The Preliminary Research On Free DNA Of Sheep’s Peripheral Blood Was Applied To The Fetal Gender Identification And Genotyping By HRM

ObjectiveFree DNA in peripheral blood is mainly refers to exist in plasma/serumcirculating system of small fragments of DNA. Its findings in the diagnosis of somediseases and non invasive prenatal diagnosis has broad application prospects. Thisstudy applies three methods from sheep extraction free DNA in peripheral bloodplasma/serum, establish efficient peripheral blood free DNA extraction method, andthe PCR amplification of SRY early fetus gender identification and statistical analysisof accuracy of sex determination different gestation period and different gestationperiod SRY gene expression.Method:1、Sheep in peripheral blood of free DNA extraction and SRY detection. Usingthe heating method, phenol chloroform method and kit from the500mu Lplasma/serum free DNA extraction. According to the characteristics of the free DNAfragment size design of amplification product size of168bp respectively detectprimers and178bp SRY gene primer for ordinary PCR detection.2、Different gestation period fetus gender identification. Using the kit to extractthe free DNA in plasma samples, design two pairs of primers of nested PCR,optimizing amplification system. The SRY gene specific amplification and statisticalresults in all stages of pregnancy and childbirth result analysis.3、Using enzyme digestion and HRM technology of plasma DNA in sheep FecBgene classification effect.Results:1、Using the heating method, phenol chloroform method, qiagen method is toextract free DNA, PCR detected by agarose gel electrophoresis after168bp fragment.At the same time by using PCR amplification to178bp of SRY gene fragment.2、Through the analysis of the results showed that positive detection rateincreases with the passage of the pregnancy period.3wk-a6wk within a total of17samples including SRY positive test results for11, birth outcomes of10males, SRYdetection rate was88.2%(10/11).23samples above6wk, a positive samples for17SRY detection, birth outcomes of17males SRY detection rate of88.2%(15/17).3、 HRM technology can greatly shorten the conventional analysis of the test oftime, reduce the cost and its accuracy is very high.Conclusion:1、Different extraction methods are effective to extract free DNA in peripheralblood serum and plasma of sheep, which can be used for the detection of SRY gene..2、It is feasible that pregnancy sheep free DNA in peripheral blood for early fetusgender identification.3、plasma DNA can be used for genotyping of the research.