[mti] Re: Unusual regular PCR bands in non targets
- From: "simon jarman" <simon.jarman@xxxxxxxxx>
- To: mti@xxxxxxxxxxxxx
- Date: Tue, 27 Mar 2007 15:50:23 +1000
It is possible that the bands are concatamerised primers. If there is
sequence complementarity between the primers they can prime from each
other and prime from the products of previous rounds, producing
ever-longer ladders. Does the pattern show up in the negative control
as well as in the non-target species?
We had something vaguely similar when amplifying ancient DNA of
copepods from lake sediments that we speculated may have actually been
concatamerised degraded DNA. The short DNA fragments that you get from
purifying ancient / dietary DNA could potentially act as primers upon
each other. If the banding doesn't show up in the negative control,
this could be the case. In the sediment DNA study we were able to get
rid of the effect by using a nested PCR approach.
On 3/27/07, Peters, Kristian (PIRSA - SARDI)
<peters.kristian@xxxxxxxxxxxxxxxx> wrote:
Good work Simon organising this discussion group. I am sorry I have no answers
as yet. I also have a question. I have designed a species specific primer that
targets mtDNA of a marine species used in a feeding trial. It works fine
amplifying the target sequence, but non targets produce unusual light regular
banding (much like bp ladder but finer) throughout the entirety when run on a
2.0% gel. I don't think it is a contaminant, but something else in the
reaction. We have changed over reagents to determine the problem with no
success. Has anyone experienced this in their PCR products?
Cheers
Kristian Peters
SARDI Aquatic Sciences
0428993524
________________________________
From: mti-bounce@xxxxxxxxxxxxx on behalf of simon jarman
Sent: Fri 23/03/2007 11:17 AM
To: mti@xxxxxxxxxxxxx
Subject: [mti] pseudogenes and chimaeras
Hello,
Thank you all for subscribing. There are now more than 30 people on
the list, which is a good starting point for a discussion group.
I have a question. Have any of you identified pseudogenes or chimaeric
sequences when using universal-type primers to amplify animal mitochondrial
genes from dietary samples?
--
Dr Simon Jarman
simon.jarman@xxxxxxxxx
(+61)0408580923
--
Dr Simon Jarman
simon.jarman@xxxxxxxxx
(+61)0408580923
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