Griffin:Nested RT-PCR

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% approx. 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
• The amount of cDNA utilized in RT-PCR reactions may vary depending on the nature of the RNA template; typically, 5 ul of the cDNA of reverse transcribed total RNA, 20 ul of the cDNA resulting from reverse transcribed poly(A)+ RNA, or 20
• Addition of Gelatin (0.01 mg/ml final) stabilizes Taq DNA polymerase during the PCR reaction, yielding more amplification product.

cDNA Synthesis (Reverse Transcription)

In a standard RT-PCR assay, varying amounts (1

• Prepare a solution containing:

a) 1 ?l oligo (dT)12–18 (500 ?g/ml)

b) 1 ng-5 ?g total RNA

c) 1 ?l 10 mM dNTPs

d) and add RNase-free water to a final volume of 12 ?l

• If extensive secondary structure is potentially present in the RNA, the RNA sample may be denatured at +70°C for 5 min before adding it to the reaction minimize RNA secondary structure, and placed on ice for 5 min before adding it to the reaction.

a) 4 ?l 5x reverse transcriptase buffer

b) 2 ?l 0.1 M DTT

c) 1 ? RNase inhibitor

Reverse Transcritpion Reaction

• Incubate at +30°C for 10 min minutes to anneal primer and template.

• Add 1 ?l reverse transcriptase (200 units) and incubate at 42° C for 60 minutes to extend the primer and then terminate the reaction by incubating at 70° C for 15 minutes. The RNA is subsequently reverse transcribed, resulting in cDNA synthesis.

NOTE: (As an optional step add 1 ?l RNase H (2 unit/?l) and incubate at 37° C for 20 minutes)

First PCR reaction

• Prepare a solution containing:

a) 5 ?l 10x PCR buffer (with or without MgCl2)

b) 5 ?l 25 mM MgCl2 (It may be necessary to vary the MgCl2 concentration, 2.5 mM final concentration recommended.)

c) 1 ?l 10 mM dNTP

d) 1 ?l primer pair A

e) 1 ?l Taq DNA polymerase

f) 2 ?l cDNA and add water to 50 ?l

First PCR amplification parameters

Perform 15–35 cycles of PCR. Annealing and extension conditions are primer and template dependent and must be determined empirically for each template-primer pair. Tm values for PCR primers generally range between 55-60 C

• Denaturation 96°C, 1.5 minute
• Annealing 57°C, 1 minute
• Polymerization (35 cycles) 72°C, 2 minutes
• Link; Extension (1 cycle) 70°C, 5 minutes
• Link to a 4°C Soak file.

NOTE: Addition of Gelatin (0.01 mg/ml final) stabilizes Taq DNA polymerase, yielding more amplification product.

Second (nested) PCR reaction

• Prepare a solution containing:

a) 5 ?l 10x PCR buffer (with or without MgCl2)

b) 5 ?l 25 mM MgCl2 (It may be necessary to vary the MgCl2 concentration, 2.5 mM final concentration recommended.)

c) 1 ?l 10 mM dNTP

d) 1 ?l primer pair B

e) 1 ?l Taq DNA polymerase

f) 1–5 ?l first PCR product and add water to 50 ?l

Second PCR amplification parameters

Perform 15-25 cycles of PCR. Annealing and extension conditions are primer and template dependent and must be determined empirically for each template-primer pair. Tm values for PCR primers generally range between 55-60 C

• Denaturation 96°C, 1.5 minute
• Annealing 57°C, 1 minute
• Polymerization (35 cycles) 72°C, 2 minutes
• Link; Extension (1 cycle) 70°C, 5 minutes
• Link to a 4°C Soak file.

NOTE: Addition of Gelatin (0.01 mg/ml final) stabilizes Taq DNA polymerase, yielding more amplification product.

• PCR products are visualized by agarose gel electrophoresis stained with an appropriate dye.

References

Roche Applied Science Cat. No. 11 483 188 001

Santa Cruz Biotechnology, Inc.

Sambrook, J., Fritsch, E.M. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 14.16, 14.20.