Separation of specific DNA fragment
ThisIsNot at MyEmail.com
Fri Jul 28 01:44:59 EST 2000
Look up "PNA-clamping during PCR". PNAs are DNA oligo 'mimics' that
hybridize sequence-specifically to DNA (ds or ss) with greater rate
and affinity than single-stranded oligonucleotide, and are more
sensitive to base mismatches than single stranded DNA oligos.
A PNA-clamp will bind just downstream to your primer preventing
amplification of the DNA......
....unless the the PNA-clamp is not specific for that short sequence
(SNP). That is, if there is a single base mismatch, the PNA clamp
will NOT bind and amplification of the DNA frag occurs. These
amplified DNA frags, representing single-nucleotide polymorphisms in a
population, can be cloned and sequenced individually.
On 26 Jul 2000 15:45:44 GMT, benedik at uh.edu (Michael Benedik) wrote:
>Do any of you bright and witty people in cyberland have good ideas
>about how to do this.
>What I wish to do is to look at the repertoire of mutations in a
>specific gene in a population of cells. So ideally I would like to
>isolate the same DNA fragment from each cell and sequence it.
>Obviously PCR on the population of cells won't work, because that won't
>represent the diversity. I could do single cell PCR, but I am not sure
>how well that really works. I could make a library from the population
>and screen for a number of clones with the same fragment (without
>amplifying the library) but that is probably too much work (large
>genome). Are there any ways of digesting the DNA from a population of
>cells and separating out one specific fragment which I could then
>clone? Or something similar.
>Thanks for any input.
>Department of Biochemical Sciences
>University of Houston
>email:...benedik at uh.edu...
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