Rapid Amplification of mRNA Ends By PCR (RACE)

Buffers

10X PCR buffer

100 mM Tris.Cl (pH 8.3)
500 mM KCl
15 mM MgCl2

10X tailing buffer

1 M potassium cacodylate
250 mM Tris.Cl (pH 7.6)
10 mM CoCl2
2 mM DTT
3'-end Amplification of mRNA by the PCR

Synthesize the first strands of cDNAs using a (dT)17-adaptor primer.

Dissolve 1-5 ug of poly (A)+ RNA or 10-20 ug of total RNA in 10 ul of water (DEPC treated and autoclaved beforehand). Heat at 65°C for 5', chill on ice.

Add 2 ul of 10X PCR buffer, 2 ul of dNTP's (each 10 mM), 2 ul of DTT (10 mM), 0.5 ul of RNasin, 3 ul of (dT)17-adaptor (100ng/ul) and 1 ul of AMV reverse transcriptase.

Incubate the reaction at 42°C for 1-2 hr, then heat at 95°C for 5'. Dilute the reaction mixture with 180 ul of TE (cDNA poll).

Amplification of the target cDNA.

1-10 ul aliquots from above cDNA poll, add 10 ul of 10X PCR buffer, 4 ul of adaptor (50 ng/ul), 4 ul of gene-specific primer (50 ng/ul), 10 ul of dNTP's (2 mM each), water to 100 ul. Then add 0.5 ul of Taq polymerase.

Do 40 cycles (cycle conditions depend on primer, size of the PCR product etc.)

Load 10 ul aliquots of PCR products plus 5 ul of sucrose loading dye on a agarose gel.

Southern blot analysis can be further performed using a internal gene-specific probe.

5'-end Amplification of mRNA by the Nested PCR

Synthesize the first strand of cDNA using a gene-specific primer 1.

Reverse transcribe RNA as above, but substitute 3 ul of gene-specific primer 1 (50 ng/ul) for (dT)17- adaptor.

Remove excess primer 1 by a centricon-100 spin filter

After heating the RT mixture at 95°C for 5', dilute with 2 ml of water, and transfer to a centricon-100, centrifuge at 3.5K (SW34 rotor) for 45'. Repeat and collect retained liquid.

Tail the 3' end of the target cDNA

Heat 15 ul aliquots of above first-strand cDNA at 65°C for 5', chill on ice.

Add 2 ul of 10X tailing buffer, 2 ul of 10 mM dGTP (or dATP), 1 ul of terminal transferase.

Incubate the reaction mixture at 37°C for 10-15'.

Heat at 65°C for 10'. Then add 200 ul of TE, extract by phenol/chloroform.

Dilute the mixture with 2 ml water. Transfer to a centricon-100 spin filter and Centrifuge at 3.5 krpm for 45'.

Amplification of the tailed cDNA by the PCR.

Do the PCR as above, using 10 ul of the tailed cDNA, 4 ul of gene-specific primer 2 (50 ng/ul), and 4 ul of (dC)15-adaptor (if the template was tailed by dGTP) or 4 ul of (dT)17-adaptor and 4 ul of adaptor (if the template was tailed by dATP). Primers concentration is 50 ng/ul.

NOTES:

  1. Primers:

(dC)15-adaptor(#1504): 5'>AAA AGA TCT GTC GAC CCC CCC CCC CCC CC<3'

(dT)17-adaptor(#1394): 5'>GAC TCG AGT CGA CAT CGA TTT TTT TTT TTT TTT TT<3'

  1. In the nested PCR, gene-specific primer 2 should located in the first-strand cDNA.

Reference:

Frohman, M.A., Dush, M.K., & Martin, G.R. (1988) Proc. Natl. Acad. Sci. USA 85, 8998-9002.