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sect25

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

1. 3'-end Amplification of mRNA by the PCR

(a) 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 ad 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).

(b) 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.

2. 5'-end Amplification of mRNA by the Nested PCR

(a) 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.

(b) 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.

(c) 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'.

(d) 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 adapter (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'

adaptor(#1395): 5'>GAC TCG AGT CGA CAT CG<3'

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

Reference

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

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