The "megaprimer" method (1) based on polymerase chain reaction (PCR) is one of the simplest and most versatile procedures of site-specific in vitro mutagenesis available to date. The method utilizes three oligonucleotide primers and two rounds of PCR performed on a DNA template containing the cloned gene that is to be mutated. The rationale of the method is shown schematically in Fig. 1 where A...

Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations....

High risk human papillomaviruses (HR-HPVs) are associated with increased risk of normal cervical cells developing to dysplasia and cervical carcinoma. Therefore, HR-HPV DNA testing can predict an endpoint of cervical carcinogenesis that is earlier than the development of cervical abnormalities. Not only the sensitivity of methods but also the amount of HPV DNA are very important and might be pa...

We attempted to develop a PCR-based marker that detects various segments of rye chromosome incorporated into wheat. We designed three sets of PCR primers based on the nucleotide sequence data of a rye repetitive sequence previously reported. One of the primer sets amplified a clear ca. 1.4 kb fragment in a rye cultivar but not in any form of wheat, diploid, tetraploid or hexaploid. We used this...

Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030 Ever since it was shown that PCR could simultaneously amplify multiple loci in the human dystrophin gene, (1) multiplex PCR has been firmly established as a general technique. A short list of multiplex PCR applications now includes pathogen identification, gender screening, linkage analysis, forensic studies, tem...

This paper describes the use of real-time PCR for the confirmation of microarray data. Current publication guidelines require that all microarray results are confirmed by an independent gene expression profiling method. Real-time PCR is the method of choice for most researchers but it is not without drawbacks. The first step in confirming array results by real-time PCR is selection of gene-spec...

Backgrounds: Canine parvovirus (CPV) has been incriminated as a primary pathogen related to acute hemorrhagic enteritis in dogs. Three major antigenic variants of CPV (CPV-2a/2b/2c) have so far been identified. Objectives: This study was carried out to investigate the frequency of CPV-2 and its variants (CPV-2a/2b/2c) in a population of healthy and diarrheic dogs in the north west of Iran. Meth...

Fungi constitute an important group in soil biological diversity and functioning. However, characterization and knowledge of fungal communities is hampered because few primer sets are available to quantify fungal abundance by real-time quantitative PCR (real-time Q-PCR). The aim in this study was to quantify fungal abundance in soils by incorporating, into a real-time Q-PCR using the SYBRGreen®...

The most studied single nucleotide polymorphisms of the VDR gene are Bsm1, Apa1, Taq1 and Fok1. Previously, many approaches have been used to study SNPs in VDR gene including restriction fragment length polymorphism (RFLP), Single Amplification refractory mutation system PCR (Single-ARMS-PCR), and sequencing of the VDR gene. The objective of the study was to develop a multiplex ARMS-PCR system ...