摘要
本试验建立了用聚合酶链反应（Ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）体外扩增牛Ｙ染色体特异ＤＮＡ序列的方法。在牛、山羊和绵羊Ｙ染色体特异ＤＮＡ同源序列高度保守区设计一对引物，两条引物均为２０ＮＴ，对公牛Ｙ染色体特异的３０７ｂｐ序列进行体外扩增。从屠宰的成年公牛、母牛肝提取ＤＮＡ，用作ＰＣＲ扩增的模板。ＰＣＲ反应总体积为５０μＬ，各成分含量为：２５ｍｍｏ１／ＬＴｒｉｓ－ＨＣＩ（ｐＨ８．２），２５ｍｍｏｌ／Ｌ（ＮＨ４）２ＳＯ４、２ｍｍｏｌ／ＬＭｇＣｌ２、０．１ｍｇ／ｍＬｇｅｌａｔｉｎ、５％Ｆｏｒｍａｍｉｄｅ、ｌμｇ模板、引物各为２５ｐｍｏｌ、ｄＮＴＰ各为２００μｍｏｌ／Ｌ、ＤＮＡ聚合酶２ＩＵ。反应在微机控制ＰＣＲ仪上进行，采用９２．５℃变性，５５℃退火，７２℃延伸，共３５个循环。扩增产物在２％琼脂糖凝胶上电泳，用ｐＧＥＭ－７ｚｆ（＋）Ｈａｅ Ⅲ作分子量标准。电泳结果，公牛个体样品显示清晰可见的特异ＤＮＡ扩增带，而母牛个体样品无扩增片段出现。扩增产物用ＰｓｔⅠ酶解后出现３条区带，与预期结果一致。
A polymerase chain reaction (PCR)-based method for sex determination ofbovine by
amplification of bovine Y chromosome-specific sequence was developed.Apair of primers of 20
bases in length that recognize bovine Y chromosome-specific se-quence were designed to
target a sequence of 307 base pairs in length. DNA from maleand female bovine were
extracted. The amplification was performed by using 35 cyclesof denaturation at 92.5
℃,annealing at 55℃ and extention at 72℃. Amqlified DNAproducts were resolved by agarose
mini-gel electrophoresis and visualized by ethidiumbromide staining and ultraviolet
illumination.The results showed that a specific amplifi-cation fragment of the expected size was
detected in the sample from male bovine but didnot appear in the sample from female bovine.
Cut by Pst Ⅰ,the amplified fragment wasdivided into three smaller fragments,which was
observed through electrophresis.Thisindicated that there really exist Pst Ⅰ site in the
amplified fragment, which was con-curred with our design.
出处
《中国兽医学报》
CAS
CSCD
1994年第2期127-129,共3页
Chinese Journal of Veterinary Science
基金
吉林省科委青年基金
