Griffin:Nested RT-PCR

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What is nested PCR?

The amplification of RNA requires the conversion of the RNA substrate into DNA. This is achieved through the use of a reverse transcriptase such as AMV RT (avian myeloblastis virus reverse transcriptase) or M-MuLV RT (moloney murine leukemia virus reverse transcriptase). The resulting cDNA can be used as a template for a standard PCR.

Nested PCR means that two pairs of PCR primers were used for a single locus. The first pair generates an amplicon within the locus as seen in any PCR experiment. The second pair of primers (nested primers) bind within the first amplicon and produce a second PCR product that will be shorter than the first one. The logic behind this strategy is that if the wrong locus were amplified by mistake, the probability is very low that it would also be amplified a second time by a second pair of primers.

Primer Tm Values

Tm values for PCR primers generally range between 55-60 C (19-21 nt, GC% ~55%). The A and B nested primer sets share similar base pair length, GC% and Tm values.

Nested PCR utilizes two pairs of PCR primers for a single locus. The first primer pair A set amplifies within the locus. The second primer pair B set (nested primers) then binds within the 'A' amplicon to produce a second nested 'B' amplicon.

Reagents

  • Reverse Transcriptase
  • Deoxynucleotide Mix, dATP, dCPT, dGTP, dTTP, 10 mM each, in sterile double-distilled water, pH 8.5
  • Reaction Buffer, 10x conc., 1.05 ml; 100 mM Tris-HCl, 500 mM KCl, pH 8.3 (20°C)
  • MgCl2 Stock Solution, 2 x 1.3 ml each 25 mM MgCl2
  • Gelatin, 0.05% gelatin (w/v)
  • Oligo-p(dT)15 Primer, 0.02 A260 units/µl (0.8 µg/µl)
  • RNase Inhibitor, 50 U/µl
  • Sterile Water

PCR Optimization

  • MgCl2 concentrations may vary depending on the template, primer, and dNTP concentrations in the amplification reaction. To optimize conditions,

use a MgCl2 titration, generally between 0.5 and 10 mM.

  • Primer concentrations may vary; typical final concentrations range from 0.01 to 0.5
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