1. Substrate Nucleotide-Determined Non-Templated Addition of Adenine by Taq DNA Polymerase: Implications for PCR-Based Genotyping and Cloning
- Author
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Stella J. Nylund, Joseph B. Rayman, Soumitra Ghosh, Lowe Al, Victoria L. Magnuson, Julie I. Knapp, Francis S. Collins, Delphine S. Ally, and Zarir E. Karanjawala
- Subjects
Genotype ,Adenine ,DNA-Directed DNA Polymerase ,Biology ,Polymerase Chain Reaction ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology ,TA cloning ,law.invention ,chemistry.chemical_compound ,chemistry ,Biochemistry ,law ,Adenine nucleotide ,TaqMan ,Taq Polymerase ,TOPO cloning ,Cloning, Molecular ,Genotyping ,Alleles ,Polymerase chain reaction ,Taq polymerase ,Hot start PCR ,Biotechnology - Abstract
The Applied Biosystems PRISMTM 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 GENOTYPERTM software.
- Published
- 1996
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