PCR basics

This is a summary for the Polymerase Chain Reaction with some typical usage values. Taken from:

Molecular Cloning: A Laboratory Manual (Third Edition)
By Joseph Sambrook, Peter MacCallum Cancer Institute, Melbourne, Australia; David Russell, University of Texas Southwestern Medical Center, Dallas
2001 – 2,344 pp – ISBN 978-087969577-4
http://www.cshlpress.com/default.tpl?cart=1322569198527814047&fromlink=T&linkaction=full&linksortby=oop_title&–eqSKUdatarq=21

Chapter 8 — In Vitro Amplification of DNA by Polymerase Chain Reaction

Components

Thermostable DNA polymerase

  • Taq polymerase — 0.5-2.5 units per standard 25-50µl reaction (efficiency of ~0.7).
  • Typical contains 2×10¹² to 10×10¹² enzyme molecules.

Pair of synthetic oligonucleotides (primers)

  • Typical non limiting amount: 0.1-0.5µM (6×10¹² to 3×10¹³ molecules) — enough for 30 cycles of amplification of 1kb segment.

Deoxynucleoside triphosphates (dNTPs)

  • Equimolar amounts of dATP, dTTP, dCTP, dGTP.
  • Concentration: 200-250 µM of each dNTP (with 1.5mM MgCl2) >> 6-6.5µg of DNA in a 50µl.
  • Higher concentrations >4mM are inhibitory, but could also be lower.
  • Should be stored at -20°C in small aliquots and discarded after second cycle of freezing/thawing.

Divalent cations

  • dNTPs and oligonucleotides bind to Mg²⁺
  • Molar concentration of cation must exceed molar concentration of phosphate groups (dNTPs + primers).
  • Routinely 1.5 mM Mg²⁺ (>4.5 decreases priming).
  • Optimization by series of 0.5mM-5mM, in 0.5mM increments, and narrowing in 0.2mM.
  • Should not contain EDTA or negative íons.

Buffer to pH

  • Tris-Cl, pH between 8.3-8.8 at room temp and concentration of 10mM.

Monovalent cations

  • 50mM KCl for amplifying DNA >500bp.
  • 70-100mM improves for shorter sequences.

Template DNA

  • Single or double stranded.
  • Closed circular DNA templates are slightly less efficient.
  • If more than 10kb restriction enzymes help (when they do not cleave at the target sequence).
  • Typical 1.0µg of DNA (3×10⁵ gene copies).

Programming

Denaturation

  • Partly determined by G+C ratio (higher proportion, higher temperatures).
  • Longer strands take longer times to denaturate.
  • Recommendation: 45 seconds at 94-95°C of DNA 55% or less G+C.

Annealing of primers

  • Critical temperature!
  • If too high, poor annealing. If too low, nonspecific annealing will occur.
  • Usually 3-5°C lower than the calculated melting temperature for primer/template dissociation.
  • Trial with 2°C to 10°C degrees below melting temperature OR touchdown PCR.

Extension of primers

  • Near optimal temperature of the polymerase enzyme, Taq is 72-78°C with ~2000 nucleotides/minute.
  • 1 minute for 1000bp.

Number of cycles

  • Depends on the number of copies of template DNA.
  • 30 cycles with 10⁵ copies of target.

Inhibitors

  • Contaminants of the template DNA are normally the culprits for problems in amplification.
  • Examples: proteinase-k, phenol, EDTA, ionic detergents, heparin, polyanions, hemoglobin, bromophenol blue, xylene cyanol.
  • Solution: cleanup by dialysis, ethanol precipitation, extraction with chloroform and/or chromatography.

Contamination

  • Common problem with exogenous DNA.
  • Amplification product appearing in the negative controls (without template DNA).
  • Easier to discard all solutions, reagents and recipients and decontaminate instruments, than trying to find the source.
  • Keep traffic in the PCR lab area to a minimum.
  • Wear gloves and change them frequently; use face masks and head caps.
  • Different set of reagents for PCR.
  • Centrifuge microfuge tubes with reagents before opening in the flow hood.
  • Dilutions of DNA at the bench and take needed amounts to PCR.
  • Do not take tubes with amplified DNA to PCR area.

For basic PCR protocol (and troubleshooting tips) see page 8.18.

Reverse transcriptase-PCR

  • Amplify cDNA sequences from mRNA.
  • First enzimatic conversion of RNA to single-stranded cDNA by oligodeoxynucleotide (oligo(dT)) primer binding to poli-A mRNA tail and extended by reverse transcriptase DNA polymerase (which will be amplified by the PCR).
  • When possible primers should bind to different exons of the RNA.
  • Positive and negative controls should be done (eg, negative: without template, positive: standard synthetic RNA from a mutated DNA (trickier).

Rapid amplification of 5′ cDNA ends

  • Sequence specific primer binds to RNA and leads to the first strand of cDNA.
  • RNA and first primers are removed and a homopolymeric tail is added to the 3′ end of the cDNA (with terminal transferase).
  • Use oligo(dT) to prime the second strand of cDNA (at poly-A tail).
  • Use a sequence specific and a oligo(dT) primer to amplify the double-strand cDNA.
  • Finish by cleaving the adaptors with restriction enzymes.

Rapid amplification of 3′ cDNA ends

  • Oligo(dT) adaptor primer is annealed to mRNAs and first-strand synthesis is achieved with reverse transcriptase and dNTPs.
  • Sequence specific primer executes the second-strand synthesis.
  • Amplification continues with sequence specific primers and complementary primer to the adaptor sequence.

Rapid characterization of cloned DNA in prokaryotic vectors

  • Transformed bacterial cells are picked from colonies and transferred to PCR mixtures with primers, but without Taq.
  • Reaction mixtures are boiled to liberate template DNA and inactivate nucleases and proteases.
  • Taq is then added and 30 cycles of standard PCR is run.
  • Successful amplification yields a DNA fragment whose size can be estimated with electrophoresis and identity confirmed by sequencing.