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Articles by Bo Lin
Total Records ( 3 ) for Bo Lin
  Dong Wang , Bo Lin , Huabin Zhu , Haisheng Hao , Chengjiang Wu , Weihua Du and Xueming Zhao
  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.
  Dong Wang , Huabin Zhu , Jiaming Guo , Bo Lin , Linbo Zhang , Haisheng Hao , Weihua Du and Xueming Zhao
  Sort reanalysis using flow cytometry is the most common method for determining the purity of X or Y enriched semen. The high cost of this technique (including the required expensive, proprietary machine) limits efforts to improve the technique and to promote develop applications for the sorted semen. In this study, the sperm sex (the presence of the X or Y chromosome) was identified by both rapid PCR and flow cytometry reanalysis. The rapid PCR results showed that the percentages of X and Y sperm were 48 and 52% in unsorted semen, 92 and 8% in X-enriched semen and 17 and 83% in Y-enriched semen, respectively. Reanalysis of the DNA content of the sorted samples revealed that the X and Y sperm frequencies were 92 and 8% in X-enriched semen and 15 and 85% in Y-enriched semen, respectively. The sex ratio of unsorted semen analyzed by PCR did not significantly deviate from the expected ratio of 1:1 and there was no significant difference between the sex ratios of sorted semen samples determined by PCR and flow cytometry reanalysis. These results indicate that we have established an effective, reliable and rapid PCR method to verify the purity of sorted semen. This method should contribute greatly to the improvement of sperm sorting techniques and the development of applications for sorted semen.
  Huabin Zhu , Bo Lin , Jun Chen , Haisheng Hao , Xueming Zhao , Shujing Li , Weihua Du , Tong Qin , Yan Liu and Dong Wang
  A rapid and simple PCR sex identification of embryo is very important for bovine embryo transferring. Many sex identification methods using duplex PCR were established according to Sry gene. But the identification process was affected greatly by more primers interaction. In order to decrease the interference from more primers, researchers explored a simple and rapid PCR Method. The sequences of Amelogenin alleles located at both sex chromosomes were downloaded from GenBank. A pair of sex specific primers was designed to span the 63 bp longer insertion sequence in X chromosome. Bovine samples of blood, fibroblasts and demi-embryos were sexed with these primers. Two-temperature PCR cycling program was used in which the extension step was deleted while the denaturizing and annealing steps were shortened to 1 sec. The results shown ideal identification were obtained and observable amplification were also obtained using even single fibroblast. About 20 bovine embryos were identified by this PCR cycling program and 15 embryos (9 females and 6 males) were transferred. The sexing results were confirmed by the anatomically proven sex after parturition, respectively. The comparison of amplification results between blood samples of bovine and human shows the excellent, specificity to bovine. Thus, a simple, rapid and effective PCR sex identification method was established.
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