发信人: musician (自由心), 信区: Biology
标 题: Re: A question on PCR efficiency
发信站: Unknown Space - 未名空间 (Fri Mar 5 14:19:59 2004), 站内信件

everything you mentioned is considered, good comments, esp the last one
for example, unknown SNP/mutation in your primer region will
certainly change PCR efficiency. Or DNA modification might change
primer binding. If you are a biologist, you would think of these first.
DNA 2nd structure is certainly another thing to consider, esp. since my
amplicons are short (~70-150 bp). My student spent quite some time on
this aspect (quite some $ on software as well) and results are dubious.
For DNA 2nd structure,
you can calculate all the thermodynamcis you want based on template,
primer concentrations, ionic strength etc. But in the real experiment,
binding kinetics might be more relevant since the molecules might not
have sufficient time to reach thermodynamics equilibrium.
It will take some major effort to rule out biological artifacts such
as DNA modification/mutation.

【 在 alucart (相见不如怀念) 的大作中提到: 】
: just some wild guess.
: did you ever think about the secondary structure change caused by the snp?
: or some other factors, such as modification on the dna?
: did you try to use pcr product to pcr,see if there is some difference?
: 【 在 musician (自由心) 的大作中提到: 】
: : primer is at least 20 bp away from the mutation site,
: mislead/confine
: have


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※ 修改:．musician 于 Mar 5 14:24:45 修改本文．[FROM: 128.197.]
※ 来源:．Unknown Space - 未名空间 mitbbs.com．[FROM: 128.197.]