(none)

Sham Nair snair at mt.net.au
Mon Sep 27 15:35:56 EST 1999


This is a multi-part message in MIME format.

------=_NextPart_000_0004_01BF0878.D8271180
Content-Type: text/plain;
	charset="iso-8859-1"
Content-Transfer-Encoding: quoted-printable

Dear Netters, I would like some advice regarding PCR with degenerate =
primers.  I have designed primers based on the amino acid sequence of a =
protein I am working with.  I have used inosine at positions of 4-fold =
degeneracy.  Overall, the degeneracy of the primers range from 64 - 256. =
 Using these primers, I tried to amplify the gene sequence encoding the =
protein of interest from genomic DNA extracted from the host organism (a =
marine chordate).  To-date, all attempts at amplification have been =
unsuccessful (ie amplified sequences do not correspond to the amino acid =
sequence when translated).  These attempts have included standard =
cycling parameters at various annealing temperatures, touchdown PCR and =
step-up PCR (including hot start). The primers were designed so that =
nesting of primary amplification products can be carried out.  The basic =
protocol was to carry out the PCR, ligate the unpurified reaction =
products to pGEM-T vector and the transform into E. coli.  Recombinants =
are then subject to PCR screening.  Purified plasmids are sequenced =
using vector specific primers. Interestingly, many of the amplified =
products contain the same primer sequence (ie reverse primer) at both =
ends (the size of the amplified product in approximately 300 bp)!  With =
the assumption that the target sequence might contain a hairpin =
loop-type structure, I included DMSO in the amplification mix, but with =
no success.
What can I do to overcome the problems and successfully amplify the =
product?  Have others faced similar problems and overcome them?  Any =
advice is greatly appreciated.  Thank you.


Regards,
Sham Nair

School of Biological Sciences,
Macquarie University,
North Ryde,
NSW 2109,
Australia
snair at mt.net.au
snair at rna.bio.mq.edu.au


------=_NextPart_000_0004_01BF0878.D8271180
Content-Type: text/html;
	charset="iso-8859-1"
Content-Transfer-Encoding: quoted-printable

<!DOCTYPE HTML PUBLIC "-//W3C//DTD W3 HTML//EN">
<HTML>
<HEAD>

<META content=3Dtext/html;charset=3Diso-8859-1 =
http-equiv=3DContent-Type>
<META content=3D'"MSHTML 4.72.2106.6"' name=3DGENERATOR>
</HEAD>
<BODY bgColor=3D#ffffff>
<DIV><FONT color=3D#000000 face=3D"Times New Roman" size=3D2><FONT =
size=3D3>Dear=20
Netters, I would like some advice regarding PCR with degenerate =
primers.&nbsp; I=20
have designed primers based on the amino acid sequence of a protein I am =
working=20
with.&nbsp; I have used inosine at positions of 4-fold degeneracy.&nbsp; =

Overall, the degeneracy of the primers range from 64 - 256.&nbsp; Using =
these=20
primers, I tried to amplify the gene sequence encoding the protein of =
interest=20
from genomic DNA extracted from the host organism (a marine =
chordate).&nbsp;=20
To-date, all attempts at amplification have been unsuccessful (ie =
amplified=20
sequences do not correspond to the amino acid sequence when =
translated).&nbsp;=20
These attempts have included standard cycling parameters at various =
annealing=20
temperatures, touchdown PCR and step-up PCR (including hot start). <FONT =

color=3D#000000 face=3D"Times New Roman" size=3D2><FONT size=3D3>The =
primers were=20
designed so that nesting of primary amplification products can be =
carried=20
out.&nbsp; The basic protocol was to carry out the PCR, ligate the =
unpurified=20
reaction products to pGEM-T vector and the transform into E. coli.&nbsp; =

Recombinants are then subject to PCR screening.&nbsp; Purified plasmids =
are=20
sequenced using vector specific primers. </FONT></FONT>Interestingly, =
many of=20
the amplified products contain the same primer sequence (ie reverse =
primer) at=20
both ends (the size of the amplified product in approximately 300 =
bp)!&nbsp;=20
With the assumption that the target sequence might contain a hairpin =
loop-type=20
structure, I included DMSO in the amplification mix, but with no=20
success.</FONT></FONT></DIV>
<DIV><FONT color=3D#000000 face=3D"Times New Roman" size=3D3>What can I =
do to overcome=20
the problems and successfully amplify the product?&nbsp; Have others =
faced=20
similar problems and overcome them?&nbsp; Any advice is greatly=20
appreciated.&nbsp; Thank you.</FONT></DIV>
<DIV><FONT color=3D#000000 face=3D"Times New Roman" =
size=3D3></FONT>&nbsp;</DIV>
<DIV><FONT color=3D#000000 face=3D"Times New Roman" =
size=3D3></FONT>&nbsp;</DIV>
<DIV><FONT color=3D#000000 face=3D"Times New Roman" =
size=3D3>Regards,</FONT></DIV>
<DIV><FONT color=3D#000000 face=3D"Times New Roman" size=3D3>Sham =
Nair</FONT></DIV>
<DIV><FONT color=3D#000000 face=3D"Times New Roman" =
size=3D3></FONT>&nbsp;</DIV>
<DIV><FONT color=3D#000000 face=3D"Times New Roman" size=3D3>School of =
Biological=20
Sciences,</FONT></DIV>
<DIV><FONT color=3D#000000 face=3D"Times New Roman" size=3D3>Macquarie=20
University,</FONT></DIV>
<DIV><FONT color=3D#000000 face=3D"Times New Roman" size=3D3>North =
Ryde,</FONT></DIV>
<DIV><FONT color=3D#000000 face=3D"Times New Roman" size=3D3>NSW =
2109,</FONT></DIV>
<DIV><FONT color=3D#000000 face=3D"Times New Roman" =
size=3D3>Australia</FONT></DIV>
<DIV><FONT color=3D#000000 face=3D"Times New Roman" size=3D3><A=20
href=3D"mailto:snair at mt.net.au">snair at mt.net.au</A></FONT></DIV>
<DIV><FONT color=3D#000000 face=3D"Times New Roman" size=3D3><A=20
href=3D"mailto:snair at rna.bio.mq.edu.au">snair at rna.bio.mq.edu.au</A></FONT=
></DIV>
<DIV><FONT color=3D#000000 face=3D"Times New Roman"=20
size=3D3></FONT>&nbsp;</DIV></BODY></HTML>

------=_NextPart_000_0004_01BF0878.D8271180--




More information about the Methods mailing list