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Polymerase Chain Reaction (PCR)

The PCR technique was described in 1985, by the early 1990's the protocol was widely used by labs as a regular technique1). This successful method is used to carry out in vitro replication of target nucleic acid sequences, which is an extremely sensitive system for the amplification and detection of specific nucleic acid sequences.

The researcher ingenuity gives PCR a lot of uses, but some applications are very common, such as:

  • To amplify DNA sequences for their use in other application such as plasmid construction and gene cloning2).
  • To detect signature sequences and single out a particular species out of many samples, which could go from prehistoric samples of tissues preserved somehow to different strains of Vibrio cholerae3)4).
  • To detect specific genetic polymorphisms which are signatures for congenital illnesses or particular types of cells such as tumor cells5)6)7)8).
  • To have a quantitative estimate of the amount of transcript from a gene which a cell type has at a certain developmental stage9).

Theory

PCR is a simple yet powerful idea. It builds (polymerizes) a DNA molecule in-vitro through a series of cycles. With each cycle the amount of template sequences for the reaction doubles amplifying target molecule exponentially. This is easier to understand graphically

10)

  • 1st. DNA template is denatured at 94°C for 30s.
  • 2nd. The DNA strands anneal with the primers 55°C for 30s.
  • 3rd. The polymerase adds the required nucleotides according to the template 72°C about 60s (time depends on the size of the expected product 1kbp/min).
  • 4th. Synthesis of new (green) templates of different sizes (1st cycle).
  • 5th. Primers anneal to the new templates and the polymerase stops when it reaches the other primer (2nd cycle).
  • 6th. Each cycle brings the synthesis of new templates, all with the same length, these “grow” exponentially (remaining cycles).

PCR Standard Protocol

WARNING: Contamination can be a major problem.

  • Reagents used for PCR should be pipetted using filtered tips.
  • Making aliquots of the reagents will help avoiding contamination

BEFORE doing the reaction have these ready, these solutions are the required reactive and solutions.

  • Create primer sequences.
  • Program the PCR machine.
  • Typically, PCR is done with annealing temperatures 50°C and 60°C, for oligos that are 18-22 nucleotides.
  • Prepare the PCR stock solutions. If not already done,
  • Dissolve the two primers with, sterile, H2O. Use a filter-tip pipette.

PCR REACTION

  • Set up PCR reaction, adding the polymerase last.
  • Water 25μl final volume
  • 2.5μl 10X Perkin-Elmer buffer
  • 2μl 1.25mM dNTPs
  • 0.375μl 0.1 M MgCl2 (for 1.5mM)
  • Template DNA (2-20ng)
  • 1μl of each primer at 10μM
  • 0.2μl polymerase (5 units/μl)
  • NEGATIVE CONTROL. ALWAYS run a no-DNA control, just add a similar volume of water as the volume used when you added the DNA template.
  • POSITIVE CONTROL. If you want to be extra-careful you could set a reaction with known template and primers which ALWAYS WORK (i.e. Use pUC18 plasmid as template, with the M13 forward and reverse primers; expect a product of 100bp in length).
  • Put the tubes into the thermocycler. The program should run for about 100~150min (it depends on the cycler and the reaction requirements). Here is a typical program; some primers may work better at other annealing temperatures.
  • 96°C Denaturation 30 seconds 1 cycle
  • 94°C Denaturation 30 seconds ˥
  • 55°C Anneal for 30 seconds | 35 cycles
  • 72°C Extend for 60 seconds ˩
  • 72°C Extend for 120 seconds 1 cycle.
  • 4°C Cool down 1 forever.
  • Run out 1/2 or less of the PCR reaction on an agarose or polyacrylamide gel.
1) , 2) Persing, D. H. (1991). Polymerase chain reaction: trenches to benches. Journal of clinical microbiology 29(7), 1281-€“1285.
3) Baron, S., S. Chevalier, and J. Lesne (2007). Vibrio cholerae in the environment: a simple method for reliable identification of the species. Journal of health, population, and nutrition 25(3), 312-€“318.
4) Rohland, N., D. Reich, S. Mallick, M. Meyer, R. E. Green, N. J. Georgiadis, A. L. Roca, and M. Hofreiter (2010). Genomic DNA sequences from mastodon and woolly mammoth reveal deep speciation of forest and savanna elephants. PLoS biology 8(12).
5) Bashir, A., Q. Lu, D. Carson, B. J. Raphael, Y.-T. T. Liu, and V. Bafna (2010). Optimizing PCR assays for DNA-based cancer diagnostics. Journal of computational biology: a journal of computational molecular cell biology 17(3), 369-€“381.
6) Mayall, F., G. Jacobson, R. Wilkins, and B. Chang (1998). Mutations of p53 gene can be detected in the plasma of patients with large bowel carcinoma. Journal of clinical pathology 51(8), 611-€“613.
7) Lee, H. H., H. T. Chao, H. T. Ng, and K. B. Choo (1996). Direct molecular diagnosis of CYP21 mutations in congenital adrenal hyperplasia. Journal of medical genetics 33(5), 371-€“375.
8) Patel, K. P., Q. Pan, Y. Wang, R. W. Maitta, J. Du, X. Xue, J. Lin, and H. Ratech (2010). Comparison of BIOMED-2 versus laboratory-developed polymerase chain reaction assays for detecting t-cell receptor-gamma gene rearrangements. The Journal of molecular diagnostics: JMD 12(2), 226€-237.
9) Paakkanen, R., H. Vauhkonen, K. T. Eronen, A. Järvinen, M. Seppänen, and M.-L. L. Lokki (2012). Copy number analysis of complement C4A, C4B and C4A silencing mutation by real-time quantitative polymerase chain reaction. PloS one 7(6).
10) By Madprime (Own work) [CC0, GFDL (http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) or CC-BY-SA-2.5-2.0-1.0 (http://creativecommons.org/licenses/by-sa/2.5-2.0-1.0)], via Wikimedia Commons

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