2 Rounds of PCR

chai_z at wehi.edu.au chai_z at wehi.edu.au
Mon Mar 8 20:49:10 EST 1993


In article <9303031439.AA03200 at horvak.hrl.dk>, hww at horvak.hrl.dk (Henrik Wengholt) writes:
> Artifact after 2 rounds of PCR
> 
> When the product obtained after one round (30 to 40 cycles) of polymerase 
> chain reaction (PCR) is subjected to a second round of PCR with the same
> primers the result is often a smear rather than the expected product. The
> smear as well as nonspecific bands are seen with most primer sets after the 
> second round of PCR even without added template (this is probably not due to
> contamination). What is the cause of the phenomenon and how can it be
> avoided ?
> P.s. In my case it is not possible to use internal (to the first primer set)
> primers for the second round of PCR. 
> 
> -- 
> Henrik Wengholt                      email:      hww at hrl.dk
> Hagedorn Research Laboratory            ph:      +45-31-68-08-60

The smear is usually a result of nonspecific binding of the primers to the
template DNA which could be caused by many factors, including too much
template, too much primers, too much dNTPs and too low annealing temp., etc.
You did not say how you set your second round PCR. However, it seems that you
had too much template in your 2nd round PCR reaction. 

Another possibility for smear formation is too many cycles run. After a certain 
numbers of cycles (30?), the further reactions will use up the primers in the
reaction very quickly. After denaturing, the PCR products (ss) will likely
anneal to the original template which is much bigger (plasmid, genomic DNA,
etc) and extend further to form bigger products (smear). Alternatively, the PCR
products have more chances to anneal to each other end by end to form bigger
products if there is too much DNA.

Hope these help.


Zhonglin Chai
Melbourne



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