Mr. T. Weissensteiner
tweissen at hgmp.mrc.ac.uk
Thu Jun 19 10:39:10 EST 1997
Tony Killeen (akilleen at umich.edu) wrote :
> I'm trying to identify a single nucleotide polymorphism
> in mammalian DNA using a site mutagenesis PCR/restriction
> enzyme approach. The polymorphism falls into a region of
> secondary structure due to AT basepairing.
> However, using template from heterozygous subjects (A/C)
> I see a marked preference for amplification of the allele
> containing the C. My interpretation of the data right now
> is that alleles with C are better templates, and knowing
> from other work that this region has secondary structure,
> my gues is that this is because the Taq polymerase has
> an easier time copying the template with less secondary
Usually, such problems seem to be typical for GC-rich DNA
sequences, but here are my few guesses on how you might be
able to improve your PCR :
If possible, avoid poor sample DNA and excessive PCR cycles as
this combination will increase preferential amplification
Next, any conditions destabilizing dsDNA and stabilizing ssDNA
should help, as long as they do not critically reduce primer
annealing (primers with a GC-rich 3' end seem to be less
Instead of reducing magnesium (a cofactor for Taq) you may do
better in avoiding the monovalent ions (K+ or NH4+) in your
PCR-buffer. Only if this doesn't help I would add DMSO or
foramide, which destabilize dsDNA, but might also inhibit Taq.
I wrote a paper about buffer optimization in PCR coamplifications
where one of the amplimers can form more stable secondary dsDNA
structure (1st reference below). In contrast to your "difficult"
sequence, mine was rather GC-rich. So if you or any other netter
with a similar problem should try the method I would be curious
to hear how it worked.
Jenner Institute for Vaccine Research
Berks., RG20 7NN
Fax : 0044 (0)1635 577901
email : tweissen at hgmp.mrc.ac.uk
Some literature on preferential amplification (no comprehensive list)
T Weissensteiner & JS Lanchbury : Strategy for controlling
preferential amplification and avoiding false negatives in PCR typing.
Biotechniques (1996) 21 : 1102- 1108
GL Mutter & KA Boynton : PCR bias in amplification of androgen
receptor alleles, a trinucleotide repeat marker used in clonality
Nucleic Acids Research (1995) 23 : 1411 - 1418
PS Walsh, HA Erlich & R. Higuchi : Preferential amplifiaction of
alleles: mechanisms and solultions
PCR Methods and Applications (1992) 1 : 241 - 250
Q Chou : Minimizing deletion artifacts during Taq DNA polymerase
PCR by E. Coli SSB.
Nucleic Acids Research (1992) 20 : 4371
W-H Shen & B Hohn : DMSO improves PCR amplification of DNA with
complex secondary structure.
Trends in Genetics (1992) 8 : 227
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