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...

PCR amplification and detection of GC rich sequences in DNA is a challenge due to formation secondary structures which resist denaturation, thereby stalling Taq polymerases as well affecting primer annealing. Presently, high fidelity polymerase used for amplifying long GC-rich fragments, while dimethyl sulfoxide (DMSO) has also been suggested an additive Polymerase Chain Reaction (PCR) mix avoi...

Oligonucleotide-directed mutagenesis is an important method for modifying specific bases of a DNA sequence (8). Current approaches include combining inverse polymerase chain reaction (PCR) with long PCR to introduce mutations directly into an intact plasmid template (1,5,10). However, the use of Taq DNA polymerase in these protocols results in a high rate of base mis-incorporation (3,7). Moreov...

Chemical modifications to DNA, such as 2' modifications, are expected to increase the biotechnological utility of DNA; however, these modified forms of DNA are limited by their inability to be effectively synthesized by DNA polymerase enzymes. Previous efforts have identified mutant Thermus aquaticus DNA polymerase I (Taq) enzymes capable of recognizing 2'-modified DNA nucleotides. While these ...

A multiplex PCR (mPCR) assay was developed for the detection of multiple Salmonella serotypes in different kind of food products. To increase specificity of this molecular method, three sets of oligonucleotide primer were used to detect the most prevalent salmonella species. PhoP primers specific to the PhoP/PhoQ loci of coliform pathogenic bacteria such as Salmonella, Escherichia coli and citr...

The method described here is derived from that of Engelke et al. (1), and uses the same cloned form of Ther7nus aquaticus (Taq) DNA polymerase to produce this enzyme in E. coli. The modified purification method described here is quite simple, however it is important to note that factors such as the bacterial strain used, induction time and protein concentration during isolation have been opfimi...

A streamlined version of direct dideoxy sequencing is presented that includes template preparation as well as sequencing protocols. The method is used routinely to sequence double-stranded PCR products after minimal purification with one of the primers used in amplification. Either 35S or 32P labeling can be used with equally good results.