the DNA preparation must be free from inhibitors of PCR. 

6. Even degraded DNA that is unsuitable for cloning can be used for PCR. This has permitted meaningful analysis of DNA preserved in archaeological specimens, such as, bones.

7. PCR does not require difficult to store and costly restriction enzymes, DNA ligase, and vector DNA, which are essential for gene cloning; this drastically reduces the cost of PCR as compared to that of gene cloning.

8. PCR does not use radioactivity; this makes the procedure rather safe. It does use UV radiation and the mutagen ethedium bromide; therefore, appropriate caution must be exercised while handling these agents.

9. Allele-specific primers can be designed to directly detect the presence of specific alleles in individuals; this can be done with astonishing ease and speed as compared to the gene cloning procedure.

10. PCR is a highly versatile technique. As a result, it has found numerous applications, some of which are briefly described in Section 3.10.

11. The PCR technique has been modified in a variety of ways (Section 3.8) to achieve specific objectives and to resolve various problems encountered during PCR. Some of the modifications enable solution to such problems that would be very difficult, if not impossible, to resolve otherwise.

LIMITATIONS OF PCR-

• The advantages of PCR are many (Section 3.11), and its applications are expanding with time (Section 3.10). But this technique has its own limitations as outlined below, and it is unlikely that it will ever replace gene cloning, which will continue to contribute significantly to molecular biology and biotechnology.

Sequence Information-

• In order to develop the PCR primers, sequence of the border regions/flanking regions of the DNA fragment to be amplified must be known. Anchored PCR allows the isolation of a fragment even when the sequence of one end of the desired fragment is known. However, in this case, the other end of the amplified fragment has to be the site where the selected restriction enzyme cuts the DNA molecule (Section 3.8.2). In addition, it may be possible to design primers using sequence information of an equivalent gene of a different organism, e.g., sequence information for a gene in mouse may be used for the isolation of the equivalent human gene. But PCR can not be used to isolate unknown genes.

 Amplicon Size-

• The DNA sequence/fragment that is amplified by PCR is commonly called amplicon. In general, PCR is able to efficiently amplify DNA sequences of up to 3 kb, but ideally this length should be less than 1 kb. This is because of the low processivity of Taq DNA polymerase, which lacks proof-reading ability.

• Therefore, it cannot remove errors committed during replication, and stalls at the mismatches, and may even give up replication. Standard PCR techniques can amplify sequences of up to 10 kb, and special techniques can even amplify 40 kb sequences. 

• In such cases, the reaction temperature is suitably lowered (to avoid high temperature damage to bases and nicks in DNA) and the pH is increased (to compensate for the lowered temperature), and a mixture of two polymerases (one having proof- reading ability, e.g., Pfu, and the other lacking it, e.g., Taq) is used. Such a procedure is called long PCR, and several companies market cocktail enzymes, e.g., TaqPlus Long PCR system marketed by Stratagene. The efficiency of PCR, however, decreases with an increase in the amplicon size, and consistent results become more and more difficult to obtain.

• Many genes, particularly human genes, are much longer than those that can be amplified by PCR. In addition, very large (upto several megabases long) DNA sequences have to isolated and multiplied; obviously, this is impossible to achieve by PCR. These objectives can be achieved only by cloning.

Error Rate during Amplification-

• All DNA polymerases commit errors during DNA replication, which they themselves correct by 'proofreading' that is based on their 3'-5' exonuclease activity and is simultaneous to their polymerase activity. Taq polymerase, the commonly used enzyme in PCR, appears to lack proofreading activity; as a result, it is unable to rectify the errors that it commits during amplification. The error rate of Taq polymerase has been estimated as 1 error per 9,000 nucleotides. But after 30 cycles of PCR, the error rate becomes 1 error per 300 bp because each cycle of PCR copies and multiplies the errors committed in the earlier cycles; as a result, errors keep on accumulating during PCR.

• When PCR products are used directly for sequencing, the correct sequence of the amplicon will be attained inspite of the high error rate. This is because the errors are distributed randomly; therefore, for every molecule that has an error at a particular nucleotide position, there will be many molecules having the correct base at that position. As a result, the error will not create confusion. But when PCR products are cloned, the situation is not so comfortable. Any given recombinant clone will contain a single random molecule from among the millions obtained by PCR. Whether this molecule contains errors as well as the number of errors it may contain can not be ascertained. 

• Therefore, results from experiments carried out with cloned PCR products are not completely reliable. In view of this, the amplification products of PCR should be studies directly rather than being clones. In case cloning has to be resorted to for some reason, it is advisable to use a polymerase like Vent polymerase that has proofreading activity as well.

Sensitivity to Inhibitors-

• PCR amplification is sensitive to several inhibitors that may be present in the DNA preparation. Examples of inhibitors include phenolics (commonly present in plants), humic acids (often found in archaeological specimens), haem breakdown products (they form complexes with Mg2+ that is required for polymerase function), etc. Whenever inhibitors are encountered, DNA purification procedures should be modified so as to eliminate these inhibitors.

Limits on Exponential Amplification-

• The exponential amplification expected from PCR does not continue indefinitely: it proceeds up to 20 cycles or so; most PCRs then enter linear phase that soon culminates in the plateau phase (i.e., no amplification). Linear and plateau phases commence because reagent concentrations become limiting,the enzyme tires of repeated heating and high concentration of the amplification products tend to reanneal together than bind to primers. In case longer cycles of amplification are considered necessary these problems need to be addressed to.

Artefacts-

• PCR procedure can often generate artefacts. For example, if a primer is not fully extended, i.e., the replication of the amplicon is not completed, before denaturation step begins, partially synthesized polynucleotide chains will be generated. This can happen when the DNA polymerase has a low processivity or it meets a damaged base in the template.

•  The partially synthesized chains could act as primer in the subsequent amplification cycles. In such a case, a chain partially synthesized using one chromosome (= DNA duplex) as template could serve as primer for the amplification of the homologous chromosome and produce 'hybrid amplicons' (Fig. 3.25). This could be a problem in some studies especially when the locus of interest is in a heterozygous state. But this problem is expected to be rare if primer concentrations are carefully balanced.

Contamination-

• Since PCR technique is extremely sensitive, it is prone to yield erroneous results due to contaminating DNA. The contaminating DNA may originate from contaminant organisms present in the biological source an airborne cellular debris or from a contaminated reagent.  By far the most important sources of spurious templates are the products of previous PCR reactions. It is possible to prevent these problems by using good laboratory techniques and adequate control.