发信人: tops (digging), 信区: Biology
标 题: Re: A question on PCR efficiency
发信站: Unknown Space - 未名空间 (Fri Mar 5 19:35:20 2004), 站内信件

I have been working on splicing of a weak intron for some times.
When I tried to measure the portion of spliced mRNA by RT-PCR,
I actually observed similar phenomina as you got here.

In my assay, I place 2 primers around the intron (89bp) to run RT-PCR,
hoping that by measuring the ratio of the two PCR products (spliced
and unspliced) on agarose gel, I could get the efficiency of the splicing.

But we found that is not really true. When we compare PCR assay with
RNase Protection Assay, obviously in the PCR assay, there is a bias
for the spliced form.

In order to find out what was the problem, we tested quite several
conditions, for exmples, RT temperature, PCR annealing temperature,
primers etc.

It turns out RT temperature and annealing temperature played big role.
the higher the RT temperature and annealing temperature, the less the
bias.

So I believe the secondary structures of the RNA (in reverse transcription)
and of the DNA (in PCR) are the main reason for the bias. Presumably,
there must be some differences in the secondary structure of my
spliced form and unspliced form, one of the two forms is more likely
to pause or stall the respective reactions.

I think doing a similar competing PCR (both SNP in a PCR tube) in an
annealing temperature gradient is a reasonable step to test if
DNA secondary structure is involved in this bias. Of course, one needs
to have a restriction site in one of the polymorphisms in order to
resolve the PCR products on gels.

【 在 musician (自由心) 的大作中提到: 】
: When you amplify a polymorphic human DNA fragment (single nucleotide
: polymorphism, in our case), sometimes one allele is much more
: preferrably amplified by PCR - for whatever reason.
: We have a few hundred of these cases, along with thousands of cases
: where the two alleles are amplified with same kinetics (not surprisingly,
: since the two alleles have virtually identical sequence except a single
: point mutation).
: Disclaimer, this is an onging side project for me, the project kind of
: stalled after we tried a few things. If you can come up with a new
: idea that works, you can be a co-author of that. Please let me know if you
: want more info on this.


--
实验总是做不完的,
老婆可只有一个...

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