Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: implications for PCR-based genotyping and cloning.
The Applied Biosystems PRISM fluorescence-based genotyping system as well as the Invitrogen TA Cloning vector system are influenced by the tendency of Taq DNA polymerase to add an adenine nucleotide to the 3' end of PCR products after extension. Incomplete addition of adenine to a majority of PCR product strands creates problems in allele-calling during genotyping and potentially diminishes the cloning efficiency of such products. Experiments reported here show that certain terminal nucleotides can either inhibit or enhance adenine addition by Taq and that PCR primer design can be used to modulate this activity. The methods we propose can substantially improve allele-calling for problematic microsatellite markers when using GENOTYPER software.
National Center for Human Genome Research, National Institutes of Health, Bethesda, MD, USA. magnuson@nchgr.nih.gov
V L
VL
Magnuson
D S
DS
Ally
S J
SJ
Nylund
Z E
ZE
Karanjawala
J B
JB
Rayman
J I
JI
Knapp
A L
AL
Lowe
S
S
Ghosh
F S
FS
Collins
D000225:Adenine / Q000378:metabolism*; D000483:Alleles; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000494:pharmacology*; D005838:Genotype; D016133:Polymerase Chain Reaction; D019914:Taq Polymerase
D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D000225:Adenine