Background: Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein’s structure and function. Objectives: We introduced a nested-SOE-PCR (N –SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR.   Materials and Methods: Genomic DNA from Bacillus thermo...

Anaerobic carbon monoxide dehydrogenase (CODH) is a key enzyme in the Wood-Ljungdahl (acetyl-CoA) pathway for acetogenesis performed by homoacetogenic bacteria. Acetate generated by gut bacteria via the acetyl-CoA pathway provides considerable nutrition to wood-feeding dictyopteran insects making CODH important to the obligate mutualism occurring between termites and their hindgut microbiota. T...

Selecting specific primers is crucial for polymerase chain reaction (PCR). Nonspecific primers will bind to unintended genes and result in nonspecific amplicons. MFEprimer is a program for checking the specificity of PCR primers against the background DNA. In this chapter, we introduce: (1) the factors that affect the specificity of primers; (2) the principle of MFEprimer and its settings; (3) ...

We investigated the utility of PCR to detect Burkholderia cepacia directly in sputum samples at two cystic fibrosis (CF) centers serving children and adults. Following liquefaction of the sputa by using N-acetyl-L-cysteine, DNA was isolated and analyzed by PCRs with three different primer pairs directed toward bacterial rRNA loci. Two primer pairs were putatively specific for B. cepacia. The ot...

Escherichia coli O86 belongs to the enteropathogenic E. coli (EPEC) group, some strains of which are pathogens of humans, wild birds and farm animals. The O-antigen gene cluster of E. coli O86 was amplified by long-range PCR using primers based on the housekeeping genes galF and gnd, and then sequenced. Genes involved in GDP-Fuc and N-acetyl-galactosamine (GalNAc) synthesis and genes encoding g...