direct PCR amplification using library

To: Nikolai troianovsk_s at MSDISK.WUSTL.EDU
Mon Aug 28 18:38:16 EST 1995


 >
Subject: Re:direct PCR amplification using library
>
Date: 28 Aug 1995 22:46:20 +0100
>
Sender: lpddist at mserv1.dl.ac.uk
>
Message-ID: <41tdfc$nrh at mserv1.dl.ac.uk>
>
MIME-Version: 1.0
>
Original-To: methods at dl.ac.uk

Sender: lpddist at mserv1.dl.ac.uk wrote:
>

>
> I am looking for a detailed protocol for dirct PCR using cDNA library
>
> as the DNA source. 

 Hi, Dinakar.

Just my $0.02.

It is the second time I cloned the gene of interest from both cDNA library
and simply pool of cDNA. In both cases I got nothing trying amplify the
gene from cDNA library. And in both cases I got successful cloning using
pool of cDNA as a template. Here is the story which might be helpful (I
hope).

The firs time it was human protein kinase A alpha isoform. The set of
primers was  verified to be specific and satisfied with the criteria of
conventional wisdom (at/gc ratio, melting point, and etc.). At first
attempt I used lambda cDNA library. Yes I got an amplificate of expected
m/w along with other bands. I separated the suspicious band, restriction
analysis showed that it was artifact. Then I decided to use pool of cDNA.
At first round of amplification I`v got nothing (looks as negative
control). Then I took this negative amplificate and after diluting it 1/100
subjected to second round of amplification (in both cases at first I used
mixture of polimerases Taq + DeepVent, while in the second round I used
DeepVent only). After the second round, the nice (fat) band of expected m/w
appeared. Restriction analysis showed that it contains the specific site. I
cloned it and sequenced. It was what I was needed.

Jus week ago I cloned another gene 3Kb in length. The set of primers covers
full length of the gene, as well as another set of primers containing
unique site splits gene in two halfs 1700 and 1300 bp. Again the first time
I used the same cDNA library (because we run out of cDNA). Again I got
amplificate of expected m/w and again contaminated with other bands. And
again they (bands) were artifacts. One case was interesting, since I got
and amplificate of double m/w using only one sense primer. Cutting band
with enzyme which site should be at band`s end results in splitting band in
two halfs. That is, the original band was palindrome!!!, apparently the
result of cloning into phage library. Then I got again cDNA (this time it
was ordered from Clontech). The first round of amplification yields set of
poorly amplified bands of lower or higher m/w. However, using amplificate
from first round in nested PCR I got what I need.

The bottom line. So far my attempts to use cDNA library as a source fail.
The use of uncloned pool of cDNA seems more reliable. Even if in the first
round of amplification there is nothing, second round will yields. What is
more important, the resulting product doesn`t contains mismatches, all
unique sites preserved, and you are doing sequencing being 100% sure that
this is what you need.

Pardon me for using to much space.

Nikolai.
http://128.252.119.253.





More information about the Methods mailing list