[Microbiology] Re: a question about PCR

Bob bbx107.XYZ at excite.XYZ.com
Thu Jun 8 21:44:54 EST 2006


On Thu, 8 Jun 2006 19:29:15 +0800, "Fan Cheung"
<cecilia801537 at gmail.com> wrote:

>I am a novice in this field. Can anybody give me the answer to the
>following question?
>During the PCR process,the primer that is complementary to the desired
>sequence is very important,right? 

right


>Then if the original DNA contains
>another sequence which also has complementary parts to the primer but
>not our target ,how can we ensure the final ampication is what we are
>looking for?

Well, that is up to you to check.

In part, this depends on whether you are dealing with known sequences
or unknown sequences. With known sequences, part of primer design is
to design primers that are specific for the intended sequence. This
may include a prediction as to the expected length of amplified
product.

With unknown DNA, you have to be extra careful to analyze the product
and show it is what you intended. Sequencing it is often appropriate.

Remember, you amplify what is between two primers. So simply having
one primer that has an extraneous binding site per se does not give an
extra discrete product. And sometimes, it may even be interesting that
a primer pair gives an extra product. Perhaps there is a 2nd copy of
the gene, or of a related gene. 

In any case, good question. And the bottom line answer is that you
must consider the possibility, and must ensure that your product is
what you intended.

bob



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