Blog

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

Part I

Polymerase Chain Reaction is one of the most important ingenious scientific research tools of the 20th century in molecular biology that allows exponential amplification of short DNA sequences within a longer double stranded DNA molecule. It was invented by Kary Mullis in association with Fred Faloona, Henry A. Erlich, and Randall K. Saiki in the year 1983, while he was working in Emeryville, California for Cetus Corporation. Mullis summarized the procedure: “Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat”. [1] In 1993 K.Mullis won the chemistry Nobel prize for developing PCR.

Principle

Polymerase Chain Reaction or PCR is an in vitro technique based on the principle of DNA polymerization reaction. It relies on thermal cycling consisting of repeated cycles of heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA using thermostable DNA polymerase, primer sequence (complementary to target region) and dNTPs. It thus can amplify a specific sequence of DNA by as many as one billion times. Most PCR methods can amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.

Components

The basic components and reagents required to set up a 100 ul PCR reaction are:

1. Microfuge tube.

These are small cylindrical plastic conical containers with conical bottoms with a snap cap. They are made up of polypropylene, thus can withstand a wide range of temperature.

2. Thermal cycler.

It is an apparatus used to amplify segments of DNA. It has a thermal block with holes where tubes holding the PCR reaction mixtures can be inserted. The cycler works on the principle of Peltier effect, which raises and lowers the temperature of the block in a pre-programmed manner by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration.

3. DNA template.

The reaction solution should contain at least (1e5-1e6 target molecules).

4. Primer.

These are oligonucleotides that define the sequence to be amplified. Two primer that are complementary to the 3′ (three prime) ends of each of the sense and anti-sense strand of the DNA target (Tm 52-58 degree centigrade preferred). Primers with melting temperatures above 65 degree centigrade have a tendency for secondary annealing. The GC content (the number of G’s and C’s in the primer as a percentage of the total bases) of primer should be 40-60%. [2]

Formula for primer Tm calculation:

Melting Temperature Tm(oK) = {ΔH/ ΔS + R ln(C)}, Or Melting Temperature Tm(oC) = {ΔH/ ΔS + R ln(C)}-273.15 where

ΔH (kcal/mole): H is the Enthalpy. Enthalpy is the amount of heat energy possessed by substances. ΔH is the change in Enthalpy. In the above formula the ΔH is obtained by adding up all the di-nucleotide pair enthalpy values of each nearest neighbor base pair.

ΔS (kcal/mole): S is the amount of disorder a system exhibits is called entropy. ΔS is change in Entropy. Here it is obtained by adding up all the di-nucleotide pair’s entropy values of each nearest neighbor base pair. An additional salt correction is added as the Nearest Neighbor parameters were obtained from DNA melting studies conducted in 1M Na+ buffer and this is the default condition used for all calculations.

ΔS (salt correction) = ΔS (1M NaCl) + 0.368 x N x ln([Na+])

Where
N is the number of nucleotide pairs in the primer (primer length -1).
[Na+] is salt equivalent in mM.

The primer annealing temperature is defined by the formula:

Ta = 0.3 x Tm (primer) + 0.7 Tm (product) -14.9

where, Tm(primer) = Melting Temperature of the primers

Tm(product) = Melting temperature of the product

5. Tris-HCl.

The recommended buffer solution is 10 to 50 mM Tris-HCl (pH 8.3-8.8) at 20 degree centigrade.

6. MgCl2.

It is the cofactor of the enzyme. It is beneficial to optimize the magnesium ion concentration. The magnesium ion affects the primer annealing, strand dissociation temperatures of template and PCR product, product specificity, formation of primer-dimer artifacts and enzymatic activity and fidelity. Taq DNA polymerase requires free magnesium that binds to template DNA, primers, and dNTPs.

7. KCl.

KCl is to be used for the reaction to facilitate primer annealing.

8. Gelatin or bovine serum.

Aautoclaved gelatin or nuclease-free bovine serum albumins are included to help stabilize the enzyme.

9. Distilled water.

Autoclaved distilled water was used. The volume depends on the reaction.

10. Deoxyneuclotide triphosphates.

These are the DNA building blocks. Dntp (TTP-thymidine triphosphate), dCTP (deoxycyctidine triphosphate), dATP (deoxyadenosine triphosphate) and dGTP (deoxyguanosine triphosphate) solutions neutralized to pH 7.0. Primary stock solution are diluted to 10 mM, aliquoted, and stored at -20 degree C. A working stock containing 1 mM each dNTP is recommended. The stability of dNTPs during repeated cycles of PCR is such that approximately 50% remains as dNTP after 50 cycles (Corey Levevenson, personal communication). dNTP concentrations between 20 and 200 uM is best for the reaction. The 4 dNTP should be at equivalent concentrations to minimize mis-incorporation error.

11. DNA polymerase.

It is an enzyme that catalyzes the reaction. Taq DNA polymerase isolated from Thermus aquaticus growing in hot springs acts best at 72 degree centigrade and the denaturation temperature of 90 degree centigrade does not destroy its enzymatic activity. Other thermostable enzyme like Pflu DNA polymerase isolated from Pyrococcus furiosus and Vent polymerase isolated from Thermococcus litoralis, were discovered and were found to be more efficient. A recommended concentration of Taq polymerase (Perkin-Elmer Cetus) is between 1 and 2.5 units (SA=20 units/pmol) per 100 uL reaction. However enzymatic activity will vary with respect to individual target templates or primers.

To set up a 25 or 50 uL reaction the concentration of the reagent are as follows:

Reagent 10X buffer 2.5 uL 5 uL dNTP 0.5 1 Forward primer 1 2 Reverse primer 1 2 Taq polymerase 0.15 0.3 water 18.85 uL 38.7 uL DNA (30-50 ng) 1 1 Total volume 25 uL 50    uL

Procedure

Polymerase chain reaction is built on 20-40 repeated cycles where the temperature changes in each cycle. The cycling starts with a single temperature step (called hold) at a high temperature (>90 degree Centigrade), and followed by one hold at the end for final product extension or for brief storage. [3] The various steps of PCR are:

1. Initialization step.

It is the first step of the cycle which consists of raising the temperature of the reaction to 94–96 °C or 98 °C if extremely thermostable polymerases are used, which is held for 1–9 minutes. This process activates the DNA polymerase used in the reaction. [4]

2. Denaturation step.

It consists of heating the reaction to 94-98 degree centigrade for 20-30 seconds. This helps in breaking of the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.

3. Annealing step.

The mixture is now cooled to a temperature of 50–65 degree centigrade for 20-40 seconds which helps in annealing of the primers to the single-stranded DNA template. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence that permits annealing of the primer to the complementary sequences in the DNA. As a rule, these sequences are located at the 3′-end of the two strands of the segment to be amplified.The duration of annealing step is usually 1 min during the first as well as the subsequent cycles of PCR. Since the primer concentration is kept very high relative to that of the template DNA, primer-template hybrid formation is greatly favored over re-annealing of the template strands. [5]

4. Extension/elongation step.

It is a DNA polymerase dependent process. Taq polymerase has its optimum activity temperature at 75-78 degree centigrade. The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80 degree centigrade. The temperature is now so adjusted that the DNA polymerase synthesizes the complementary strands by utilizing the 3′-OH of the primer. The primers are extended towards each other so that the DNA segment lying between the two primers is copied; this is ensured by employing primers complementary to the 3′-ends of the segment to be amplified. The duration of primer extension is usually 2 min at 72°C. Taq polymerase usually amplifies DNA fragments of up to 2 Kb; special reaction conditions are necessary for the amplification of longer segments. As a thumb rule, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute, leading to exponential (geometric) amplification of the specific DNA fragment. [6][7]

5. Final elongation.

This step is performed at a temperature of 70-74 degree centigrade for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended. [5]

6. Final hold.

In this step the mixture is allowed to cool to a temperature of 4-15 degree centigrade for short-term storage of the reaction. [5]

PCR Stages

1. Exponential amplification.

As a result of each cycle, the number of copies of the desired segment becomes twice the number present at the end of the previous cycle.

The more times the three PCR cycles are repeated the more DNA you can obtain. This is because every cycle of a PCR reaction theoretically doubles the amount of target copies, so we expect a geometric amplification. In other words PCR is an exponential process.

One could use this formula to calculate the theoretical output of any input:

Y = X (1 + efficiency) ⁿ Y=amount of amplification target

X=input copy number

n =number of cycles

Efficiency factor is given for each cycle in the kit

2. Leveling off stage.

The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.

3. Plateau stage.

The term “plateau effect” is used to describe the attenuation in the exponential rate of product accumulation that occurs during late PCR cycles. The plateau effect is affected by:

• Utilization of substrates (dNTPs or primers).
• Stability of reactants (dNTPs or enzyme).
• End-product inhibition (pyrophosphate, duplex DNA).
• Competition for reactants by nonspecific products or primer-dimer.
• Reannealing of specific product at concentrations above 1E8 M (may decrease the extension rate or processivity of Taq DNA polymerase or cause branch-migration of products strands and displacement of primers.
• Incomplete denaturation/strand separation of product at high concentration.

Books on Polymerase Chain Reaction (PCR)

Check out these books on PCR (polymerase chain reaction)

References

1. Mullis, Kary (1990). “The unusual origin of the polymerase chain reaction”. Scientific American 262 (4): 56-61, 64-65
2. “PCR Primer Design Guidelines” (http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html)
3. Rychlik W, Spencer WJ, Rhoads RE (1990). “Optimization of the annealing temperature for DNA amplification in vitro”. Nucl Acids Res 18 (21): 6409–6412
4. Sharkey, D. J., Scalice, E. R., Christy, K. G., Atwood, S. M., Daiss, J. L. (1994). “Antibodies as Thermolabile Switches: High Temperature Triggering for the Polymerase Chain Reaction”. Bio/Technology 12 (5): 506–509.
5. “Polymerase chain reaction” (http://en.wikipedia.org/wiki/Polymerase_chain_reaction)
6. Chien A, Edgar DB, Trela JM (1976). “Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus” J. Bacteriol 174 (3): 1550-1557
7. Lawyer, F., Stoffel, S., Saiki, R., Chang, S., Landre, P., Abramson, R., Gelfand, D. (1993). “High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5′ to 3′ exonuclease activity”. PCR methods and applications 2 (4): 275-287
8. Doris M. Kuehnelt, Elisabeth Kukovetz, Herwig P. Hofer, and Rudolf J. Schaur. 1994. “Quantitative PCR of Bacteriophage lambda DNA Using a Second-Generation Thermocycler” Genome Res. 1994 3: 369-37
9. G. Zangenberg, R. K. Saiki, and R. Reynolds. “MULTIPLEX PCR: OPTIMIZATION GUIDELINES”
10. Gupta PK. 1999.”Polymerase chain reaction (PCR) and Gene Amplification” Pp.70-83 in ELEMENTS OF BIOTECHNOLOGY. 1st ed. Rastogi Publications
11. Norman Arnheim, Tom White, and William E. Rainey. 1990. “The virtually unlimited uses of PCR in evolutionary biology, zoology, botany, animal behavior, conservation biology, environmental science, and ecology” BioScience 4:174-182, March 1990
12. Aimee E. Belanger, Angel Lai, Marcia A. Brackman, and Donald J. LeBlanc. 2002. “PCR-Based Ordered Genomic Libraries: a New Approach to Drug Target Identification for Streptococcus pneumoniae” ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2002, p. 2507-2512
13. SINGH BD.1998.”Recombinant DNA technology” Pp. 12-93 in BIOTECHNOLOGY.1st ed. Kalyani Publications
14. Singh OP, Goswami Geeta, Nanda N, Raghavendra K, Chandra D,  and Subbarao SK. 2004. “An allele-specific polymerase chain reaction assay for the differentiation of members of the Anopheles culicifacies complex.”
15. Claude Pirmez, Vale Ria Da Silva Trajano, Manoel Paes-Oliveira Neto, Alda Maria Da-Cruz, Sylvio Celso Gonc¸Alves-Da-Costa, Marcos Catanho, Wim Degrave, and Octavio Fernandes: 1999. “Use of PCR in Diagnosis of Human American Tegumentary Leishmaniasis in Rio de Janeiro, Brazil” J Clin Microbiol. 1999 Jun; 37(6):1819-23
16. Carsten Goessl et al. “Detection of prostate cancer using methylation-specific PCR”
17. Athale UH, Shurtleff SA, Jenkins JJ, Poquette CA, Tan M, Downing JR, Pappo AS. “Use of reverse transcriptase polymerase chain reaction for diagnosis and staging of alveolar rhabdomyosarcoma, Ewing sarcoma family of tumors, and desmoplastic small round cell tumor. 2001.” J Pediatr Hematol Oncol. 2001 Feb;23(2):99-104
18. Yin W, Wang X, Ding Y, Peng H, Liu YL, Wang RG, Yang YL, Xiong JH, Kang SX. 2011. “Expression of Nuclear Factor -κBp65 in Mononuclear Cells in Kawasaki Disease and its Relation to Coronary Artery Lesions.” Indian J Pediatr. 2011 Jun 18. [Epub ahead of print]
19. Brantsaeter AB, Holberg-Petersen M, Jeansson S, Goplen AK, Bruun JN.2007. “CMV quantitative PCR in the diagnosis of CMV disease in patients with HIV-infection – a retrospective autopsy based study.” BMC Infect Dis. 2007 Nov 6;7:127
20. Du WD, Chen G, Cao HM, Jin QH, Liao RF, He XC, Chen DB, Huang SR, Zhao H, Lv YM, Tang HY, Tang XF, Wang YQ, Sun S, Zhao JL, Zhang XJ.2011. “Du WD, Chen G, Cao HM, Jin QH, Liao RF, He XC, Chen DB, Huang SR, Zhao H, Lv YM, Tang HY, Tang XF, Wang YQ, Sun S, Zhao JL, Zhang XJ.” Dis Markers. 2011 Jan 1;30(4):181-90
21. Ramprasath T, Senthil Murugan P, Prabakaran AD, Gomathi P, Rathinavel A, Selvam GS.2011. “Potential risk modifications of GSTT1, GSTM1 and GSTP1 (glutathione-S-transferases) variants and their association to CAD in patients with type-2 diabetes” Biochem Biophys Res Commun. 2011 Apr 1; 407(1): 49-53. Epub 2011 Feb 23
22. Rapeah Suppian, Zainul Fadziruddin Zainuddin, Mohd Nor Norazmi. 2006. “CLONING AND EXPRESSION OF MALARIA AND TUBERCULOSIS EPITOPES IN MYCOBACTERIUM BOVIS BACILLE CALMETTE-GUERIN” Malaysian Journal of Medical Sciences, Vol. 13, No. 1, January 2006:13-20
23. Chow WH, McCloskey C, Tong Y, Hu L, You Q, Kelly CP, Kong H, Tang YW, Tang W. 2008. “Application of isothermal helicase-dependent amplification with a disposable detection device in a simple sensitive stool test for toxigenic Clostridium difficile.” J Mol Diagn. 2008 Sep;10(5):452-8. Epub 2008 Jul 31
24. Engelstad H, Carney G, Saulis D, Rise J, Sanger WG, Rudd MK, Richard G, Carr CW, Abdul-Rahman OA, Rizzo WB. 2011. “Large contiguous gene deletions in Sjogren-Larsson syndrome” Mol Genet Metab. 2011 May 30. [Epub ahead of print]
25. Ravi Kumar A, Sathish V, Balakrish Nair G, Nagaraju J. 2007. “Genetic characterization of Vibrio cholerae strains by inter simple sequence repeat-PCR” FEMS Microbiol Lett. 2007 Jul;272(2):251-8. Epub 2007 May 22
26. N Boeckx, M W J C Jansen, C Haskovec, P Vandenberghe, V H J van der Velden and J J M van Dongen. 2005 “Identification of e19a2 BCR-ABL fusions (mu-BCR breakpoints) at the DNA level by ligation-mediated PCR” Leukemia. 2005 Jul;19(7): 1292-5
27. Isenbarger TA, Finney M, Rios-Velazquez C, Handelsman J, Ruvkun G. 2008. “Miniprimer PCR, a new lens for viewing the microbial world.” Appl Environ Microbiol. 2008 Feb;74(3):840-9. Epub 2007 Dec 14.
28. Calvo B, Bilbao JR, Urrutia I, Eizaguirre J, Gaztambide S, Castano L.1998. “Identification of a novel nonsense mutation and a missense substitution in the vasopressin-neurophysin II gene in two Spanish kindreds with familial neurohypophyseal diabetes insipidus” J Clin Endocrinol Metab. 1998 Mar;83(3):995-7
29. Schiavoni G, Di Pietro M, Ronco C, De Cal M, Cazzavillan S, Rassu M, Nicoletti M, Del Piano M, Sessa R.2010 “Chlamydia pneumoniae infection as a risk factor for accelerated atherosclerosis in hemodialysis patients.” J Biol Regul Homeost  Agents. 2010 Jul-Sep; 24 (3):367-75