degenerate oligo design
drmax at casbah.acns.nwu.edu
Wed Dec 8 21:53:55 EST 1993
In article <2e5ulq$t8g at news.mic.ucla.edu>,
Ron Kagan <rkagan at ewald.mbi.ucla.edu> wrote:
>In article <9312071027.AA01255 at net.bio.net> Tom MacAllister,
>N490047 at UNIVSCVM.CSD.SCAROLINA.EDU writes:
>> I wish to design a degernate oligo based on peptide sequence for
>>screening a genomic library. I wondered if anyone could give me an idea
>>to the relative importance of number of possible combinations vs number
>>known bases. I have the follosing options:
>> 20mer 2048 combinations 14 known bases
>> 17mer 512 combinations 12 known bases
>> 14mer 128 combinations 9 known bases
>>Is it better to go with more known bases and hope that a lot of the
>>combinations will hybridize anyway? My feeling is that the longer oligo
>>give me more room to mess with hybridization conditions.
>I think that any of the above combinations would give you a large number
>of false positives, depending on how complex the genomic library that you
>are screening is
>(e.g.: human vs. yeast). I would go with Jon Gary's suggestion if your
>organism of choice has a well-defined codon bias. otherwise, try the
>17-mer in all combinations.
>rkagan at ewald.mbi.ucla.edu
Question: Do you only have the 7 amino acids that you are using as your
pattern for the oligo or do you have others? If you have more of the
peptide sequence you could make 2 degenerate oligos using inosines and PCR
up a short stretch to use as a probe. You could do this from genomic DNA
(even from your phage library) as long as you pick a stretch that is
unlikely to have a huge intron. I get good results with as little as
200 bases and random priming.
You don't even have to clone this piece since you can sequence directly
from the PCR product (assuming you get a single product of course) to check
that it is correct and then random prime it (gel purify first to get rid of
the primers) or even make a very hot PCR probe from 1ul of your original
reaction and more primer.
I have a question: How do inosines react when using a TMAC wash buffer for
screening plaques with an oligo? Do they constitute a mismatch?
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