CA microsatellite repeat PCR

Martin Kennedy mkennedy at chmeds.ac.nz
Tue May 25 17:31:36 EST 1993


Hi,

A methods question for all you CA-repeat guru's:  during the structural
analysis of a gene on 1q32-1q42 I tripped over a CA repeat, and thought it
worth working it up as a possibly informative STS.  We first used one
gamma-labelled  primer on a handful of genomic DNAs, and ran out the products
on a sequencing gel.  The products were of the appropriate sizes, but horribly
smeared - ie no definition of individual bands.  We denatured them carefully in
formamide dye mix etc, and didn't use intensifier screens on exposure.  Does
anyone have any idea what may have caused the smearing?

Second problem is that we switched to direct incorporation of 35 S dATP, using
2uM dCTP,dGTP,TTP, and 0.2uM dATP,  with 25 cycles of PCR.  This time we got
nicely resolvable products, but only in about 50% of the genomic DNA samples
tested - we have titrated MgCl2 and it doesn't make much difference.  We're
about to repeat it all with direct incorporation of 32 P dCTP, and bumping up
the cycles to 30, but I thought it wise to seek some advice from the experts. 

Any idea why the reactions are incocnsistent? (all the genomic DNAs we're using
have been fine on Southern blots, so the quality should be ok). 

Is it better to keep the dNTP concentrations the same, say at 2uM, rather than
reducing the conc of the cold dNTP equivalent to the labelled one?

Is there a consensus "favoured" method for CA repeat amplification, or does
everyone use a different variant?

Thanks for your help,

-- 
Cheers,

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
NN   NNNN              Christchurch, New Zealand              ZZZZZZZ



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