{"id":143,"date":"2020-12-02T10:53:41","date_gmt":"2020-12-02T01:53:41","guid":{"rendered":"https:\/\/www.promega.co.jp\/resources\/faq\/?p=143"},"modified":"2021-02-15T17:14:45","modified_gmt":"2021-02-15T08:14:45","slug":"q47","status":"publish","type":"post","link":"https:\/\/www.promega.co.jp\/resources\/faq\/amplification-quantification\/pcr-and-rt\/total\/pcr-info\/q47","title":{"rendered":"PCR\u306e\u6700\u9069\u5316\u306e\u305f\u3081\u306b\u7279\u306b\u91cd\u8981\u306a\u30d1\u30e9\u30e1\u30fc\u30bf\u30fc\u306f\u4f55\u3067\u3059\u304b\uff1f"},"content":{"rendered":"\n

PCR\u306f\u30af\u30ed\u30fc\u30cb\u30f3\u30b0\u3001\u30b5\u30d6\u30af\u30ed\u30fc\u30cb\u30f3\u30b0\u3001DNA\u30b7\u30fc\u30af\u30a8\u30f3\u30b7\u30f3\u30b0\u3001\u907a\u4f1d\u5b50\u767a\u73fe\u306e\u89e3\u6790\u3001\u6b20\u5931\u5909\u7570\u3001\u5909\u7570\u306e\u5c0e\u5165\u306a\u3069\u3092\u542b\u3080\u591a\u304f\u306e\u30a2\u30d7\u30ea\u30b1\u30fc\u30b7\u30e7\u30f3\u3067\u6709\u52b9\u306a\u624b\u6cd5\u3067\u3059(1-3)\u3002PCR\u5897\u5e45\u3092\u78ba\u5b9f\u306b\u884c\u3046\u305f\u3081\u306b\u6700\u9069\u5316\u3092\u5fc5\u8981\u3068\u3059\u308b\u91cd\u8981\u306a\u30d1\u30e9\u30e1\u30fc\u30bf\u30fc\u306f\u3001\u92f3\u578b\u306e\u9078\u629e\u304a\u3088\u3073\u7cbe\u88fd\u5ea6\u3001\u30de\u30b0\u30cd\u30b7\u30a6\u30e0\u6fc3\u5ea6\u3001\u30d7\u30e9\u30a4\u30de\u30fc\u30c7\u30b6\u30a4\u30f3\u304a\u3088\u3073\u6fc3\u5ea6\u3001\u30cc\u30af\u30ec\u30aa\u30c1\u30c9\u306e\u6fc3\u5ea6\u3001\u8010\u71b1\u6027\u9175\u7d20\u306e\u30bf\u30a4\u30d7\u304a\u3088\u3073\u6fc3\u5ea6\u3001\u30b5\u30a4\u30af\u30eb\u306e\u8a2d\u5b9a\u306a\u3069\u304c\u6319\u3052\u3089\u308c\u307e\u3059\u3002

\u92f3\u578b<\/strong><\/em>\uff1a \u30b2\u30ce\u30e0\u3001\u30a6\u30a4\u30eb\u30b9DNA\u3001\u30d7\u30e9\u30b9\u30df\u30c9\u306e\u3088\u3046\u306a\u30d9\u30af\u30bf\u30fc\u3001\u9006\u8ee2\u5199\u306b\u3088\u3063\u3066\u5408\u6210\u3055\u308c\u305fcDNA\u306a\u3069\u591a\u7a2e\u985e\u306eDNA\u304c\u5897\u5e45\u306e\u92f3\u578b\u3068\u3057\u3066\u4f7f\u308f\u308c\u307e\u3059\u3002\u30b2\u30ce\u30e0\u306e\u3088\u3046\u306a\u8907\u96d1\u306a\u69cb\u9020\u306e\u92f3\u578b\u3067\u306f\u7d041ng\uff5e1\u03bcg\u304c\u4f7f\u308f\u308c\u307e\u3059\u3002\u30d7\u30e9\u30b9\u30df\u30c9\u3084cDNA\u306e\u3088\u3046\u306a\u8907\u96d1\u3067\u306a\u3044\u6a19\u7684\u3067\u3042\u308b\u92f3\u578b\u306f\u4e00\u822c\u7684\u306b10pg\uff5e100ng\u3092\u5fc5\u8981\u3068\u3057\u307e\u3059(2)\u3002

\u30de\u30b0\u30cd\u30b7\u30a6\u30e0<\/strong><\/em>\uff1a \u53cd\u5fdc\u6db2\u4e2d\u306e\u30de\u30b0\u30cd\u30b7\u30a6\u30e0\u6fc3\u5ea6\u306f\u91cd\u8981\u3067\u3042\u308a\u3001\u4e00\u822c\u7684\u306b1.0\uff5e5.0\uff4dM\u306e\u7bc4\u56f2\u3067\u6700\u9069\u5316\u3055\u308c\u307e\u3059\u3002\u3057\u304b\u3057\u3001\u3053\u306e\u6fc3\u5ea6\u306f\u8010\u71b1\u9175\u7d20\u306e\u7a2e\u985e\u3068\u540c\u3058\u3088\u3046\u306b\u30cc\u30af\u30ec\u30aa\u30c1\u30c9\u306e\u6fc3\u5ea6\u306b\u4f9d\u5b58\u3057\u3066\u5909\u5316\u3057\u307e\u3059(2,3)\u3002\u30de\u30b0\u30cd\u30b7\u30a6\u30e0\u6fc3\u5ea6\u306f\u30d7\u30e9\u30a4\u30de\u30fc\u306e\u30a2\u30cb\u30fc\u30ea\u30f3\u30b0\u3001PCR\u7523\u7269\u306e\u7279\u7570\u6027\u3001\u9175\u7d20\u306e\u6d3b\u6027\u3084\u5fe0\u5b9f\u6027\u306b\u5f71\u97ff\u3059\u308b\u53ef\u80fd\u6027\u304c\u3042\u308a\u307e\u3059(2)\u3002PCR\u3067\u306f\u7dcfdNTP\u6fc3\u5ea6\u304b\u3089\u3055\u3089\u306b0.5\uff5e2.5mM\u306e\u30de\u30b0\u30cd\u30b7\u30a6\u30e0\u3092\u542b\u3080\u5fc5\u8981\u304c\u3042\u308a\u307e\u3059(2)\u3002<\/p>\n\n\n\n

\u30d7\u30e9\u30a4\u30de\u30fc<\/strong><\/em>\uff1a PCR\u306b\u4f7f\u3046\u305f\u3081\u306e\u30d7\u30e9\u30a4\u30de\u30fc\u3092\u30c7\u30b6\u30a4\u30f3\u3059\u308b\u6642\u306b\u3044\u304f\u3064\u304b\u306e\u8003\u616e\u3059\u3079\u304d\u70b9\u304c\u3042\u308a\u307e\u3059\u3002\u305d\u308c\u3089\u306b\u306f\u30aa\u30ea\u30b4\u30cc\u30af\u30ec\u30aa\u30c1\u30c9\u306e\u9577\u3055\u3001Tm\u5024\u3001\u914d\u5217\u306e\u7269\u7406\u7684\u306a\u7279\u5fb4\u3001\u30d7\u30e9\u30a4\u30de\u30fc\u306e\u76f8\u4e92\u4f5c\u7528\u306e\u53ef\u80fd\u6027\u3001\u5897\u5e45\u7523\u7269\u306e\u9577\u3055\u3001\u6a19\u7684\u3068\u306a\u308b\u92f3\u578b\u914d\u5217\u4e0a\u3067\u306e\u4f4d\u7f6e\u306a\u3069\u304c\u542b\u307e\u308c\u307e\u3059(1-3)\u3002\u30d7\u30e9\u30a4\u30de\u30fc\u306e\u6fc3\u5ea6\u306f\u4e00\u822c\u7684\u306b0.1\uff5e0.5\u03bcM\u3067\u4f7f\u308f\u308c\u307e\u3059\u3002\u30d7\u30e9\u30a4\u30de\u30fc\u306e\u6fc3\u5ea6\u304c\u9ad8\u3059\u304e\u305f\u5834\u5408\u3001\u30df\u30b9\u30d7\u30e9\u30a4\u30df\u30f3\u30b0\u3092\u8d77\u3053\u3057\u305f\u308a\u3001\u4eba\u9020\u7684\u306aPCR\u7523\u7269\u3068\u547c\u3070\u308c\u308b\u975e\u7279\u7570\u7684\u306a\u7523\u7269\u3092\u7523\u751f\u3057\u307e\u3059\u3002\u4e00\u822c\u7684\u306a\u30d7\u30e9\u30a4\u30de\u30fc\u306f18\uff5e28 nucleotides\u306e\u9577\u3055\u3001Tm\u5024\u306f55\uff5e80\u2103\u3092\u793a\u3057\u3001G\/C\u542b\u91cf\u306f45\uff5e55%\u3067\u3059(1,2)\u3002<\/p>\n\n\n\n

\u30cc\u30af\u30ec\u30aa\u30c1\u30c9<\/strong><\/em>\uff1a \u30cc\u30af\u30ec\u30aa\u30c1\u30c9\u306e\u6eb6\u6db2\u306fpH7.0\u306b\u8abf\u6574\u3057\u3001\u6700\u7d42\u6fc3\u5ea6\u306f\u305d\u308c\u305e\u308c20\uff5e250\u03bcM\u306b\u306a\u308b\u3088\u3046\u306b\u52a0\u3048\u307e\u3059(2)\u3002\u3053\u306e\u6fc3\u5ea6\u3067\u306f\u53ce\u91cf\u3001\u7523\u7269\u306e\u7279\u7570\u6027\u3001\u9175\u7d20\u306e\u5fe0\u5b9f\u6027\u306b\u304a\u3044\u3066\u81f3\u9069\u5316\u3055\u308c\u305f\u30d0\u30e9\u30f3\u30b9\u3067\u5f97\u3089\u308c\u307e\u3059\u3002\u5404dNTP\u306f\u53d6\u308a\u8fbc\u307f\u306e\u30df\u30b9\u3092\u6e1b\u3089\u3059\u305f\u3081\u306b\u7b49\u30e2\u30eb\u6fc3\u5ea6\u3067\u52a0\u3048\u3066\u304f\u3060\u3055\u3044\u3002dNTPs\u306f\u904a\u96e2\u306e\u30de\u30b0\u30cd\u30b7\u30a6\u30e0\u30a4\u30aa\u30f3\u3092\u30ad\u30ec\u30fc\u30c8\u3059\u308b\u306e\u3067\u3001Mg2+<\/sup>\u3092\u52a0\u3048\u3066\u88dc\u6b63\u3057\u306a\u3044\u5834\u5408\u306f\u904e\u5270\u306edNTPs\u3092\u52a0\u3048\u306a\u3044\u3067\u304f\u3060\u3055\u3044\u3002<\/p>\n\n\n\n

\u9175\u7d20<\/strong><\/em>\uff1a PCR\u7528\u306b\u591a\u7a2e\u985e\u306e\u8010\u71b1\u6027DNA\u30dd\u30ea\u30e1\u30e9\u30fc\u30bc\u304c\u8ca9\u58f2\u3055\u308c\u3066\u3044\u307e\u3059\u3002\u3053\u308c\u3089\u306e\u30dd\u30ea\u30e1\u30e9\u30fc\u30bc\u3067\u306f\u8010\u71b1\u6027\u3001\u5fe0\u5b9f\u5ea6\u3001\u6700\u9069\u306a\u53cd\u5fdc\u6761\u4ef6\u304c\u7570\u306a\u3063\u3066\u3044\u307e\u3059\u3002\u8010\u71b1\u6027DNA\u30dd\u30ea\u30e1\u30e9\u30fc\u30bc\u306e\u4e00\u822c\u7684\u306a\u6982\u8981\u306f\u6587\u732e1\u306b\u8a73\u3057\u304f\u8a18\u8f09\u3055\u308c\u3066\u3044\u307e\u3059\u3002Pfu\u306e\u3088\u3046\u306a\u8010\u71b1\u6027DNA\u30dd\u30ea\u30e1\u30e9\u30fc\u30bc\u306f3\u2019\u21925\u2019\u65b9\u5411\u3078\u306e\u30a8\u30ad\u30bd\u30cc\u30af\u30ec\u30a2\u30fc\u30bc\u6d3b\u6027\u3092\u6301\u3063\u3066\u3044\u308b\u305f\u3081\u3001\u5e73\u6ed1\u672b\u7aef\u3092\u6301\u3064PCR\u7523\u7269\u3092\u4f5c\u6210\u3057\u307e\u3059\u3002\u4e00\u65b9\u3001\u30a8\u30ad\u30bd\u30cc\u30af\u30ec\u30a2\u30fc\u30bc\u6d3b\u6027\u3092\u6301\u305f\u306a\u3044Taq\u306e\u3088\u3046\u306a\u305d\u306e\u4ed6\u306e\u8010\u71b1\u6027\u9175\u7d20\u306f1\u500b\u306e\u30a2\u30c7\u30ce\u30b7\u30f3\u3092\u4ed8\u52a0\u3055\u308c\u305fPCR\u7523\u7269\u3092\u4f5c\u6210\u3057\u307e\u3059(4)\u3002\u4e00\u822c\u7684\u306b100\u03bcl\u306e\u53cd\u5fdc\u306b\u306f0.5\uff5e1.25 units\u306e\u9175\u7d20\u304c\u4f7f\u308f\u308c\u307e\u3059(2,3)\u3001\u3057\u304b\u3057\u3001\u7279\u6b8a\u306a\u30a2\u30d7\u30ea\u30b1\u30fc\u30b7\u30e7\u30f3\u3067\u306f\u9175\u7d20\u306e\u8ffd\u52a0\u304c\u5fc5\u8981\u306a\u5834\u5408\u304c\u3042\u308a\u307e\u3059\u3002\u9175\u7d20\u6fc3\u5ea6\u304c\u4f4e\u3059\u304e\u308b\u3068\u3001\u5145\u5206\u91cf\u306e\u7523\u7269\u304c\u4f5c\u6210\u3055\u308c\u307e\u305b\u3093\u3002\u9ad8\u3059\u304e\u308b\u5834\u5408\u3001\u975e\u7279\u7570\u7684\u306a\u30d0\u30c3\u30af\u30b0\u30e9\u30a6\u30f3\u30c9\u306e\u30d0\u30f3\u30c9\u304c\u4f5c\u6210\u3055\u308c\u307e\u3059(2)\u3002<\/p>\n\n\n\n

\u30b5\u30a4\u30af\u30ea\u30f3\u30b0<\/strong><\/em>\uff1a PCR\u306e\u30b5\u30a4\u30af\u30eb\u6761\u4ef6\u306f\u201c\u5909\u6027\u201d\uff64\u201c\u30a2\u30cb\u30fc\u30ea\u30f3\u30b0\u201d\uff64\u201c\u4f38\u9577\u201d\u304b\u3089\u306a\u308b\u9023\u7d9a\u306e\u30b9\u30c6\u30c3\u30d7\u309220\uff5e30\u56de\u7e70\u308a\u8fd4\u3057\u307e\u3059\u300292\uff5e95\u2103\u306710\uff5e60\u79d2\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30b7\u30e7\u30f3\u3059\u308b\u3053\u3068\u3067\u92f3\u578bDNA\u306e\u5909\u6027\u3092\u884c\u3044\u3001\u7d9a\u3044\u3066\u30d7\u30e9\u30a4\u30de\u30fc\u306e\u30a2\u30cb\u30fc\u30ea\u30f3\u30b0\u30b9\u30c6\u30c3\u30d7\u3092\u884c\u3044\u307e\u3059\u3002\u4e00\u822c\u7684\u306b\u30a2\u30cb\u30fc\u30ea\u30f3\u30b0\u306f\u30d7\u30e9\u30a4\u30de\u30fc\u306eTm\u5024\u306e\u00b15\u2103\u306e\u7bc4\u56f2\u5185\u306e\u6e29\u5ea6\u306710\uff5e60\u79d2\u9593\u884c\u3044\u307e\u3059\u3002\u9175\u7d20\u306b\u3088\u308b\u4f38\u9577\u30b9\u30c6\u30c3\u30d7\u306f\u4e00\u822c\u7684\u306b72\uff5e74\u5ea6\u306730\uff5e60\u79d2\u9593\u884c\u308f\u308c\u307e\u3059\u3002\u3053\u308c\u3089\u306e\u6761\u4ef6\u306f\u76ee\u7684\u306ePCR\u7523\u7269\u306e\u9396\u9577\u3084\u7528\u3044\u308b\u9175\u7d20\u306e\u7279\u7570\u6027\u306b\u3088\u3063\u3066\u5909\u66f4\u3055\u308c\u307e\u3059\u3002\u5168\u9577\u306e\u7523\u7269\u3092\u4f5c\u6210\u3059\u308b\u305f\u3081\u306b\u6700\u521d\u306e\u5909\u6027\u30b9\u30c6\u30c3\u30d7\u3068\u6700\u5f8c\u306e5\uff5e10\u5206\u306e\u4f38\u9577\u53cd\u5fdc\u304c\u6709\u7528\u306a\u5834\u5408\u304c\u3042\u308a\u307e\u3059\u3002<\/p>\n\n\n\n

\u3053\u306e\u57fa\u672c\u7684\u306a\u5897\u5e45\u306e\u30d7\u30ed\u30d5\u30a3\u30fc\u30eb\u3092\u5143\u306b\u3057\u3066\u591a\u304f\u306e\u30d0\u30ea\u30a8\u30fc\u30b7\u30e7\u30f3\u3092\u884c\u3046\u3053\u3068\u304c\u3067\u304d\u307e\u3059\u3002\u305d\u308c\u3089\u306e\u30d0\u30ea\u30a8\u30fc\u30b7\u30e7\u30f3\u306f\u4f7f\u308f\u308c\u3066\u3044\u308bPCR\u306e\u30a2\u30d7\u30ea\u30b1\u30fc\u30b7\u30e7\u30f3\u306b\u3088\u308a\u307e\u3059\u3002\u6700\u8fd1\u306e\u30a2\u30d7\u30ea\u30b1\u30fc\u30b7\u30e7\u30f3\u306b\u306f\u3001\u30c7\u30a3\u30d5\u30a1\u30ec\u30f3\u30b7\u30e3\u30eb\u30c7\u30a3\u30b9\u30d7\u30ec\u30a4(5)\u3067\u7406\u8ad6\u7684\u306b\u5168\u3066\u306emRNA\u3092\u5897\u5e45\u3059\u308b(\u7d9a\u3051\u3066cDNA\u3092\u4f5c\u6210\u3059\u308b)\u305f\u3081\u306edegenerate\u30d7\u30e9\u30a4\u30de\u30fc\u3092\u4f7f\u3046\u3082\u306e\u3084\u3001\u5e0c\u5c11\u306a\u76ee\u7684\u306e\u914d\u5217\u3092\u691c\u51fa\u3057\u305f\u308a\u3001\u975e\u7279\u7570\u7684\u306a\u7523\u7269\u306e\u5897\u5e45\u3092\u6e1b\u5c11\u3055\u305b\u308b(6)\u305f\u3081\u306enested\u30d7\u30e9\u30a4\u30de\u30fc\u3092\u4f7f\u3046\u7b2c2\u30e9\u30a6\u30f3\u30c9\u306ePCR\u306e\uff12\u3064\u304c\u3042\u308a\u307e\u3059\u3002<\/p>\n\n\n\n

\u5897\u5e45\u3055\u308c\u306b\u304f\u3044\u92f3\u578bDNA\u306f\u3001DMSO(7)\u3001betain(7)\u3001formamide(8)\u3001single-stranded DNA binding protein(9)\u306a\u3069\u306e\u8a66\u85ac\u3092\u6dfb\u52a0\u3059\u308b\u3053\u3068\u3067\u6539\u5584\u3059\u308b\u3053\u3068\u304c\u3067\u304d\u307e\u3059\u3002<\/p>\n\n\n\n

\u53c2\u8003\u6587\u732e<\/strong><\/p>\n\n\n\n

  1. Griffin, H.G. and Griffin, A.M. (1994) PCR Technology: Current Innovations, CRC Press, Inc., Boca Raton, FL.<\/li>
  2. Innis, M.A. et al. (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA.<\/li>
  3. Mullis, K.B. et al. (1994) The Polymerase Chain Reaction, Birkhauser, Boston, MA.<\/li>
  4. Clark, J.M. (1988) Nucl. Acids Res. 16, 9677.<\/li>
  5. McClelland, M., Mathieu-Daude, F. and Welsh, J. (1995) Trends Genet. 11, 242.<\/li>
  6. Mitsuhashi, M. (1996) J. Clin. Lab. Anal. 10, 285.<\/li>
  7. Frackman, S., Kobs, G., Simpson, D. and Storts, D. (1998) Promega Notes 65, 27.<\/li>
  8. McConlogue, L., Brow, M.A. and Innis, M.A. (1988) Nucl. Acids Res. 16, 9869.<\/li>
  9. Oshima, R.G. (1992) BioTechniques 13, 188.<\/li><\/ol>\n","protected":false},"excerpt":{"rendered":"

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