The Specificity Analysis And Application Of The On/off Switch In Sex Idenfication

Aim: The objective of this experiment is to study the application of molecular on/offswitch in Y chromosome identification.Methods: Five ml of blood samples were collected from individuals with normalsex phenotype. The blood samples were treated with 0.5 heparin to prevent from coagulation. Genomic DNA was extracted using phenol/chloroform and the DNA was examined by agarose gel electrophoresis, quantitatively determined by the ratio of 260/280 by UV spectrometry. The DNA aliquots were prepared before storage in freezer. Specific primers were designed according to gene sequence and SNP sites of Y chromosome. Samples of known sex were determined by regular PCR mediated by Taq polymerase and by Pfu polymerase respectively, with X chromosome specific primer X1, Y chromosome specific primer Y02, and homologue sequence of Y chromosome and autosomal chromosome specific primer Y2.Gender identification of these DNA samples was also determined by the combination of real-time PCR and the on/off switch with primers of Y02 and Y 2. The samples of known sex were determined by regular PCR and inverse nested PCR with specific primers targeting M45, M89, M172 SNP sites in Y chromosome, flanking with the common tails. The results between Taq polymerase and Pfu polymerase were compared. Furthermore, 40 DNA samples that were kept blinded from individuals with normal sex phenotype were determined with primer Y02.Result: In regular PCR: both male and female DNA samples yielded amplifying products using primer X1 and primer Y2; only male DNA samples yielded products using primer Y02 was used. There was no significant result between Taq polymerase and Pfu polymerase observed. Male DNA samples yielded PCR products whereas female DNA samples had no PCR product, using primers M45, M89, M172.