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Polymerase Chain Reaction (PCR) is an in vitro technique based on the principle of DNA polymerization reaction by which a particular DNA sequence can be amplified and made into multiple copies. Using this technique scientists have now been able to study genes and proteins in a much better way and this technique has boosted the field of biotechnology the world over. This article is split into three parts and covers the following topics:

1. Part I: Principle, Components, Procedure and Stages of PCR
2. Part II: Validation, Optimization, Limitations and Applications
3. Part III: Variations or Types of PCR and Future prospects of PCR (Current Article)

Part III

Types of Polymerase Chain Reaction

1. Allele-specific polymerase chain reaction.

Allele-specific PCR is a variation of the polymerase chain reaction which is used as a diagnostic or cloning technique, to identify or utilize single-nucleotide polymorphisms (SNPs) (single base differences in DNA). Allele-specific PCR does require the sequence of the target DNA sequence, including differences between the alleles. Allele-specific PCR is a modification of the general PCR which is used as a diagnostic or cloning technique, to identify or utilize single-nucleotide polymorphisms (SNPs). The allele-specific PCR uses primers whose 3′ ends encompass the SNP. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. Allele-specific PCR was used to identify the identification of members of Anopheles culicifacies from non-vector species. Ovaries of mosquitoes were collected and chromosomes were processed for polytene chromosome plates (Green and Hunt 1980). Four primers were selected including two universal primers D3A and D3B used for amplification of D3 domain of 28S rDNA. Two allele specific primers ACA (forward, sequence 5′-GCCGTCCCCATACACTG-3′) and ACB (reverse, sequence 5′-CCGTAATCCCGTGATAACTT-3′), which are specific to species A/D and species B/C/E respectively, were selected for design of multiplex ASPCR. Since this ASPCR is targeted to rDNA, a multigene family, it discriminated Anopheles culicifacies species A and D (non-vector species) B, C and E (vector species). In another classic example, allele-specific PCR detected paternally inherited D1152H mutation in the presence of an excess of the corresponding wild-type sequence from cell-free fetal DNA circulating in maternal blood drawn from 11 pregnant women. Allele-specific PCR detected single nucleotide polymorphisms in CYP3A5 (A6986G) and MDR-1 (C3435T) genes in Indian population which has been associated with the toxicity of tacrolimus in case of renal transplant recipients. Hence the physicians can adjust the starting dose of tacrolimus in order to avoid the drug induced nephrotoxicity in the genotype patients. [14]

2. Hot start PCR.

The specificity and DNA yield of PCRs are often improved by the “hot start” technique and analogous method. It reduces non-specific amplification during the initial set up stages of the PCR. It inhibits the polymerase’s activity at ambient temperature, either by the binding of an antibody. In Brazil, Human American Tegumentary Leishmaniasis (ATL) occurs quite frequently. Oligonucleotides that amplify the conserved region of the minicircle molecules of Leishmania were used in a hot-start PCR. The technique successfully identified Leishmania kinetoplast DNA present in patients. [15]

3. Methylation-specific polymerase chain reaction.

It can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. It analysis uses bisulfite-treated DNA but avoids the need to sequence the area of interest. Primer pairs are designed to be “methylation-specific” by including sequences complementing only unconverted 5-methylcytosines, or “unmethylation-specific”, complementing thymines converted from unmethylated cytosines. Methylation is determined by the ability of the specific primer to achieve amplification. Prostate cancer which is very common in elderly people is associated with genetic alteration i.e. in hypermethylation of the glutathione S-transferase P1 (GSTP1) promoter. Primers specific for the methylated promoter were: 5′-6FAM-TTCGGGGTGTAGCGGTCGTC-3′ and 5′-GCCCCAATACTAAATCACGACG-3′. Primers specific for the unmethylated promoter were: 5′-HEX-GATGTTTGG GGTGTAGTGGTT GTT-3′ and 5′-CCACCCCAATACTAAATCACAACA-3′. The assay successfully detected hypermethylation of the GSTP1 promoter, not only in tissue samples from patients with prostate cancer, but also in a high proportion of serum and plasma samples, which contain much lower amounts of DNA.  [16]

4. Reverse transcription polymerase chain reaction.

It is one of the most commonly used polymerase chain reaction used in molecular biology. It is a sensitive method for the detection of mRNA expression levels. It involves two steps: RNA is first reverse transcribed into cDNA using a reverse transcriptase and then the resulting cDNA is used as templates for subsequent PCR amplification using primers specific for one or more genes. RT-PCR is widely used in expression profiling, which detects the expression of a gene. Reverse Transcription PCR is also used for insertion of eukaryotic genes into prokaryotes. The technique is used to for diagnosis, staging, and detection of metastatic disease in pediatric alveolar rhabdomyosarcoma (ARMS), Ewing sarcoma family of tumors (ESFT), and desmoplastic small round cell tumors. [17] Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of TNF-alpha and MCP-1 mRNA in Chinese children suffering from complications of Kawasaki disease. It was found that the value NF-kBp65 (optical density) in the PBMC cell nuclei in the Kawasaki disease group was significantly higher than the control group. Detection of NF-Bp65 plays an important role in the detection and aggravation of vasculitis in Kawasaki disease. [18]

5. Quantitative polymerase chain reaction.

Quantitative PCR is used widely to detect and quantify specific DNA sequences in scientific fields that range from fundamental biology to biotechnology and forensic sciences. It quantitatively measures starting amounts of DNA, cDNA, or RNA. HIV infected patients not responding to antiretroviral therapy are at risk of cytomegalovirus (CMV) disease. Quantitative PCR had a sensitivity and specificity of 47% and 70%, respectively for detecting cytomegalovirus (CMV) in HIV infected patients. [19]

6. Multiplex-polymerase chain reaction.

It uses multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test-run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes. That is, their base pair length should be different enough to form distinct bands when visualized by gel electrophoresis. Multiplex PCR was very useful in known mitochondrial DNA mutations in Chinese patients with Leber’s hereditary optic neuropathy (LHON). This disease is a maternally transmitted disease. Thirty-two pairs of oligonucleotide probes matched with the mutations potentially linked to the disease. Primary mutations of 11778G>A, 14484T>C or 3460G>A are main variants of mtDNA gene leading to LHON in China. [20]. Glutathione S-transferases (GSTM1, GSTT1 and GSTP1) variants are associated with the development of coronary artery disease in patients with Type-2 diabetes mellitus. Multiplex PCR successfully detected GSTM1-null genotype is associated with a 2-fold increase and GSTT1-null genotype is associated with a 3-fold increase risk to develop Type-2 diabetes mellitus. Ile/Val and Val/Val genotypes of GSTP1 also showed a significant risk for Type-2 diabetes mellitus. [21]

7. Assembly polymerase chain reaction or polymerase cycling assembly.

Assembly PCR is a polymerase chain reaction variation that artificial synthesizes long DNA sequences by performing PCR on a pool of long oligonucleotides (primers) with short overlapping segments. Mycobacterium bovis bacille Calmette-Guerin (BCG) is most important live vectors for the delivery of foreign antigens to the immune system. A recombinant BCG containing a synthetic gene coding the fragment 2 of region II of EBA-175 (F2R(II)EBA) and the repeat sequence of the circumsporozoite protein NANP was constructed using assembly PCR. [22]

8. Helicase-dependent amplification polymerase chain reaction.

It is almost similar to the traditional PCR but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA helicase is used to unwind DNA. Clostridium difficile tcdA toxin gene was amplified using helicase-dependent amplification. Clostridium difficile is associated with diarrhea. [23]

9. Inverse polymerase chain reaction.

Inverse polymerase chain reaction is a variant of PCR, and is used when only one internal sequence of the target DNA is known. It is therefore very useful in identifying flanking DNA sequences of genomic inserts. Sjogren-Larsson syndrome (SLS) is an autosomal recessive disorder disease due to the mutations in the ALDH3A2 gene for fatty aldehyde dehydrogenase, an enzyme that catalyzes the oxidation of fatty aldehyde to fatty acid. It is mostly caused by large contiguous gene deletions of the ALDH3A2 locus on 17p11.2. These deletions were detected by long distance inverse PCR and microarray-based comparative genomic hybridization. [24]

10. Intersequence-specific polymerase chain reaction.

It amplifies the region between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths. It is an efficient tool in phylogenetic classification of prokaryotic genomes in general and diagnostic genotyping of microbial pathogens in particular. Vibrio cholerae pathogenic and nonpathogenic can be identified by using Intersequence-specific PCR. Vibrio cholerae strains including 15 O1 El Tor, nine O139 and 21 non-O1/non-O139 strains were analyzed using eight ISSR primers. Only two sero groups O1 and O139 were identified to cause the disease. [25]

11. Ligation-mediated polymerase chain reaction.

This technique amplifies DNA fragments between an insertion sequence element and an MspI restriction site. Ligation-mediated PCR was use to report a new case of chronic myeloid leukemia (CML) with an e19a2 transcript, the fusion at the DNA level between BCR and ABL gene. [26]

12. Miniprimer polymerase chain reaction.

It uses “miniprimers” or “smalligos” which are about 9 to 10 nucleotide s for detecting sequences beyond those detected by standard methods using longer primers and Taq polymerase. By use of several combinations of miniprimers PCRs 16S rRNA genes from Escherichia coli or Halobacterium salinarum genomic DNA were amplified. [27]

13. Solid phase polymerase chain reaction.

During the solid-phase PCR (SP-PCR), DNA oligonucleotides complementary to a soluble template are immobilized on a surface are extended in situ. Although primarily used for pathogen detection, SP-PCR has the potential for much broader application, including disease diagnostics, genotyping, and expression studies. It was used to identify nonsense mutation and a mis-sense substitution in the vasopressin-neurophysin II gene in two Spanish kindred with familial neurohypophyseal diabetes insipidus.  The nonsense mutation in exon 3 of the gene, consisting in a G to T transition at nucleotide 2101, which produces a stop signal in codon 82 (Glu) of NPII and a G279A substitution at position -1 of the signal peptide was observed in patients. [28]

14. Touchdown polymerase chain reaction.

Touchdown PCR also called Step-down polymerase chain reaction is another modification of conventional PCR that may result in a reduction of nonspecific amplification. It involves the use of an annealing temperature that is higher than the target optimum in early PCR cycles. The annealing temperature is decreased by one degree centigrade every cycle or every second cycle until a specified or ‘touchdown’ annealing temperature is reached. The touchdown temperature is then used for the remaining number of cycles. This allows for the enrichment of the correct product over any non-specific product. Chlamydia pneumoniae, a respiratory pathogen, is risk factor for patients suffering from cardiovascular diseases. The presence of Chlamydia pneumoniae DNA was determined by real-time PCR and ompA nested touchdown PCR. [29]

Future of Polymerase Chain Reaction

Polymerase chain reaction is the wave tool of the future in molecular biology. PCR technology not only overcomes the time-consuming process using conventional culture and microscopic analysis, but also has increased sensitivity, specificity. An interesting PCR is digital PCR. Digital PCR combines the amplification and quantification power of PCR with limiting dilution of template targets. This allows not only for the quantification of PCR products but also for quantification of rare initial nucleic acid targets, important in such areas as cancer and prenatal diagnostics. Digital PCR performed on microfluidic PCR devices has been used for single-copy DNA droplet PCR, aneuploidy detection and absolute quantification of point variants.  This allows not only for the quantification of PCR products but also for quantification of rare initial nucleic acid targets, important in such areas as cancer and prenatal diagnostics.  The future of PCR holds further advances in multiplexing kinetic PCR mRNA profiling designer DNA polymerases and accessory proteins. In the future, incorporation of computer in different polymerase chain reaction will help in deciphering the entire genome of various organisms and generate more information on the evolutionary relation between organisms.

Books on Polymerase Chain Reaction (PCR)

Check out these books on PCR (polymerase chain reaction)

References

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