A multiplex PCR assay consisting of 13 Rapidly Mutating Y STR loci called RM-Yplex was developed. Platinum® Taq DNA polymerase was used to amplify the 13 Y STR loci in a single reaction in approximately 2.5 hours . In order to shorten the process with reliable results, several DNA polymerases were tested with the multiplex. Phusion® Flash High Fidelity and Platinum® Taq DNA polymerases were inv...

DNA polymerase from Thermus aquaticus has become a common reagent in molecular biology because of its utility in DNA amplification and DNA sequencing protocols. A simplified method is described here for isolating the recombinant Taq enzyme after overproduction in Escherichia coli. Purification requires 8 to 10 h and entails heat treating and clearing the E. coli lysate, followed by precipitatio...

An allylic adenosine triphosphate analog (AATP) was tested as a substrate for commercially available DNA polymerases. All but one of the enzymes assayed incorporated AATP opposite thymidine (T) with concomitant termination of the elongation reaction. A concentration of only 1 microM was sufficient for complete termination of the polymerization reaction for a short template mediated by Ampli Taq...

We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) tr...

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...