jladasky at cmgm.Stanford.EDU
Sat Feb 7 20:26:44 EST 1998
In article <34DBE24F.2D189955 at columbia.edu>,
Joel Guss <jg183 at columbia.edu> wrote:
>We are trying to find a PCR to genotype mutant mice with a LINE element
>insert. The primers to detect presense of a WT gene are on either
>sides of where the insert is and work well. One of the primers used
>to detect the LINE insert is obviously in the LINE element. PCR with
>this primer, though, gives many nonspecific nonreproduceable bands
>in both wild type and mutant mice!
>This is most likely due to the presense of large numbers of these
>inserts that contain very homologous sequences thorughout the genome.
>Any primer we select even has at least three good anealing sites just
>within the one LINE element we have!
>Does anyone have any experience with this and might be able to lend
>some advice? We would prefer not to have to use Southerns to genotype
LINE elements are typically about 6 kB long, correct? I have
recently experienced something similar. I have cloned a fragment which
contains an Alu repeat. I placed one of my sequencing primers inside
the Alu repeat, and for this purpose the primer works fine. But when I
try PCR with it, I get a huge smear.
If you're feeling a little ambitious, you could try doing a long
PCR assay. In order to pull this off you will need to know a bit of se-
quence clear on the other side of the LINE sequence from where you're
working now. Maybe it's the same as the sequence opposite the break-
point in the wild-type animal? Do you know this already? Maybe you can
use the primers that you're using for the wild-type analysis, just with
long PCR conditions. If not, I would just genotype using a Southern
blot -- it will take you more work to isolate the new sequence you need
and develop a reliable long PCR. But, if you have this information, try
a long PCR; in the animals that have the insert, you will get a 6 kB+
band. In animals that lack the insert, you will get the band you already
have, probably a few hundred bases. Heterozygous animals might be dif-
ficult to call, because the small fragment will amplify much more effici-
ently than the large fragment and will compete for polymerase, dNTP's and
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw. - John Ladasky
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