The synthesis of a gene coding for horseradish peroxidase (HRP, isozyme c; EC 1.11.1.7) is described using a polymerase chain reaction (PCR)-mediated gene synthesis approach developed in our laboratory. In this approach, all the oligonucleotides making up the gene are ligated in a single step by using the two outer oligonucleotides as PCR primers and the crude ligation mixture as the target. Th...

The determination of unknown DNA sequences around a known locus has important applications in molecular genetics, specifically in genomic walking and genome mapping. Several PCR-based methods have been reported to address this issue, but they often involve multiple, time-consuming steps. We have previously described a technique known as restriction site PCR (RS-PCR) that allows sequence acquisi...

backgrounds and aims: coxsakievirus b3 (cvb3), one of the six coxsakievirus b serotypes, is a member of the enterovirus genus within the picornaviridae family. cvb3 is an important pathogen of viral myocarditis, which accounts for more than 50% of viral myocarditis cases. the genome of cvb3, like that of other entroviruses, is a single-stranded, sense, polyadenylated rna molecule with 7400 nucl...

A nested polymerase chain reaction (PCR) was developed to detect genomic DNA of Piscirickettsia salmonis, the causative agent of an epizootic disease in salmonids. The nested PCR assay, which used general bacterial 16s rDNA primers in the flrst amplification reaction, and P salmonisspecif~c primers in a second reaction, allowed detection of less than 1 P salmonis tissue culture infect~ous dose ...

Massively parallel sequencing is rapidly emerging as an efficient way to quantify biodiversity at all levels, from genetic variation and expression to ecological community assemblage. However, the number of reads produced per sequencing run far exceeds the number required per sample for many applications, compelling researchers to sequence multiple samples per run in order to maximize efficienc...

With the rise of next-generation sequencing methods, it has become increasingly possible to obtain genomewide sequence data even for nonmodel species. Such data are often used for the development of single nucleotide polymorphism (SNP) markers, which can subsequently be screened in a larger population sample using a variety of genotyping techniques. Many of these techniques require appropriate ...

Several site-directed mutagenesis methods have been developed and are being currently used in research. All these methods apply three or four primers, including one or two bearing the appropriate mutation, and most use PCR to amplify the desired sequence and to incorporate the mutation (1–6). Although several developments have improved the robustness and reliability of the method, all of them r...

in the present research, molecular detection of bovine leukocyte adhesion deficiency (blad) and complex vertebral malformation (cvm)in a population of iranian holstein cows has been carried outusing milk somatic cells by polymerase chain reaction-restriction fragment length polymorphism(pcr-rflp). the blad and cvm are monogenic and autosomal recessive heredity lethal syndrome in holstein-friesi...

Figure 1. Diagram of a split-marker gene replacement strategy using fusion-PCR (A) FPCR step 1 amplification of flanking regions and partial marker segments. Dotted tails indicate the overlap sequences on selected primers which allow fusion in step 2. (B) FPCR step 2 flanking regions are fused to the marker segments at the overlap sights, forming two constructs. The fusion products are then amp...