3'RACE PCR

3' R apid A mplification of c DNA E nds (RACE ) PCR

This technique is used to obtain the 3'end of a cDNA, it requires some sequence information internal to the mRNA under study. The sequence information obtained from this technique can be utilised to obtain full length cDNA clones using the 5'RACE technique.

The method I've used is loosely based on that given in 'PCR protocols: A guide to methods and applications', Academic Press Inc.

The protocol given uses SuperScript II reverse transcriptase (Life Technologies) and Taq polymerase from Boehringer Mannheim. You will need to adjust the reagents according to the enzymes you use.

Reagents:

Sterile water (high purity, RNase-free)

5 x SuperScript RTase buffer (Life Technologies)

100mM DTT

2.5mM dNTPs

RNase inhibitor (Pharmacia)

SuperScript RTase (Life Technologies)

10 x PCR buffer (Boehringer Mannheim, contains 15mM MgCl2 )

500mM dNTPs

Mineral oil

RNA : Both total and polyA RNA are suitable for this technique. I recently compared the two and found the polyA RNA reaction to have the edge, but only just. I do recommend using polyA RNA for 5'RACE though.

Primers:-

T17 Adapter primer (T17AP): GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT

Adapter primer (AP): GACTCGAGTCGACATCG

Gene specific primer: Designed from known sequence, ideally it should an 18-22-mer with an annealing temperature of around 56℃, where a G/C = 4℃ and an A/T = 2℃.

Protocol

1. Place approx. 1ug of total RNA (~10ng polyA RNA) in an RNase-free eppendorf, make up to 10μl with pure water. Add 1μl of T17AP (0.5ug/ul).

2. Heat this to 70℃ for 10 minutes, then place on ice for 5 minutes.

3. Spin pulse the tube and then add the following reagents:

4μl 5X SuperScript RTase buffer

2μl 100mM DTT

1μl 2.5mM dNTPs

1μl RNase inhibitor

1μl SuperScript RTase.

4. Mix and incubate at 37℃ for 2 hours.

5. Use a serial dilution of the reverse transcription reaction: 2μl of undiluted cDNA, 2μl of 10 x diluted cDNA and 2μl of 100 x diluted cDNA in the PCR reaction. Set up the PCR as follows:

35.5μl pure water

5μl 10 x PCR buffer (Boehringer Mannheim)

5μl 500uM dNTPs (some enzymes require a higher concentration of dNTPs (2mM), for example Pfu polymerase (though I'd recommend against the use of this enzyme)

1μl Adapter primer (25uM)

1μl Gene specific primer (25uM)

2μl cDNA template (various dilutions)

6. Mix and overlay with mineral oil.

7. Perform the following PCR cycles:

a. 94℃ 3 minutes

b. 72℃ 3 minutes (at this point add 0.5μl of Taq polymerase)

c. 94℃ 30 seconds

d. 56℃* 1 minute

e. 72℃ 2 minutes (longer if expected fragment size is greater than 2kb)

f. Cycle steps c. - e. 35 times

g. 72℃ 10 minutes.

* Varies according to the melting temperature of your gene specific primer. The annealing temperature in the PCR may require some adjustment for optimal yield of PCR product.

8. Load all or part of the reaction on an agarose gel. Don't be too surprised if you get a faint background smear as well as your prominent band.