The QuikChange Multi Site-Directed Mutagenesis Kit is a simple and efficient method for introducing point mutations at up to five sites simultaneously in plasmid DNA templates. Here we used the QuikChange Multi kit with degenerate (one codon) primers to introduce all possible amino acids at selected sites in the lacZ gene. In reactions employing two or three degenerate primers, diverse librarie...

Degenerate PCR primers, UP-1 and UP-2r, for the amplification of DNA gyrase subunit B genes (gyrB) were designed by using consensus amino acid sequences of gyrases from Escherichia coli, Pseudomonas putida, and Bacillus subtilis. In addition to the degenerate sequences, these primers have sequences at the 5' end which allow direct sequencing of amplified PCR products. With these primers, DNA se...

A polymerase chain reaction (PCR) primer sequence is called degenerate if some of its positions have several possible bases. The degeneracy of the primer is the number of unique sequence combinations it contains. We study the problem of designing a pair of primers with prescribed degeneracy that match a maximum number of given input sequences. Such problems occur, for example, when studying a f...

BACKGROUND The advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. Such methods are used to identify T-DNA insertion sites and so are especially relevant for organisms where large T-DNA insertion libraries have been created,...

Microsatellites are popular genetic markers in molecular ecology, genetic mapping and forensics. Unfortunately, despite recent advances, the isolation of de novo polymorphic microsatellite loci often requires expensive and intensive groundwork. Primers developed for a focal species are commonly tested in a related, non-focal species of interest for the amplification of orthologous polymorphic l...