Amplification, detection, and automated sequencing of gibbon interleukin-2 mRNA by Thermus aquaticus DNA polymerase reverse transcription and polymerase chain reaction

Inbred strains of rats and mice have long been used to study basic mechanisms of human disease. Our knowledge of the rodent and human immune systems has increased in recent years, largely because of the availability of reagents and techniques specific for these species. In contrast, outbred animals, including domestic companion and food animals, have not been used routinely as experimental models for human disease, largely because reagents and assays necessary for basic research in immunology and physiology have not been available. Here, using consensus cytokine nucleic acid sequences, we adapt a previously described reverse transcription-quantitative competitive polymerase chain reaction technique to measure interleukin 2 (IL2), IL4, IL6, IL10, IL12, interferon γ, tumor necrosis factor α, and glyceraldehyde-3-phosphate dehydrogenase mRNA expression in the cow, cat, dog, horse, and pig. We demonstrate that the assay is sensitive, accurate, and reproducible.

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Optimal conditions and specific characteristics of Vent exo– DNA polymerase in ligation-mediated polymerase chain reaction protocols

Biochemistry and Cell Biology ◽

10.1139/o04-134 ◽

2005 ◽

Vol 83 (2) ◽

pp. 147-165 ◽

Cited By ~ 7

Author(s):

François Vigneault ◽

Régen Drouin

Keyword(s):

Polymerase Chain Reaction ◽

Dna Polymerase ◽

Pyrococcus Furiosus ◽

Pcr Amplification ◽

Polymerase Activity ◽

Optimal Dosage ◽

Chain Reaction ◽

Thermus Aquaticus ◽

Thermococcus Litoralis ◽

Polymerase Chain

An optimized procedure for the ligation-mediated polymerase chain reaction (PCR) technique using Thermococcus litoralis exo– DNA polymerase (Vent exo–) was developed. The optimal dosage of Vent exo– at the primer extension and PCR amplification steps as well as the optimal DNA quantity to use were established. We showed that Vent exo– can efficiently create the blunt-ended termini required for subsequent linker ligation. Vent exo– proves to be more efficient than Pyrococcus furiosus exo– (Pfu exo–) for this task. Vent exo– resolves highly GC-rich sequence substantially better than Thermus aquaticus DNA polymerase (Taq) and with a similar efficiency as Pfu exo–. The DNA/DNA polymerase activity ratio is significantly higher for Vent exo– than for Pfu exo–, which is reflected by the sensibility of Vent exo– in efficiently amplifying genomic DNA. Furthermore, the range of efficiency of Vent exo– demonstrates the importance of conducting evaluative testing to identify the optimal dosage of use of this polymerase to obtain successful PCR amplification. Optimal MgSO4 concentrations to use with Vent exo– were established. Our results show that Vent exo– DNA polymerase produces bands of uniform and strong intensity and can efficiently be used for the analysis of DNA in living cells by ligation-mediated PCR.Key words: Vent exo– DNA polymerase, Pfu exo– DNA polymerase, DNA sequence context, ligation-mediated polymerase chain reaction (PCR), PCR buffer.

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