| Milk and egg production are gender characteristics, and they show large differences in the growth and development of male and female. So the sex control technology is an important technique in animal production. Currently, sperm separation technology on different DNA content in X, Y sperms is the best repeatability and most scientific and effective method, but the separation evaluation system has not yet established. At the same time, among many technology methods developed in embryo gender identification which is another gender control forms, PCR has been the research focus because of its stable results and simple operation. However, the previous PCR technology is mostly based on sry gene in Y chromosome and it is detected and analyzed through double PCR, nested PCR methods which is not only cumbersome and high demanding for the operating techniques, but also needs long time to do the amplification and the subsequent detection analysis, which increases the response time of embryo in vitro. Therefore, it is necessary to do some further research on all the steps of PCR to establish a simple, rapid, efficient and low cost of practical PCR embryo sex identification technology and a scientific and efficient evaluation system corresponding to the X, Y sperm separation technology.Conventional PCR cycling program typically includes denaturing, annealing and extension, but study shows the existence of the extension step and its duration time is related to the length of the amplified fragment. When the amplified fragment is small, the three temperature gradient PCR can be replaced by two temperature gradient PCR, so amplification process can be significantly reduced. Therefore, the PCR method could be a rapid, simple, low-cost technique and be applied widely in many biology fields by exploring its amplification potential and optimizing the amplification conditions. Based on this, in this study, using two temperature gradient PCR, DNA templates prepared from bovine blood, sperm and hair follicle were successfully amplified with fragments from 207 bp to 1413 bp but failed in 1561 bp. Study on the amplification condition of 1561 bp fragment showed that the extension time could be decreased by increasing of the denaturation and annealing time, the reason of which needs to be further studied. Factors influencing the yield and detecting efficiency of the product were also optimized using the two-step PCR. The best result was obtained with 1.0 mM magnesium ion, 1.0 unit polymerase and 30 cycle number. The comparison of the amplification results among different PCR equipments and different Taq polymerase products indicated that the two-step PCR might be achieved with any common Taq polymearase products, in any PCR equipments and for any fragments less than 1413bp, which indicated that it was the kinetic rate of the polymerase and not the special regents, such as Taq polymerase, or special equipments that helps and limits the amplification rate and the amplified length of the fragment. The sex identification of the bovine blood and fibroblast samples indicated that the two-step PCR method could be used not only in both simple PCR and double PCR amplification, but also in fields such as bovine sex dermination. Compared to the common PCR, the costing time of the two-step PCR was shortened from 1.5 hours ~ 2.0 hours to 25 minutes ~ 35 minutes, which is less than half of its previous time.The characteristics of primer template in sex identification determine PCR amplification types. Instead of sex identification according to the gender-specific sequence in Y chromosome, in this study, a pair of gender-specific primers was designed through comparing the amelogenin DNA sequences (AML) in X and Y chromosomes. Bovine blood, semen DNA template were firstly amplified to verify the validity of the primers, then 100 single sperms isolated from the conventional semen, separated X-sperm and Y-sperm individually were amplified using this primers in rapid PCR method, and clear identifiable specific product bands were obtained. The statistical results of X, Y sperm ratio in conventional semen, separated X-sperm and Y-sperm showed that the ratio of X,Y sperms is 1:1 in conventional frozen semen,the percent of X-sperm in sex typing X-sperms is 84.0%,the percent of Y-sperm in sex typing Y-sperms is 88.0%. The fact that there is no much difference between statistical results and theoretical ratio for conventional semen and re-separation results for sex typing sperms indicates that the rapid PCR can be used for sex identification of bovine sperm and purity evaluation for the sex typing sperms. Since then, our laboratory established a simple, low cost, high reliability, rapid purity detection of the sex typing semen, which makes the purity analysis of sexing semen is independent of the large exclusive equipment such as flow cytometry and can be a common test and analysis method in the general laboratory. This technique pays the way for further research on the sperm separation and provides strong technical support for the popularization and application of sexing semen.The established simple PCR was also used for sex identification of embryo. First, bovine fibroblast cells were amplified using rapid PCR method, and the double PCR method was used as a positive control. The results showed that this method can accurately detect the sex of fibroblasts. In the research of embryonic sex identification using double PCR method as positive control, good amplification results were obtained using this simple PCR method, thus this method was verified by double PCR method. The reaction system was optimized and designed a simple and effective kit, which simplifies the experimental operations and makes it more convenient for field detection analysis. Test results of the reagent kit showed that the kit will help save production costs, improve production efficiency and thus has great significance in bovine sex determination and sex control. The establishment of this method also has higher applications value in forensic identification.
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