September 16, 1999 ECK
This technique is based on an observation made independently by John Weis ("'Race no more': an alternative approach to cloning the 5' end of transcripts", NAR 16, 3427-3428, 1994) and by me. He was doing many cycles of primer extension to map the end of cDNA, and I was attempting to reamplify megaprimers made in a previous round of PCR.
At low frequency during primer extension, high-temperature resistant polymerases, such as AmpliTaq and Pfu, will occasionally read across a molecule of primer at the end of a template, as though it were a continuation of the template. This places a complementary copy of the primer at the end of the completed strand, providing primer binding sites at both ends of the double-stranded template and enabling PCR.
Because of the low frequency of the event, many cycles are required to get a product. This number is typically 50 to 100. As opposed to other methods of reading from known to unknown regions, this method uses highly stringent conditions and results in few false bands due to double mispriming. However, ends are needed and can be provided by appropriately restricting the DNA.
John used AmpliTaq (Perkin-Elmers) and I used Pfu. I give the protocol for the latter. I was not able to reproduce the technique with Promega Taq. Both of us used air thermal cyclers from Idaho Technology, which provide steep ramps and rapid cycles. This helps to maintain stringency, decreasing the number of misprimed bands.
8 L H2O
2 L DMSO
2 L 10 x MPfuBB
2 L 4 dNTP's
2 L primer @ 5 M
2 L restricted genomic DNA, diluted 10-2 - 10-3
2 L Pfu @ 0.5 u/L
20 L total
For details on doing PCR in an air cycler, see the "Standard PCR" protocol.
Typical air cycler settings
50 - 100 cycles at ramp = 9.9 of
0" x 94,
0" x 45-65, depending on primer Tm,
30" x 72
5' x 72
Do several reactions, each with a differently restricted DNA. Use enzymes with frequent sites, such as Sau3A or AluI and don't cut to completion (10' at 37 is good enough). Don't waste time trying to optimize partial digests or purify a window of template sizes. PCR will find the correct templates and geometrically amplify them for you. As a negative control, use uncut DNA. As a positive control, reamplify an existing PCR fragment using only one of the amplifying primers (preferably the same one you are using in the experiment).
10 x MPfuBB
200 mM Tris, pH8.8 [200 L 1 M]
100 mM KCl [100 L 1M]
100 mM (NH4)2SO4 [100 L 1M]
20 mM MgSO4 [20 L 1M]
1 % Triton X-100 [10L neat]
2.5 mg/mL BSA (crystalline, not acetylated!) [25 L 100 mg/mL]
5 % Ficoll [200 L 25%]
1 mM Cresol Red [10 L 100 mM]
H2O [325 L; final vol. = 1 mL]
dGTP, dATP, dTTP, dCTP combined, each at 2 mM