Fw: How many primers in RAPD?

Deanne Bell dbell at qnis.net
Wed Mar 7 13:07:32 EST 2001


Hi M.Shi

What exactly are you wanting to prove with this experiment?  Are you trying
to find a marker that you can link to the transgenic trait?  If that is the
case, I think what you should really be asking is "how many polymorphic
bands or "markers" should I use to show the difference?"  After
amplification you will need to prove that the marker(s) can be linked to
only those plants that are transgenic and is (are) not due to any
inheritable genetic characteristics.
 
Because RAPD primers amplify "random" portions of the genome, you will
probably need to screen LOTS of primers until you find one that happens to
amplify within your transgenic region.
 
Just to throw in my 2 cents - A technique which may be more direct is ISSR
(Inter Simple Sequence Repeats)  the primers are ~ 20mers based on simple
sequence repeats, so you are actually amplifying areas between the simple
repeats.  The University of British Columbia sells a set of 100 primers for
about $250 (telephone  604  822-4570).  Here is a reference if you are
interested:
Ewa Zietkiewicz, Antoni Rafalski, and Damian Labuda.  Genome Fingerprinting
by Simple Sequence Repeat (SSR)-Anchored Polymerase Chain Reaction
Amplification.  Genomics 20, 176-183 (1994)

Deanne Bell
Molecular Markers Lab Technician
USDA Agricultural Research Service
2021 S. Peach Ave.
Fresno, CA 93727
e-mail: dbell at qnis.net
phone: 559 453-3170
fax: 559 453-3088



----------
> From: "Bright" <biosci at eyou.com>
> To: methods at hgmp.mrc.ac.uk
> Subject: How many primers in RAPD?
> Date: Wednesday, March 07, 2001 4:50 AM
> 
> Hello,
> 
>    I wanna use RAPD to analyse wheat and its transgenic progeny,how many
> random primers should I use to show the difference?
> 
>    Thanks.
> 
> M.Shi
> 
> 
> 
> 
> 
> --http://www.eyou.com
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> 
> 
> 
> ---
> 
> 


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