Discrimination between the amplification of mRNA and contaminating genomic DNA is a common problem when performing a reverse transcriptase-polymerase chain reaction (RT-PCR). Even after treatment of the samples with DNAse, it is possible that negative controls (samples in which no reverse transcriptase was added) will give positive results. This indicates that there was amplification of DNA, wh...

Microsatellites are abundant in eukaryotic genomes and have high rates of strand slippage-induced repeat number alterations. They are popular genetic markers, and their mutations are associated with numerous neurological diseases. However, the minimal number of repeats required to constitute a microsatellite has been debated, and a definition of a microsatellite that considers its mutational be...

Abstract The phosphoprotein gene of the paramyxoviruses encodes multiple protein products. P, V, and W proteins are generated by transcriptional slippage. This process results in insertion non-templated guanosine nucleosides into mRNA at a conserved edit site. P is an essential component viral RNA polymerase encoded faithful copy majority paramyxoviruses. However, some cases, non-essential V de...

To achieve the production of a thermostable DNA polymerase free from bacterial DNA contamination, we developed eukaryote-made thermostable DNA (Taq) polymerase. The novel eukaryote-made thermostable DNA polymerase resolves the problem of contaminating bacterial DNA in conventional bacterially made thermostable DNA polymerase as a result of its manufacture and incomplete purification. Using euka...

BACKGROUND: Clinical specimens are routinely fixed in 10% buffered formalin and paraffin embedded. Although DNA is commonly extracted from fixed tissues and amplified by PCR, the effects of formalin fixation are relatively unknown. Formalin fixation is known to impair PCR, presumably through damage that blocks polymerase elongation, but an insidious possibility is error prone translesion synthe...

The highly thermostable DNA polymerase from Thermus aquaticus (Taq) is ideal for both manual and automated DNA sequencing because it is fast, highly processive, has little or no 3'-exonuclease activity, and is active over a broad range of temperatures. Sequencing protocols are presented that produce readable extension products greater than 1000 bases having uniform band intensities. A combinati...

The use of glass–silicon chips for PCR analysis has been widely reported in the last decade, but there have been few systematic efforts to pin down the biochemical problems such systems bring forth. Here we report a systematic analysis of material-related inhibition and adsorption phenomena in glass–silicon PCR-chips. The results suggest that the previously reported inhibition of PCR by silicon...

We previously described in a paper published in BIOS an undergraduate lab activity involving the gene cloning, expression, and purification of Thermus aquaticus (Taq) DNA polymerase, an enzyme used in the polymerase chain reaction (PCR). Based on the large number of requests for biological materials and questions about the protocols this paper invoked, we explored methods to simplify the protei...

Kary B. Mullis developed a revolutionary method name polymerase chain reaction (PCR) in 1983, which can synthesize new strand of DNA complementary to the template and produce billions copies fragment only few hours. Denaturation, annealing, extension are three primary steps involved PCR process, generally requires thermocyclers, template, pair primers, Taq polymerase, nucleotides, buffers, etc....

Few techniques are available to detect DNA lesions in cultured cells at the nucleotide level (8). One such method is primer extension of genomic DNA (5) that may be improved using linear amplification by repeated PCR cycles (1,11). This method has been used successfully to map the genomic sites of topoisomerase II (2,3,7) and topoisomerase I activity (10). DNA topoisomerases modulate DNA struct...