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Journal of Animal and Veterinary Advances
Year: 2011  |  Volume: 10  |  Issue: 6  |  Page No.: 715 - 722

Study of the Amplification Capacity of A Two-Temperature PCR and its Application in Bovine Sex Identification

Dong Wang, Bo Lin, Huabin Zhu, Haisheng Hao, Chengjiang Wu, Weihua Du and Xueming Zhao    

Abstract: To study the amplification potential and establish a two-temperature PCR sex identification method, DNA templates prepared from bovine blood, sperm and hair follicles were amplified through two-temperature PCR in this study. Fragments from 207-1413 bp could be amplified successfully while fragments of 1500 bp in length could not be amplified. In further studies we found that an extension step was necessary to amplify longer fragments and as the extension time decreased, the denaturation and annealing times were increased. After optimization we found the best two-temperature PCR to be a reaction using 1.0 mmol L-1 Mg2+, 1.0 unit polymerase and 30 cycles. The amplification results using different equipment and Taq polymerases indicated that the two-temperature PCR can be used for any fragment equal to or <1413 bp with any common Taq polymerase. The kinetic rate of the polymerase was determined to be the factor limiting the amplification length and rate. Additionally, the PCR timing was shortened from 1.5-2.0 h to 30-38 min with the two-temperature PCR. The sex identification of bovine blood, fibroblasts and blastmeres were carried out by turns using this rapid PCR and a rapid sex identification method of cattle embryo was establishment. Both amplification time and cost were saved greatly with this method and we found that the two-temperature PCR was sensitive and fast enough for widespread use in many research areas such as genetics, sex identification, forensics and clinical medicine.

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