Methods for anchored PCR/RACE
cytogen at chmeds.ac.nz
Sat Dec 5 19:44:05 EST 1992
This message was posted a month or two back, but I have had some more enquiries
so here it is again:
I have had several enquiries about anchored PCR, so here is the method in
detail. It is based on those described by Frohman et al., 1988 (PNAS 85, 8998)
and Loh et al., 1989 (Science 243, 217), and it seems to work well, at least
with RNA from mammalian cell lines. Thanks to Neil Dear, at LMB Cambridge,
who showed me this method:
5' Anchored PCR
A. cDNA synthesis:
1. 5ug total RNA (or 1ug polyA+)
0.5ug oligo-dT or a gene-specific 3' primer
1ul RNasin (or other RNase inhibitor)
Water to 13ul final vol
--> 65oC for 5 min, then place on ice.
1ul RNasin (again)
4ul of 5X buffer (0.25M Tris-HCl, pH8.3; 0.75M KCl; 50mM MgCl2, 25mM
DTT, 2.5mM all dNTPs)
1ul of Reverse Transcriptase
--> 42oC for an hour
I use LifeTech/BRL Superscript for the reverse transcription; Anglian Biotech
SuperRT (AMV) also works well - haven't tried any others. I use the buffer
supplied with the enzyme, and add in my own dNTPs (to 0.5mM final conc in rx).
3. Dilute reaction to 1ml with water. Load into a Centricon 30
microconcentrator and spin in fixed rotor such as Sorvall SS34
(other types of spin-dialysis units will probably do
as well, but make sure the MW cut-off is 30K, not 10K). I see Amicon
now have a microfuge - sized unit that uses the same membrane as
te Centricon 30, and should work fine.
4. Add another 1ml water to the concentrated sample, and spin again.
Repeat this step one more time. Each successive spin will take longer
to concentrate the sample. Using an SS34 rotor, I do successive 5KRPM
spins of 10', 20' and 45'. Final volume of sample is about 40ul.
B. Tailing of 5' end (for 5' RACE only).
1. 34ul "Centricon'd" cDNA
35ul 2X Tailing buffer (0.4M K cacodylate, 50mM Tris-HCl, pH6.9, 4mM
DTT, 1mM CoCl2, 500ug/ml BSA)
1ul 10mM dGTP
1ul (10-20 units) terminal deoxynucleotidyl transferase (TdT)
--> 37oC for at least 20 min; --> 65oC for 10 min.
2. Add water to 100ul final vol. Store tailed cDNA at -20oC.
The tailing buffer is a curse to prepare. Check out your enzyme suppliers:
LifeTech/BRL and Promega both supply TdT with buffer. Pharmacia (last time I
ordered), didn't supply buffer. Pharmacia TdT is reputed to be the best. I
currently use this enzyme in Promega buffer - when I finish the Pharmacia
enzyme I'll start on the Promega TdT.
C. PCR reaction:
1. Use 5ul tailed cDNA in 50ul reaction with C-forward primer (see below)
and 3' gene-specific primer (internal to that used for cDNA synthesis,
if one used instead of oligo-dT at step A1).
Conditions for PCR will vary, but for what it is worth, I use Promega Taq, in
the buffer they supply (contains 1.5mM MgCl2 final conc) plus 0.2mM dNTPs, at
the following parameters:
72o/2' for 40 cycles, plus final chase of 10' at 72o.
I incorporate restriction sites at the 3' end of both primers, and clone all
products directly from the PCR mix (often only a smear rather than a discrete
band is visible). The 5' primer terminates at its 3' end in either A,G or T,
not C; this minimizes the length of the polyC-tract. The 5' primer I use for
5'-GGAATTCGCGGCCGC(18)A/G/T-3' This includes sites for EcoR1 and Not1.
3' Anchored PCR:
A cDNA synthesis:
Exactly as above, except use an oligo-dT primer that terminates in A/C/G, not
T, to prime cDNA synthesis. This primer should also have a longish polylinker
tract at its 5' end. A primer which anneals only to this region, and doesn't
contain oligo-dT sequence, is then used for the PCR reaction.
I use the following primer for cDNA synthesis:
This includes an EcoR1,Xho1 and Not1 site at its 5' end.
No need to tail this cDNA; just dilute to 100ul with water and use 5ul in a
PCR reaction with your 5' gene-specific primer, and the following 3' primer:
This is much more specific than using the first oligo-dT - containing primer.
Finally, anchored PCR doesn't seem to work over regions much longer than about
500bp, at least in my experience. Some PCR reactions show a nice tight band,
others a smear - I've obtained genuine clones from both, by "shotgun" cloning
the entire PCR rx. I treat the PCR reaction by one phenol/CHCl3 extraction,
put through a G-50 spin column, digest with appropriate enzymes, and ligated
into plasmid vector. Then I just scccrreen lots'a minipreps! Promega "Magic
PCR prep" columns also seem to clean up the PCR products ok for cloning too.
I don't work for any of these companies, I just use their products!!
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