{"id":161,"date":"2023-03-06T02:06:41","date_gmt":"2023-03-05T18:06:41","guid":{"rendered":"http:\/\/39.105.184.3\/?p=161"},"modified":"2025-12-21T01:06:33","modified_gmt":"2025-12-20T17:06:33","slug":"%e5%b0%8f%e9%bc%a0%e5%9f%ba%e5%9b%a0%e5%9e%8b%e9%89%b4%e5%ae%9a","status":"publish","type":"post","link":"https:\/\/fanyiming.life\/?p=161","title":{"rendered":"\u5c0f\u9f20\u57fa\u56e0\u578b\u9274\u5b9a"},"content":{"rendered":"\n<h1 class=\"wp-block-heading\">Genotyping<\/h1>\n\n\n\n<p>\u5f53\u6d89\u53ca\u5230\u4e0d\u540c\u57fa\u56e0\u578b\u5c0f\u9f20\u7684\u6742\u4ea4\u7684\u65f6\u5019\uff0c\u5176\u540e\u4ee3\u57fa\u56e0\u578b\u9075\u5faa\u5b5f\u5fb7\u5c14\u9057\u4f20\u5b9a\u5f8b\u548c\u8fde\u9501\u4ea4\u6362\u5b9a\u5f8b\u3002\u6211\u4eec\u5f80\u5f80\u9700\u8981\u5176\u4e2d\u7684\u4e00\u79cd\u7279\u5b9a\u7684\u57fa\u56e0\u578b\u7528\u4e8e\u540e\u7eed\u5b9e\u9a8c\uff0c\u8fd9\u65f6\u5c31\u9700\u8981\u5bf9\u5c0f\u9f20\u7684\u57fa\u56e0\u578b\u8fdb\u884c\u9274\u5b9a\uff08Genotyping\uff09\u3002<\/p>\n\n\n\n<p>\u9274\u5b9a\u7684\u65b9\u6cd5\u662f\uff1a\u4ece\u5c11\u91cf\u7ec4\u7ec7\uff08\u8840\u6db2\u3001\u5c3e\u7f18\u3001\u811a\u8dbe\uff09\u4e2d\u63d0\u53d6\u57fa\u56e0\u7ec4\uff0c\u901a\u8fc7\u5728\u76ee\u7684\u4f4d\u70b9\u8bbe\u8ba1\u53ef\u4ee5\u5bf9\u4e0d\u540c\u57fa\u56e0\u578b\u8fdb\u884c\u533a\u5206\u7684\u5f15\u7269\uff0cPCR\u540e\u901a\u8fc7\u8dd1\u80f6\u6761\u5e26\u5dee\u5f02\u6216\u6d4b\u5e8f\u78b1\u57fa\u987a\u5e8f\u5dee\u5f02\u786e\u5b9aGenotype\u3002<\/p>\n\n\n\n<h1 class=\"wp-block-heading\">\u526a\u811a\u8dbe<\/h1>\n\n\n\n<p>\u526a\u811a\u8dbe\u6709\u4e24\u4e2a\u76ee\u7684\uff0c\u4e00\u662f\u63d0\u53d6DNA\u7528\u4e8egenotyping\uff0c\u4e8c\u662f\u5bf9\u5c0f\u9f20\u8fdb\u884c\u6807\u53f7\u3002<\/p>\n\n\n\n<p>\u526a\u811a\u8dbe\u7684\u65f6\u671f\u4e00\u822c\u662f\u5c0f\u9f20\u51fa\u751f\u540e10-15\u5929\u3002\u56e0\u4e3a\u5c0f\u9f20\u5728\u51fa\u751f\u540e21\u5929\u65ad\u5976\uff0c\u9700\u8981\u548c\u6bcd\u9f20\u5206\u7b3c\uff0c\u800c\u5206\u7b3c\u4e00\u822c\u662f\u6839\u636e\u5b9e\u9a8c\u76ee\u7684\u6309\u7167\u57fa\u56e0\u578b\u5206\u7684\uff0c\u6240\u4ee5\u526a\u5b8c\u811a\u8dbe\u8fd9\u51e0\u5929\u6b63\u597d\u7528\u4e8e\u505agenotyping\uff0c\u4e0b\u4e00\u6b21\u5c31\u53ef\u4ee5\u5e26\u7740genotyping\u7684\u7ed3\u679c\u53bb\u52a8\u7269\u623f\u5206\u7b3c\u4e86\u3002<\/p>\n\n\n\n<p>\u526a\u811a\u8dbe\u7684\u64cd\u4f5c\uff1a\u53f3\u624b\u6293\u7740\u5c3e\u5df4\uff0c\u8ba9\u5c0f\u9f20\u5728\u94c1\u7f51\u4e0a\u722c\u3002\u5de6\u624b\u4ece\u5c3e\u90e8\u5f00\u59cb\u634f\u4f4f\u810a\u67f1\u5411\u524d\u63a8\uff0c\u76f4\u5230\u62c7\u6307\u548c\u98df\u6307\u634f\u4f4f\u53cc\u8033\uff0c\u624b\u638c\u56fa\u5b9a\u4f4f\u5c0f\u9f20\u540e\u80cc\u90e8\u76ae\u80a4\uff0c\u7ffb\u8fc7\u6765\u8179\u4fa7\u5411\u4e0a\u3002\u53f3\u624b\u62ff\u526a\u5200\u526a\u811a\u8dbe\u3002\u5982\u679c\u5c0f\u9f20\u6525\u62f3\uff0c\u8f7b\u5fae\u6447\u6643\u4f7f\u5176\u624b\u638c\uff08\u811a\u638c\uff09\u5f20\u5f00\u3002<\/p>\n\n\n\n<figure class=\"wp-block-image\"><img loading=\"lazy\" decoding=\"async\" decoding=\"async\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202303060121817.png?x-oss-process=style\/screenshots\" alt=\"image-20230306012109891\" \/><\/figure>\n\n\n\n<p>\u4e0a\u56fe\u662f\u8179\u4fa7\u5411\u4e0a\uff08\u5c0f\u9f20\u8eba\u7740\uff09\u7684\u7b80\u7b14\u753b\uff0c\u524d\u722a4\u6307\uff0c\u540e\u722a5\u8dbe\u3002\u6807\u53f7\u987a\u5e8f\u662f\uff0c\u4ee5\u5b9e\u9a8c\u8005\u89c2\u5bdf\u4ece\u5de6\u5230\u53f3\u7684\u89c6\u89d2\uff0c\u4ece\u540e\u722a\u5f00\u59cb\uff0c\u4f9d\u6b21\u4e3a\u4e2a\u4f4d\u65701~9\u548c10\uff0c\u524d\u722a\u4e3a\u5341\u4f4d\u657020~90\uff0c\u8fd9\u6837\u80fd\u8868\u793a1~100\u4e4b\u95f4\u7684\u4efb\u610f\u6574\u6570. \u6536\u96c6\u526a\u4e0b\u6765\u7684\u811a\u8dbe\u5230\u6807\u53f7\u5bf9\u5e94\u6807\u53f7\u7684EP\u7ba1\u91cc\uff0c\u4e00\u8dbe\u4e00\u7ba1\u3002<\/p>\n\n\n\n<figure class=\"wp-block-image\"><img loading=\"lazy\" decoding=\"async\" decoding=\"async\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202303060121130.png?x-oss-process=style\/screenshots\" alt=\"image-20230306012134346\" \/><\/figure>\n\n\n\n<p>\u5b9e\u9a8c\u8981\u6c42\u540c\u6027\u522b\uff0c\u4e00\u822c\u5728\u526a\u811a\u8dbe\u65f6\u5904\u6b7b\u65b0\u751f\u96cc\u9f20\uff0c\u53ea\u5bf9\u65b0\u751f\u96c4\u9f20\u6807\u53f7\u3002\u533a\u5206\u96cc\u96c4\u4e3b\u8981\u770b\u662f\u5426\u6709\u4e73\u5934\u548c\u5c0f\u9e21\u9e21\u3002<\/p>\n\n\n\n<h1 class=\"wp-block-heading\">PCR<\/h1>\n\n\n\n<p>\u57fa\u56e0\u7ec4\u63d0\u53d6\u6d41\u7a0b\uff1a<\/p>\n\n\n\n<p>\u5728\u6bcf\u4e2a\u811a\u8dbe\u7684EP\u7ba1\u4e2d\u52a0\u5165120\u03bcL A\u6db2\uff0c95\u2103\u91d1\u5c5e\u6d7410min\uff0c\u79bb\u5fc3\uff0c\u4e0a\u6e05\u52a0\u516530\u03bcL B\u6db2\uff0c\u6df7\u5300\u3002\u53d61\u03bcL\u4f5c\u4e3a25\u03bcL PCR\u4f53\u7cfb\u7684\u6a21\u677f\uff08\u5b9e\u9a8c\u5f88\u7cd9\uff0c\u79bb\u5fc3\u5b8c\u4e0d\u7528\u6362\u7ba1\u5b50\uff0c\u76f4\u63a5\u52a0B\u6db2\u5c31\u884c\uff09\u3002<\/p>\n\n\n\n<blockquote class=\"wp-block-quote is-layout-flow wp-block-quote-is-layout-flow\">\n<p>A\u6db2\uff1a50mM NaOH (0.1g\u219250mL\u6c34) B\u6db2\uff1a1M Tris + 5mM EDTA pH=8.0 (6.06g Tris + 0.093g Na2EDTA\u00b72H2O \u2192 50mL\u6c34)<\/p>\n<\/blockquote>\n\n\n\n<p>PCR\u4ea7\u7269\u8dd1\u80f6\uff08\u6216\u6d4b\u5e8f\uff09\uff0c\u6761\u5e26\u5c0f\u7684\u8bdd\u8bb0\u5f97\u75282% Agarose Gel\u3002<\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\" \/>\n\n\n\n<p>\u62ff\u5230genotyping\u7ed3\u679c\u540e\uff0c\u5728\u5c0f\u9f20\u51fa\u751f\u540e21\u5929\u65ad\u5976\u540e\u5206\u7b3c\u3002\u51fa\u751f\u540e6~8\u5468\u5c0f\u9f20\u6027\u6210\u719f\uff0c\u4e00\u822c\u75288\u5468\u9f20\u8fdb\u884c\u5b9e\u9a8c\uff0c\u6807\u914d\u662f6\u53ea\u5b9e\u9a8c\u9f20+6\u53ea\u5bf9\u7167\u9f20\u3002<\/p>\n\n\n\n<p>\u4e0b\u9762\u4e3e\u4f8b\u5b50\uff1a<\/p>\n\n\n\n<h1 class=\"wp-block-heading\">CKO\u5c0f\u9f20genotyping<\/h1>\n\n\n\n<p>\u5047\u8bbe\u6211\u4eec\u5df2\u7ecf\u62ff\u5230\u7eaf\u5408\u7684flox\u5c0f\u9f20\u548cCre\u5c0f\u9f20\u4e86\u3002\u7b2c\u4e00\u6b65\u662f\u8ba9\u8fd9\u4e8c\u8005\u6742\u4ea4\uff0c\u540e\u4ee3F1\u5e94\u4e3a\u6742\u5408flox\u548c\u6742\u5408Cre\u3002\u6211\u4eec\u5f53\u7136\u9700\u8981\u7eaf\u5408\u7684\u6572\u9664\u9f20\uff0c\u6240\u4ee5F1\u81ea\u4ea4\u3002\u6839\u636e\u5b5f\u5fb7\u5c14\u9057\u4f20\u5b9a\u5f8b\uff0cF2\u67091\/4\u7684\u6982\u7387\u5f97\u5230\u7eaf\u5408\u7684flox\uff0c\u67091\/4\u7684\u6982\u7387\u5f97\u5230\u7eaf\u5408\u7684Cre\uff0c\u90a3\u4e48\u5982\u679c\u662f\u5b8c\u5168\u81ea\u7531\u7ec4\u5408\uff0c\u5e94\u8be5\u67091\/16\u7684\u6982\u7387\u5f97\u5230\u7eaf\u5408\u7684flox\u548cCre\uff0c\u8fd9\u6837\u5c31\u5b9e\u73b0\u4e86floxed gene\u7684\u4e24\u4e2aallele\u90fd\u88ab\u6572\u4e86\u3002Cre\u6742\u5408\uff08hemizygous\uff09\u5c31\u53ef\u4ee5\u53d1\u6325\u4f5c\u7528\uff0c\u6240\u4ee5\u6211\u4eec\u67091\/4*3\/4=3\/16\u7684\u6982\u7387\u5f97\u5230\u76ee\u7684\u6572\u9664\u9f20\u3002<\/p>\n\n\n\n<p>\u56e0\u4e3aF1\u4e2d\u7684Cre\u662fhemizygous\uff0cF2\u4e2d\u6709\u53ef\u80fd\u4ea7\u751fCre\u7684nullizygous\uff0c\u4ee5\u53cafloxed gene\u7684heterozygous\u548cnullizygous\u3002\u8fd9\u4e0d\u662f\u6211\u4eec\u60f3\u8981\u7684\uff0c\u6240\u4ee5\u8fdb\u884cgenotyping\u3002<\/p>\n\n\n\n<p>\u6839\u636e\u5b9e\u9a8c\u76ee\u7684\uff0cGenotyping\u4e3b\u8981\u770b\u4e24\u70b9\uff1a\u7b2c\u4e00\uff0cflox\u8981\u6c42\u662f\u7eaf\u5408\u7684\uff0c\u6240\u4ee5\u9700\u8981\u628aflox allele (CKO allele)\u548cwt allele\u8bbe\u8ba1\u5f15\u7269\u8fdb\u884c\u533a\u5206\u3002\u7b2c\u4e8c\uff0c\u5fc5\u987b\u5b58\u5728Cre\u9176\uff0chomozygous\u548chemizygous\u90fd\u53ef\u4ee5\uff0c\u4f46\u4e0d\u80fd\u662fnullizygous\uff0c\u6240\u4ee5\u9700\u8981\u8bbe\u8ba1\u5f15\u7269\u533a\u5206Cre allele\u548cwt&#8217; allele\uff08wt\u540e\u9762\u52a0\u4e86\u4e2a&#8217;\u4ee5\u793a\u8ddfflox\u4f4d\u70b9\u7684wt\u533a\u5206\uff09\u3002<\/p>\n\n\n\n<p>\u7b2c\u4e00\u5bf9\u5f15\u7269\u8fd9\u4e48\u8bbe\u8ba1\uff0c\u7ea2\u8272\u7684\u662floxp site\uff0c\u6709loxp\u7684\u5c31\u957f\u51e0\u5341\u4e2abp\uff0c\u5c31\u662fCKO allele\uff0c\u77ed\u7684\u5c31\u662fwt allele\u3002<\/p>\n\n\n\n<figure class=\"wp-block-image\"><img loading=\"lazy\" decoding=\"async\" decoding=\"async\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202303060122395.png?x-oss-process=style\/screenshots\" alt=\"image-20230306012201631\" \/><\/figure>\n\n\n\n<p>\u7b2c\u4e8c\u5bf9\u5f15\u7269\u6d4b\u6709\u6ca1\u6709Cre\uff0c\u75283\u6761\u5f15\u7269\u6df7\u4e00\u5757PCR\u3002\u5982\u679c\u53ea\u6709&lt;wt Forword\u548ccommon&gt;\u8fd9\u4e00\u5bf9\u7684\u7247\u6bb5\uff0c\u8bf4\u660e\u662f\u7eaf\u5408wt&#8217; allele\uff0c\u5982\u679c\u53ea\u6709&lt;mutant forward\u548ccommon&gt;\u8fd9\u4e00\u5bf9\u7684\u7247\u6bb5\uff0c\u8bf4\u660e\u662f\u7eaf\u5408Cre allele\uff0c\u5982\u679c\u4e24\u6761\u5e26\u90fd\u6709\uff0c\u8bf4\u660e\u662fhemizygous\u3002<\/p>\n\n\n\n<figure class=\"wp-block-image\"><img loading=\"lazy\" decoding=\"async\" decoding=\"async\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202303060123980.png?x-oss-process=style\/screenshots\" alt=\"image-20230306012258651\" \/><\/figure>\n\n\n\n<p>\u4e0b\u9762\u662f\u6211\u6628\u5929\u9274\u5b9a\u7684\u771f\u56fe\u3002<\/p>\n\n\n\n<figure class=\"wp-block-image\"><img loading=\"lazy\" decoding=\"async\" decoding=\"async\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202303060123662.png?x-oss-process=style\/screenshots\" alt=\"image-20230306012335620\" \/><\/figure>\n\n\n\n<p>Cas9KO\u662f\u76f4\u63a5\u6572\u6389\u4e86\u4e24\u4e2a\u78b1\u57fa\uff0c\u56fe\u4e0a\u770b\u4e0d\u51fa\u5dee\u5f02\uff0c\u9700\u8981\u6d4b\u5e8f\u3002<\/p>\n\n\n\n<figure class=\"wp-block-image\"><img loading=\"lazy\" decoding=\"async\" decoding=\"async\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202303060123807.png?x-oss-process=style\/screenshots\" alt=\"image-20230306012354486\" \/><\/figure>\n\n\n\n<p>\u8fd9\u79cd\u5c31\u8bf4\u660e\uff0c\u786e\u5b9e\u662fKO\u9f20\uff0c\u53d1\u751f\u4e86Frame shift\u3002CKO\u4e2d6\u30017\u30019\u53f7\u662f\u6742\u5408\u5b50\uff0c\u610f\u5473\u7740\u6709\u4e00\u6761allele\u662ffloxed 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