The Construction Of Individual Identification On Bull By Multiple-primer PCR

It is one of methods to improve Animal population genetic performance using molecular mark of bovine semen. The study found out rapid extraction method of cattle semen DNA and constructed multiple-primer PCR system by orthogonal experimental method.11 of 29 primers were screened to be used for personal identification, and the way of individual identification with microsatellites.1 Orthogonal experimental method was used to the reagents proportion. Suitable PH value, osmotic pressure and the concentration of the ions was mediated. The use of Digestive juices of PH=8.5 can rapidly extract high quality DNA, which conclude Tris 50 mmol, EDTA 10 mmol, MgCl22.9 mmol, Protein K 100 mg/L, DTT 10 g/L, NaCl 9 g/L, urea 1 mol/L and SDS 2%. All these reagents was mixed together and digested 1 h, and 97℃10 min. Then, using phenol and chloroform method extracted DNA. The parameter of OD26o/OD28o of DNA is 1.84, which is belong tol.8-2.0 of fine DNA.2 Adjusting the common laboratory reagent proportion used to enhance PCR effect (glycerin, dimethyl sulfone, armour amide and DDT) and PH value to optimize Taq polymerase reactions environment in order to enhance its specificity. The best system of multiple-primer PCR was screened by elimination interferon. The system concluded 10×buffer (Tris-HCl 100 mmol and KCl500 mmol) PH=9.72.5μL, DDT 8 mM, MgCl2 2.9 mmol, dNTP 0.8 mmol, glycerol 5%, dimethy sulfoxide 0.25%, formamide 0.5%, template (100 ng/μL) 1μL,10 U Taq polymerase and interfering 11 pair primers which concentration of upstream and downstream is 32ppmol and its response procedures is 94℃for 10 min denaturizing step, followed by 10 PCR cycles at 94℃for 30 s, touch-down method was used from 65-61℃for 40 s,72℃for 45 S, followed by 25 PCR cycles at 94℃for 30 s,61℃for 40 s,72℃for 45 S, then extensions at 65℃for 1h and extensions 25℃for 1h. At last they were saved at 4℃forever.This study found out the method of individual identification by bull semen DNA. Using these methods, we can quickly extraction of high quality DNA and carry out multiple-primer PCR. Through the methods, we can improve the efficiency of individual identification and parentage testing in breeding bulls. So the study lays a theoretical foundation and experiment technology for Livestock individual identification.