The fungus Venturia nashicola is the causal agent of scab on Asian pears. For the rapid and reliable identification as well as sensitive detection of V. nashicola, a PCR-based technique was developed. DNA fingerprints of three closely related species, V. nashicola, V. pirina, and V. inaequalis, were obtained by random amplified polymorphic DNA (RAPD) analysis. Two RAPD markers specific to V. na...

Cleavage specificity of RNase HI was examined on model Okazaki fragments, to determine the likely role of this nuclease in lagging strand DNA replication. Each substrate was prepared by annealing a short RNA primer, made by transcription in vitro, to a single-stranded synthetic DNA template, and subsequently extending the primer by DNA polymerization. The calf thymus RNase HI makes a structure-...

Using in vivo fidelity assays in which bacterial beta-galactosidase or green fluorescent protein genes served as reporters of mutations, we have identified a murine leukemia virus (MLV) RNase H mutant (Y586F) that exhibited an increase in the retroviral mutation rate approximately 5-fold in a single replication cycle. DNA-sequencing analysis indicated that the Y586F mutation increased the frequ...

target sequence is correct, the target sequence is amplified, and if the primer pair is in the wrong orientation relative to the amplified cDNA inserts, the target sequence is not amplified. This simple, linear pre-amplification of cDNAs in the library has the potential for wide use. This approach requires not only far fewer starting materials than conventional PCR cloning but also circumvents ...

We present two examples of exponential nucleic acid amplification with the polymerase chain reaction (PCR) in the presence of only one amplification primer. Cloning and sequencing of the PCR products generated by amplification of human genomic DNA revealed that the amplified sequence contained only one primer and its complement, at the two ends of the PCR product. Although these experiments wer...

To produce large cDNA strands from biological samples containing limited numbers of template molecules, it may be necessary to minimize both nonspecific primer attachment in first-strand synthesis and secondary structure in RNA molecules. Failure to do so could result in the accumulation of shortened cDNA strands and therefore may reduce the yield of large cDNA molecules, sometimes below detect...

The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal techni...

Anaerobic ammonia oxidizing (anammox) bacteria play an important role in transforming ammonium to nitrogen gas and contribute to fixed nitrogen losses in freshwater environments. Understanding the diversity and abundance of anammox bacteria requires reliable molecular tools, and these are not yet well established for these important Planctomycetes. To help validate PCR primers for the detection...

The polymerase chain reaction (PCR) is one of the most rapidly expanding methods in molecular biology. Although the range of applications of PCR is very broad, the key points for successful performance remain the careful selection of optimal primers and the proper determination of the temperature conditions of the reaction. The program presented here, 'Primer Master', has been developed to assi...

background: identifying regional types and evaluating the frequency of pneumococcal strains has become increasingly important especially in vaccination. the purpose of this study was the identification and frequency determination of our regional serotype and evaluation of the performance of recent type specific multiplex pcr for the diagnosis of streptococcus pneumonia serotypes. methods: all i...