GC Rich PCR

[Squeezymo]

Hello all!

So I have been trying to amplify a deletion in the HbA1 and HbA2 genes (the genes that code for alpha domains in hemoglobin) and so far I've had no luck. The region is about 70% GC rich, and my amplification products (depending on the primers I've used) have ranged from 2100 bp to 1000 bp. I've tried adding DMSO and betaine. No luck. I used a Qiagen kit with something called "Q solution" (pretty much betaine) and used an exact protocol somebody else used with the same primers and had no luck. I've decreased annealing temp down to 48 and increased it to 60. I've increased initial denaturation to 95 C for 15 min. In total I've run about 20 reactions and not once got a band at the correct length, and mostly got no band at all.

Any suggestions?

[admin]

Hi there,
it is easily possible to amplify even 90% GC rich regions in PCR.

One of the easiest methods is DMSO and optimizing your PCR conditions.

Have you tried 1-5% DMSO conditions - and compared all to see which is best - is it no effect or some % difference?

Have you tried optimizing your PCR steps with a PCR machine that allows different annealing temperatures at the same time (I would stick to the high end of the pcr)? What was the result - % wise?

Have you tried touchdown pcr?

You could even try increasing denaturating step a bit higher temperature with less time needed - 15 minutes is quite long.

Have you tried 2 sets of primers to test?

[HernaicTom]

Originally Posted by Squeezymo

I've increased initial denaturation to 95 C for 15 min.

Unless you're using a Hot Start Taq, your Taq might not survive this length of denaturing step....

[davidflora]

[Only registered users see links. ] The polymerase chain reaction (PCR) technique has become an indispensable method in molecular research. However, PCR-amplification of GC-rich templates is often hampered by the formation of secondary structures like hairpins and higher melting temperatures. We present a novel method termed 'Slowdown PCR', which allows the successful PCR-amplification of extremely GC-rich (>83%) DNA targets.