{"id":9244,"date":"2019-07-04T16:01:46","date_gmt":"2019-07-04T08:01:46","guid":{"rendered":"http:\/\/mp.weixin.qq.com\/s?__biz=MzI3NjUzOTM1Mg==&mid=100010663&idx=1&sn=56b5116d3cc8cb29641c442585077489&chksm=6b7158105c06d106f783a68702853a8224f10ea634710c15e7748229fd2935571ed6c3e0f0c8#rd"},"modified":"2026-04-08T10:19:57","modified_gmt":"2026-04-08T02:19:57","slug":"1019851","status":"publish","type":"post","link":"https:\/\/www.geneclub.net.cn\/index.php\/2019\/07\/04\/1019851\/","title":{"rendered":"\u201c\u57fa\u56e02.0\u65f6\u4ee3\u201d\u7684\u57fa\u56e0\u5408\u6210\u6280\u672f\u5e72\u8d27\uff08\u4e0a\uff09"},"content":{"rendered":"

<\/p>\n

\n\n
\n
\"RmFicvQBAkgCuC54K47siaoibvbClYbpoQdmoEA7Yo9T0l4l5c9wsK8DWv13zXvBYojNDUX4HpbxOEe8enSicCuVVg\"\/<\/figure><\/div>\n\n
\u767e\u5bb6\u8c08<\/div>\n
\u751f\u547d\u5065\u5eb7\u7684\u5148\u950b\u58f0\u97f3<\/div>\n
\u5173\u952e\u8bcd\/\u57fa\u56e0\u5408\u6210<\/div>\n
\u00a0<\/div>\n
\u901a\u8fc7\u9ad8\u901a\u91cf\u6d4b\u5e8f\u7b49\u6280\u672f\u201c\u8bfb\u201dDNA\uff0c\u8ba9\u9057\u4f20\u4fe1\u606f\u89e3\u8bfb\u548c\u5e94\u7528\u5feb\u901f\u666e\u60e0\u5927\u4f17\uff0c\u4e3a\u201c\u57fa\u56e01.0\u65f6\u4ee3\u201d\uff1b\u6b63\u5728\u915d\u917f\u7684\u201c\u57fa\u56e02.0\u65f6\u4ee3\u201d\uff0c\u901a\u8fc7\u57fa\u56e0\u5408\u6210\u201c\u5199\u201dDNA\uff0c\u6df1\u5ea6\u7406\u89e3\u3001\u8bbe\u8ba1\u548c\u91cd\u6784\u751f\u7269\u7cfb\u7edf\uff0c\u8d4b\u80fd\u80b2\u79cd\u3001\u80fd\u6e90\u548c\u73af\u5883\u7b49\u3002\u9884\u89c1\u672a\u6765\uff0c\u672c\u6587\u4ece\u6280\u672f\u89d2\u5ea6\u4e00\u89c8\u57fa\u56e0\u5408\u6210\u539f\u7406\u548c\u6d41\u7a0b\u7b49\u3002<\/div>\n
* \u6b22\u8fce\u5411\u57fa\u56e0\u6167\u6295\u7a3f\uff0c\u878d\u8d44\u4fe1\u606f\u9996\u53d1\u514d\u8d39\u3002<\/div>\n
\u00a0<\/div>\n
\u5212\u91cd\u70b9<\/div>\n
1. \u57fa\u56e0\u5408\u6210\u7279\u70b9\uff1a\u8bbe\u8ba1\u3001\u6539\u9020\u3001\u9a8c\u8bc1\u3001\u4fee\u6b63\u7684\u53cd\u590d\u8bd5\u9519 \u3002<\/div>\n
2. \u6838\u5fc3\u70b9\uff1a\u57fa\u4e8e\u8ba1\u7b97\u673a\u7684DNA\u5e8f\u5217\u8bbe\u8ba1\uff0c\u6210\u672c\u3001\u9519\u8bef\u7387\u548cDNA\u5e8f\u5217\u8f6c\u5165\u7ec6\u80de\u7684\u529f\u80fd\u6d4b\u8bd5\u3002<\/div>\n
3. \u57fa\u56e0\u5408\u6210\u590d\u6742\u5ea6\uff0c\u4ece\u5355\u94fe\u5be1\u6838\u82f7\u9178\u94fe\u3001\u53cc\u94fe\u5be1\u6838\u82f7\u9178\u94fe\u3001\u539f\u6838\u57fa\u56e0\u7ec4\u5230\u771f\u6838\u57fa\u56e0\u7ec4\u3002<\/div>\n
4. \u57fa\u56e0\u5408\u6210\u6280\u672f\uff0c\u5305\u62ec\u57fa\u4e8eDNA\u8fde\u63a5\u9176\u3001\u57fa\u4e8eDNA\u805a\u5408\u9176\u4e24\u79cd\u601d\u8def\u3002<\/div>\n
5. \u57fa\u56e0\u7ec4\u5b8c\u6210\u7ec4\u88c5\u540e\uff0c\u9700\u8981\u4f9d\u6258\u57fa\u56e0\u7ec4\u7f16\u8f91\u6280\u672f\u8fdb\u884c\u9a8c\u8bc1\u5e76\u8fdb\u884c\u90e8\u5206\u6821\u6b63\u3002<\/div>\n
\u00a0<\/div>\n
\u00a0\u4e00\u00a0\u00a0\u00a0\u751f\u547d\u5065\u5eb7\u65f6\u4ee3\u7684\u5ba0\u513f\uff1a\u57fa\u56e0\u5408\u6210<\/div>\n
BT\uff08\u751f\u7269\u6280\u672f\uff09\u548cIT\uff08\u4fe1\u606f\u6280\u672f\uff09\u7684\u7ed3\u5408\uff0c\u5f62\u6210\u57fa\u56e0\u6280\u672f\u4ee5\u8d85\u6469\u5c14\u5b9a\u5f8b\u7684\u901f\u5ea6\u666e\u53ca\uff0c\u5728\u533b\u7597\u3001\u5065\u5eb7\u3001\u5927\u6570\u636e\u548c\u91d1\u878d\u5c42\u9762\u9010\u6e10\u5f62\u6210\u5171\u8bc6\uff0c\u5de5\u4e1a\u65f6\u4ee3\u7684\u6280\u672f\u9769\u547d\u4ece\u4fe1\u606f\u6280\u672f\u9010\u6e10\u8f6c\u79fb\u5230\u751f\u547d\u5065\u5eb7\u6280\u672f\u3002<\/div>\n
\u00a0<\/div>\n
\u9ad8\u901a\u91cf\u6d4b\u5e8f\u7b49\u6280\u672f\u201c\u8bfb\u201dDNA\u8ba9\u9057\u4f20\u4fe1\u606f\u89e3\u8bfb\u548c\u5e94\u7528\u5feb\u901f\u666e\u53ca\uff0c\u6b63\u5728\u915d\u917f\u7684\u201c\u57fa\u56e02.0\u65f6\u4ee3\u201d\u901a\u8fc7\u57fa\u56e0\u5408\u6210\u201c\u5199\u201dDNA\u6df1\u5ea6\u7406\u89e3\u548c\u8bbe\u8ba1\u751f\u7269\u7cfb\u7edf\uff0c\u5c06\u53d8\u9769\u80b2\u79cd\u3001\u80fd\u6e90\u548c\u73af\u5883\u7b49\u8bf8\u591a\u9886\u57df\u3002\u79d1\u5b66\u5bb6\u4eec\u53ef\u4ee5\u5bf9\u7f16\u7801\u57fa\u56e0\u3001\u4fe1\u53f7\u901a\u8def\u3001\u4ee3\u8c22\u9014\u5f84\u4e43\u81f3\u57fa\u56e0\u7ec4\u8fdb\u884c\u8bbe\u8ba1\u548c\u5408\u6210\u3002\u5404\u56fd\u653f\u5e9c\u5df2\u5c06\u5408\u6210\u751f\u7269\u5b66\u6280\u672f\u4f5c\u4e3a\u91cd\u70b9\u8d44\u52a9\u5bf9\u8c61\uff0c\u6d89\u53ca\u5408\u6210\u751f\u7269\u5b66\u6280\u672f\u7684\u521b\u65b0\u521b\u4e1a\u4e5f\u5907\u53d7\u8d44\u672c\u5e02\u573a\u7684\u9752\u7750\u3002<\/div>\n
\u57fa\u56e0\u5408\u6210\u5c5e\u4e8e\u751f\u7269\u5408\u6210\u7684\u8303\u7574\uff0c\u5177\u4f53\u5305\u62ec\u8ba1\u7b97\u673a\u8bbe\u8ba1\u3001DNA\/RNA\u5408\u6210\u3001CRISPR\u7f16\u8f91\u7b49\u6280\u672f\u3002\u5728\u6d41\u7a0b\u4e0a\u662f\u4e00\u4e2a\u4e0d\u65ad\u8bbe\u8ba1\u3001\u6539\u9020\u3001\u9a8c\u8bc1\u3001\u4fee\u6b63\u7684\u8bd5\u9519\u8fc7\u7a0b\uff0c\u4ee5\u6b64\u6765\u8bc4\u4f30\u5de5\u7a0b\u5316\u6539\u9020\u751f\u7269\u7cfb\u7edf\u7684\u9884\u671f\u529f\u80fd\u3002<\/div>\n
\u00a0<\/div>\n
\u3010\u57fa\u56e0\u5408\u6210\u6d41\u7a0b\u3011<\/div>\n
1. \u901a\u8fc7\u4f7f\u7528\u8ba1\u7b97\u673a\u8f6f\u4ef6\u8bbe\u8ba1DNA\u5e8f\u5217\uff0c\u8fd9\u4e9bDNA\u5e8f\u5217\u53ef\u6784\u6210\u751f\u7269\u7535\u8def\u6216\u901a\u8def(\u56fe1)\u3002<\/div>\n
2. DNA\u5e8f\u5217\u88ab\u5206\u5272\u6210\u66f4\u5c0f\u7684\u91cd\u53e0\u7247\u6bb5(\u901a\u5e38\u4e3a200-1500bp\u7684\u7247\u6bb5)\uff0c\u7136\u540e\u8fdb\u4e00\u6b65\u5206\u5272\u6210\u53ef\u5316\u5b66\u5408\u6210\u7684\u4e00\u7ec4\u5355\u94fe\u5be1\u6838\u82f7\u9178\u3002<\/div>\n
3. \u5355\u94fe\u5be1\u6838\u82f7\u9178\u901a\u8fc7\u5404\u79cdDNA\u7ec4\u88c5\u6280\u672f\uff0c\u6700\u7ec8\u88ab\u7ec4\u88c5\u6210\u66f4\u5927\u7684DNA\u7247\u6bb5\u5e76\u8fdb\u884c\u514b\u9686\u9a8c\u8bc1\u3002<\/div>\n
4. \u9a8c\u8bc1\u540e\u7684DNA\u5e8f\u5217\u88ab\u8f6c\u5165\u7ec6\u80de\u8fdb\u884c\u529f\u80fd\u6d4b\u8bd5\u3002<\/div>\n
5. \u6839\u636e\u529f\u80fd\u8bd5\u9a8c\u7ed3\u679c\uff0c\u5bf9\u672c\u8f6e\u8bbe\u8ba1\u5e8f\u5217\u8fdb\u884c\u66f4\u6539\uff0c\u5e76\u91cd\u590d\u6d4b\u8bd5\u5468\u671f\uff0c\u5f62\u6210\u8fed\u4ee3\u3002<\/div>\n\n
\n
\"640?wx_fmt=png\"\/<\/figure><\/div>\n\n
\u56fe1\uff1a\u57fa\u56e0\u5408\u6210\u7684\u5faa\u73af\u8fc7\u7a0b\u3002\uff08\u6765\u6e90\uff1aCold Spring Harb Perspect Biol\uff09<\/div>\n
\u00a0\u4e8c\u00a0\u00a0\u00a0\u57fa\u56e0\u5408\u6210\u7684\u53d1\u5c55\u5386\u7a0b<\/div>\n\n
\n
\"RmFicvQBAkgDGqJ25wBjOKiaY8dUKgVawQq0wQTbk5ZwXw8L4UOUdgQ6cs6SaibfocdhuNMZPlKrpM2eKQWnicT9WA\"\/<\/figure><\/div>\n\n
\u56fe2\uff1aDNA\u4eba\u5de5\u5408\u6210\u7684\u53d1\u5c55\u3002\uff08\u6765\u6e90\uff1aNature Biotechnology\uff09<\/div>\n
\u57fa\u56e0\u5408\u6210\u57281970\u5e74\u524d\u53ea\u662f\u5355\u94fe\u5be1\u6838\u82f7\u9178\u94fe\u5f62\u5f0f\u3002\u81ea1970\u5e74\u540e\uff0c\u53cc\u94feDNA\uff08>100 bp\uff09\u7684\u5408\u6210\u5f00\u59cb\u8fc5\u901f\u53d1\u5c55\u3002<\/div>\n
2002\u5e74\uff0c\u810a\u9ad3\u7070\u8d28\u708e\u75c5\u6bd2\uff08poliovirus\uff09\u57fa\u56e0\u7ec4\uff08\u7ea67,500 kb\uff09\u88ab\u6210\u529f\u5408\u6210(Cello, Paul et al. 2002)\uff0c\u6b63\u5f0f\u5f00\u542f\u57fa\u56e0\u7ec4\u5408\u6210\u65f6\u4ee3\u3002<\/div>\n
2010\u5e74\uff0cVenter \u548c Smith\u7b49\u5c06\u4eba\u5de5\u5408\u6210\u7684\u8548\u72b6\u652f\u539f\u4f53(Mycoplasma mycoides)\u57fa\u56e0\u7ec4\u8f6c\u5165\u5230\u5c71\u7f8a\u652f\u539f\u4f53\uff08Mycoplasma capricolum\uff09\u5bbf\u4e3b\u7ec6\u80de\u4e2d(Gibson, Glass et al. 2010)\uff0c\u8d70\u51fa\u4e86\u4eba\u5de5\u5408\u6210\u57fa\u56e0\u521b\u9020\u65b0\u7ec6\u80de\u7684\u5386\u53f2\u6027\u4e00\u6b65\u3002<\/div>\n
\u4ece 2011 \u5e74\u5f00\u59cb\u7684\u9175\u6bcd\u57fa\u56e0\u7ec4\u5408\u6210\u8ba1\u5212\uff08Sc2.0\uff09\uff0c\u5f53\u524d\u5df2\u7ecf\u5b8c\u6210\u4e86 2\u30013\u30015\u30016\u300110 \u548c 12 \u53f7\u67d3\u8272\u4f53\u7684\u5408\u6210\uff0c\u5176\u4e2d\u4e2d\u56fd\u79d1\u5b66\u5bb6\u505a\u51fa\u4e86\u91cd\u5927\u8d21\u732e\u3002DNA\u7684\u5408\u6210\u4e0e\u7ec4\u88c5\u672a\u6765\u7684\u53d1\u5c55\u4e5f\u8bb8\u4f1a\u5305\u62ec\u590d\u6742\u7684\u5fae\u751f\u7269\u7fa4\u843d\u6216\u7ec6\u80de\u7ec4\u7ec7\u7684\u4ece\u5934\u5efa\u7acb\u3002\u00a0<\/div>\n
\u00a0<\/div>\n
\u00a0\u4e09\u00a0\u00a0\u00a0\u5be1\u6838\u82f7\u9178\u7684\u8bbe\u8ba1\u548c\u5408\u6210\u6d41\u7a0b<\/div>\n
\u4e00\u6761\u57fa\u56e0\u5e8f\u5217\u7684\u5408\u6210\u8d77\u59cb\u4e8e\u5be1\u6838\u82f7\u9178\u7684\u8bbe\u8ba1\u548c\u5316\u5b66\u5408\u6210\uff0c\u901a\u8fc7\u5404\u79cd\u7ec4\u88c5\u7b56\u7565\u5f97\u52300.3-0.5 kb\u5de6\u53f3\u7684\u77ed\u7247\u6bb5\uff0c\u518d\u8fdb\u884c1-2 kb\u57fa\u56e0\u957f\u5ea6DNA\u5e8f\u5217\u7684\u7ec4\u88c5\u3002<\/div>\n
\u5408\u6210\u5177\u6709\u590d\u6742\u7ed3\u6784\u7684\u57fa\u56e0\u751a\u81f3\u57fa\u56e0\u7ec4\u6c34\u5e73DNA\u65f6\uff0c\u5408\u7406\u7684\u8bbe\u8ba1\u662f\u5173\u952e\u3002\u9776\u5e8f\u5217\u901a\u5e38\u9700\u8981\u901a\u8fc7\u8f6f\u4ef6\u4ece\u5934\u8bbe\u8ba1\uff0c\u5305\u62ec\u5bc6\u7801\u5b50\u4f18\u5316\u3001DNA\u4e8c\u7ea7\u7ed3\u6784\u8c03\u6574\u4ee5\u53ca\u5176\u4ed6\u53ef\u80fd\u5f71\u54cd\u7cfb\u7edf\u529f\u80fd\uff08\u5982\u86cb\u767d\u8868\u8fbe\uff09\u7684\u4e00\u7cfb\u5217\u7406\u6027\u8bbe\u8ba1\u3002<\/div>\n
\u3010\u5e38\u7528\u7684DNA\u5e8f\u5217\u8bbe\u8ba1\u8f6f\u4ef6\u3011<\/div>\n
DNAWORKS (http:\/\/mcl1.ncifcrf.gov\/dnaworks\/dnaworks2.html)<\/div>\n
GEMS\uff08http:\/\/software.kosan.com\/GeMS\uff09<\/div>\n
Oligo Cuts<\/div>\n
GeneDesign\uff08http:\/\/slam.bs.jhmi.edu\/gd\/<\/div>\n
TmPrime\uff08http:\/\/prime.ibn.a-star.edu.sg\uff09<\/div>\n
\u00a0<\/div>\n
\u4f20\u7edf\u7684\u5be1\u6838\u82f7\u9178\u5408\u6210\u4e00\u822c\u79f0\u4e3a\u67f1\u5f0f\u5408\u6210\uff0c\u57fa\u4e8e\u56db\u6b65\u6cd5\u4e9a\u78f7\u9170\u80fa\u5316\u5b66\u5408\u6210\u65b9\u6cd5(Froehler,Matteucci, 1983)\uff0c\u5728\u56fa\u76f8\u4e0a\u8fdb\u884c\u5be1\u6838\u82f7\u9178\u5408\u6210\uff0c\u73b0\u5728\u4ecd\u88ab\u5927\u591a\u5546\u4e1a\u5316DNA\u5408\u6210\u516c\u53f8\u6240\u91c7\u7528\u3002<\/div>\n
\u8be5\u8fc7\u7a0b\u5c06\u9178\u6d3b\u5316\u7684\u8131\u6c27\u6838\u82f7\u4e9a\u78f7\u9170\u80fa\u5206\u5b50\u8026\u5408\u5230\u4e00\u4e2a\u56fa\u5b9a\u5728\u56fa\u76f8\uff08\u4e00\u822c\u662f\u7845\u6750\u8d28\u8868\u9762\uff09\u7684\u8131\u6c27\u6838\u82f7\u9178\u5206\u5b50\u4e0a\u3002\u5728\u7b2c\u4e00\u4e2a\u5408\u6210\u5faa\u73af\u4e2d\uff0c\u6838\u82f7\u9178\u94fe\u4ece\u7b2c\u4e00\u4e2a\u56fa\u5b9a\u5728\u8868\u9762\u4e0a\u53d7\u4fdd\u62a4\u7684\u6838\u82f7\u9178\u5206\u5b50\u5f00\u59cb\u5ef6\u4f38\u3002\u5176\u4e2d\uff0c\u7ecf\u5e38\u91c7\u7528\u7684\u56fa\u5b9a\u5316\u8868\u9762\u4e3b\u8981\u662f\u53ef\u63a7\u5b54\u5ea6\u73bb\u7483\uff08 CPG\uff09\u6216\u8005\u805a\u82ef\u4e59\u70ef\u5fae\u73e0\uff08PS beads\uff09\u3002\u8bd5\u5242\u88ab\u6cf5\u5165\u5e76\u6d41\u7ecf\u6750\u8d28\u8868\u9762\uff0c\u8bf1\u5bfc\u5206\u6b65\u6dfb\u52a0\u7684\u6838\u82f7\u9178\u5355\u4f53\u52a0\u5165\u5230\u5be1\u6838\u82f7\u9178\u94fe\u4e0a\uff0c\u4f7f\u5be1\u6838\u82f7\u9178\u94fe\u4e0d\u65ad\u5ef6\u957f\u3002\u00a0<\/div>\n
\u00a0<\/div>\n
\u3010\u6bcf\u6dfb\u52a0\u4e00\u4e2a\u6838\u82f7\u9178\u5355\u4f53\u5230\u6838\u82f7\u9178\u94fe\u7684\u56db\u6b65\u8fc7\u7a0b\u3011<\/div>\n
\uff081\uff09\u8131\u4fdd\u62a4\uff1a\u4e00\u79cd\u5f31\u9178\u4ece\u5ef6\u957f\u7684\u6838\u82f7\u9178\u94fe\u76845\u2019\u7aef\u672b\u7aef\u79fb\u9664\u4e8c\u7532\u6c27\u4e09\u82ef\u7532\u57fa\u57fa\u56e2\uff0c\u5e76\u4ea7\u751f\u4e00\u4e2a5\u2019\u7aef\u6d3b\u6027\u7f9f\u57fa\u57fa\u56e2\u3002<\/div>\n
\uff082\uff09\u8026\u5408\uff1a\u8131\u6c27\u6838\u82f7\u4e9a\u78f7\u9170\u80fa\u5206\u5b50\u5728\u9002\u5f53\u7684\u6d3b\u5316\u5242\uff08\u5982\u56db\u5511\uff0ctetrazole\uff09\u4f5c\u7528\u4e0b\uff0c\u4f1a\u4ea7\u751f\u4e00\u4e2a\u6d3b\u6027\u5355\u4f53\u5206\u5b50\uff0c\u4e0e\u4e0a\u4e00\u6b65\u4ea7\u751f\u76845\u2019\u7f9f\u57fa\u57fa\u56e2\u8fdb\u884c\u53cd\u5e94\u3002<\/div>\n
\uff083\uff09\u76d6\u5e3d\uff1a\u672a\u8026\u5408\u76845\u2019\u7f9f\u57fa\u57fa\u56e2\u4f1a\u88ab\u5c01\u95ed\uff0c\u7ecf\u5e38\u91c7\u7528\u7684\u5c01\u95ed\u5242\u662f\u9170\u5316\u5242\u3002<\/div>\n
\uff084\uff09\u6c27\u5316\uff1a\u8fde\u63a5\u6838\u82f7\u9178\u5206\u5b50\u4e4b\u95f4\u7684\u4e9a\u78f7\u916f\u952e\u662f\u4e0d\u7a33\u5b9a\u7684\uff0c\u5bb9\u6613\u88ab\u9178\u3001\u78b1\u6c34\u89e3\uff0c\u6240\u4ee5\u9700\u8981\u88ab\u6c27\u5316\u6210\u4e3a\u66f4\u52a0\u7a33\u5b9a\u7684\u78f7\u9178\u4e09\u916f\u3002<\/div>\n
\u6574\u4e2a\u5408\u6210\u8fc7\u7a0b\u5b8c\u6210\u540e\uff0c\u91c7\u7528\u6d53\u6c28\u6c34\u5c06\u5be1\u6838\u82f7\u9178\u94fe\u4ece\u56fa\u76f8\u4e0a\u5207\u9664\uff0c\u5be1\u6838\u82f7\u9178\u94fe\u4e0a\u7684\u4fdd\u62a4\u57fa\u56e2\u4f1a\u88ab\u540c\u65f6\u79fb\u9664\uff0c\u6b8b\u7559\u6700\u521d\u56fa\u5b9a\u5728\u8868\u9762\u7684\u90a3\u4e00\u4e2a\u6b8b\u57fa\u3002<\/div>\n
\u00a0<\/div>\n
\u76ee\u524d\uff0c\u57fa\u4e8e\u4e9a\u78f7\u9170\u80fa\u5408\u6210\u6cd5\u7684DNA\u5168\u81ea\u52a8\u5408\u6210\u4eea\u5df2\u7ecf\u53ef\u4ee5\u5c06\u5355\u6b21\u5408\u6210\u7684\u901a\u91cf\u4ece1\u6839\u63d0\u9ad8\u52301,536\u6839\u5e8f\u5217(Cheng, Chen, Kao, et al., 2002)\u3002\u4e9a\u78f7\u9170\u80fa\u65b9\u6cd5\u5408\u6210\u5be1\u6838\u82f7\u9178\u7684\u6210\u672c\u4e5f\u5728\u9010\u6e10\u964d\u4f4e\u3002\u67f1\u5f0f\u5408\u6210\u901a\u5e38\u4ee510\u81f31000 nmol\u7684\u89c4\u6a21\u5408\u6210\u5be1\u6838\u82f7\u9178\uff0c\u6bcf\u4e2a\u78b1\u57fa\u7684\u6210\u672c\u57280.05\u81f30.17\u7f8e\u5143\u4e4b\u95f4\u3002\u8fd9\u4e9b\u5be1\u6838\u82f7\u9178\u901a\u5e38\u53ef\u4ee5\u5408\u6210\u591a\u8fbe100nt\uff0c\u9519\u8bef\u7387\u57280.5%\u6216\u4ee5\u4e0b\uff0c\u6bcf\u4e2a\u5355\u4f53\u8026\u5408\u6548\u7387\u901a\u5e38\u53ef\u8fbe99%\u3002<\/div>\n
\u00a0<\/div>\n
\u7136\u800c\uff0c\u968f\u7740\u5be1\u6838\u82f7\u9178\u957f\u5ea6\u7684\u589e\u52a0\uff0c\u5168\u957f\u5be1\u6838\u82f7\u9178\u7684\u4ea7\u91cf\u901a\u5e38\u4f1a\u4e0b\u964d\u3002\u5e76\u4e14\uff0c\u5408\u6210\u5be1\u805a\u7269\u7684\u968f\u673a\u53bb\u560c\u5464\u4f1a\u5f71\u54cd\u5168\u957f\u5be1\u6838\u82f7\u9178\u7684\u4ea7\u91cf\uff0c\u7279\u522b\u662f\u5728\u5408\u6210\u5468\u671f\u7684\u9178\u8131\u4fdd\u62a4\u6b65\u9aa4\u4e2d\uff0c\u817a\u560c\u5464\u5bb9\u6613\u53d7\u5230\u53bb\u560c\u5464\u5f71\u54cd\uff0c\u5e76\u6700\u7ec8\u4fc3\u4f7f\u5be1\u6838\u82f7\u9178\u4e3b\u5e72\u7684\u65ad\u88c2\uff0c\u4ece\u800c\u964d\u4f4e\u5168\u957f\u5be1\u6838\u82f7\u9178\u7684\u4ea7\u91cf\u3002\u5bf9\u4e8e\u8f83\u957f\u7684\u5be1\u6838\u82f7\u9178\u5408\u6210\u6765\u8bf4\uff0c\u8be5\u95ee\u9898\u5c24\u5176\u7a81\u51fa(Efcavitchand Heiner 1985;Septak 1996;LeProust et al.2010)\u3002<\/div>\n
\u00a0<\/div>\n
\u5be1\u6838\u82f7\u9178\u5408\u6210\u8d28\u91cf\u7684\u8fdb\u4e00\u6b65\u964d\u4f4e\u662f\u7531\u4e8e\u5728\u5408\u6210\u8fc7\u7a0b\u4e2d\u5f15\u5165\u968f\u673a\u7a81\u53d8\uff0c\u4e3b\u8981\u662f\u5355\u78b1\u57fa\u7f3a\u5931\u7c7b\u578b\u3002\u8fd9\u7c7b\u9519\u8bef\u4e3b\u8981\u6765\u81eaDMT\u4fdd\u62a4\u57fa\u56e2\u7684\u4e0d\u5b8c\u5168\u53bb\u9664\u6216\u5408\u6210\u5468\u671f\u4e2d\u8026\u5408\u548c\u5c01\u76d6\u6b65\u9aa4\u7684\u7efc\u5408\u6548\u7387\u4f4e\u4e0b\u3002\u5b8c\u5168\u53bb\u9664\u5408\u6210\u9519\u8bef\u662f\u4e0d\u592a\u53ef\u80fd\u7684\uff0c\u56e0\u4e3a\u4efb\u4f55\u5316\u5b66\u53cd\u5e94\u7684\u6548\u7387\u90fd\u65e0\u6cd5\u8fbe\u5230100%\u3002\u4f46\u5408\u6210\u5de5\u827a\u7684\u6539\u8fdb\u4ecd\u7136\u80fd\u63d0\u9ad8\u5be1\u6838\u82f7\u9178\u7684\u5408\u6210\u957f\u5ea6\u548c\u8d28\u91cf(LeProust et al.2010\u5e74)\u3002\u00a0<\/div>\n
\u00a0<\/div>\n
\u5f53\u524d\u4e3b\u6d41\u7684DNA\u5408\u6210\u4f9b\u5e94\u5546\u4e00\u822c\u662f\u57fa\u4e8e\u67f1\u5f0f\u5408\u6210\u7684\u5be1\u6838\u82f7\u9178\u3002\u56e0\u4e3a\u67f1\u5f0f\u5be1\u6838\u82f7\u9178\u6210\u5206\u5355\u4e00\uff0c\u4ea7\u91cf\u9ad8\uff0c\u9519\u8bef\u7387\u4f4e\uff0c\u5bf9\u4e8e\u57fa\u56e0\u5408\u6210\u6765\u8bf4\u7ec4\u88c5\u66f4\u5bb9\u6613\uff0c\u4fdd\u771f\u6027\u4e5f\u66f4\u9ad8\uff0c\u8fd9\u5c31\u6781\u5927\u964d\u4f4e\u4e86\u64cd\u4f5c\u7684\u96be\u5ea6\u548c\u7ea0\u9519\u7684\u6210\u672c\u3002\u4f46\u67f1\u5f0f\u5408\u6210\u5b58\u5728\u663e\u800c\u6613\u89c1\u7684\u7f3a\u9677\uff0c\u6bd4\u5982\u5408\u6210\u901a\u91cf\u4f4e\u3001\u6210\u672c\u9ad8\uff0c\u65e0\u6cd5\u652f\u6301\u5927\u89c4\u6a21\u57fa\u56e0\u7684\u5408\u6210\u3002<\/div>\n
\u00a0<\/div>\n
\u00a0\u56db\u00a0\u00a0\u00a0\u57fa\u56e0\u5408\u6210\u7684\u6280\u672f\u6d41\u7a0b<\/div>\n
\u4eba\u5de5\u5408\u6210DNA\u4e00\u822c\u8d77\u59cb\u4e8e\u9884\u5148\u5408\u6210\u7684\u5be1\u6838\u82f7\u9178\uff08\u4e00\u822c60-120 nt\uff09\u3002\u867d\u7136\u66f4\u957f\u7684\u6838\u82f7\u9178\u94fe\uff08\u957f\u81f3300-600 nt\uff09\u53ef\u4ee5\u76f4\u63a5\u5408\u6210\uff0c\u4f46\u662fDNA\u7684\u5316\u5b66\u5408\u6210\u968f\u7740\u957f\u5ea6\u5ef6\u957f\uff0c\u5408\u6210\u4ea7\u7387\u4f1a\u6025\u5267\u964d\u4f4e\uff0c\u56e0\u6b64\u76f4\u63a5\u5408\u6210\u957f\u94feDNA\u7684\u65b9\u6cd5\u5e76\u6ca1\u80fd\u5f97\u5230\u63a8\u5e7f\u3002<\/div>\n
\u5728\u8fc7\u53bb\u51e0\u5341\u5e74\u91cc\uff0c\u5c06\u5be1\u6838\u82f7\u9178\u7ec4\u88c5\u6210\u4e3a\u57fa\u56e0\u957f\u5ea6DNA\u7684\u65b9\u6cd5\u5728\u4e0d\u65ad\u53d1\u5c55\u3002\u5176\u4e2d\u57fa\u4e8e\u8fde\u63a5\u9176\u7684\u7ec4\u88c5\u548c\u57fa\u4e8e\u805a\u5408\u9176\u94fe\u5f0f\u53cd\u5e94\u7684\u7ec4\u88c5\u662f\u6700\u4e3b\u8981\u7684\u4e24\u79cd\u65b9\u6cd5\uff08\u56fe3\uff09\u3002<\/div>\n\n
\n
\"RmFicvQBAkgDGqJ25wBjOKiaY8dUKgVawQr47FFvaGVDkM6W6fKxqb323rtwic3KqGlsppXfds7cNLEEmkospZeBA\"\/<\/figure><\/div>\n\n
\u56fe3\uff1a\u9176\u4ecb\u5bfc\u7684\u57fa\u56e0\u5408\u6210\u3002\uff08\u6765\u6e90\uff1aMolecular Biosystems\uff09<\/div>\n
1\uff09 \u57fa\u4e8eDNA\u8fde\u63a5\u9176\u7684\u7ec4\u88c5\u65b9\u6cd5<\/div>\n
\u6700\u65e9\u7684\u4f9d\u8d56\u8fde\u63a5\u53cd\u5e94\u7684\u57fa\u56e0\u5408\u6210\u53ef\u4ee5\u8ffd\u6eaf\u52301960s\u540e\u671f\uff0cGupta\u7b49\u4eba\u4f7f\u7528\u5be1\u6838\u82f7\u9178\u5408\u6210\u6280\u672f\u548cDNA\u8fde\u63a5\u9176\u4ecb\u5bfc\u7684\u7ec4\u88c5\u6280\u672f\uff0c\u5408\u6210\u4e86\u4e00\u676130bp\u7684\u9175\u6bcd\u4e19\u6c28\u9178tRNA\u57fa\u56e0\u7247\u6bb5\u3002\u4e8c\u5341\u5e74\u540e\uff0c\u7b2c\u4e00\u6761\u7f16\u7801\u4eba\u80f0\u5c9b\u7d20A\u7684\u57fa\u56e0\uff0863bp\uff09\u88ab\u4eba\u5de5\u5408\u6210\u51fa\u6765\uff0c\u5408\u6210\u65b9\u6cd5\u5c31\u57fa\u4e8eDNA\u8fde\u63a5\u9176\u3002\u516b\u5341\u5e74\u4ee3\u4e00\u79cd\u79f0\u4e3a\u201c\u9e1f\u67aa\u6cd5\u8fde\u63a5\uff08Shotgun Ligation\uff09\u201d\u7684\u65b0\u578b\u7ec4\u88c5\u65b9\u6cd5\u5f00\u53d1\u51fa\u6765\u3002\u8fd9\u79cd\u65b9\u6cd5\u5c06\u76ee\u6807\u57fa\u56e0\u5207\u5272\u6210\u4e3a\u591a\u6761\u7247\u6bb5\uff0c\u76f8\u90bb\u7247\u6bb5\u4e4b\u95f4\u5b58\u5728\u91cd\u53e0\u533a\u57df\uff0c\u800c\u6bcf\u6761\u7247\u6bb5\u7531\u591a\u6761\u78f7\u9178\u5316\u7684\u5be1\u6838\u82f7\u9178\u7ec4\u6210\uff08\u5982\u56fe4\uff09\u3002<\/div>\n\n
\n
\"RmFicvQBAkgDGqJ25wBjOKiaY8dUKgVawQJgwRtQ8iaUHL0Maaicrd5GYNsm8kiaCy1InKkJyok1AG1Rbe1yg35Dc2w\"\/<\/figure><\/div>\n\n
\u56fe4\uff1a\u8fde\u63a5\u9176\u4ecb\u5bfc\u7684\u57fa\u56e0\u5408\u6210\u65b9\u6cd5\u3002\uff08\u6765\u6e90\uff1aSynthetic Biology\uff09<\/div>\n
\u968f\u7740\u8010\u70ed\u6027DNA\u8fde\u63a5\u9176\uff08Thermostable DNA ligase\uff09\u7684\u53d1\u73b0\uff0c\u57fa\u4e8eLCR\u7684\u57fa\u56e0\u7ec4\u88c5\u65b9\u6cd5\u53d8\u5f97\u66f4\u52a0\u65b9\u4fbf\u6613\u884c\u3002\u4f7f\u7528\u8010\u70ed\u8fde\u63a5\u9176\u7684\u4e00\u4e2a\u663e\u8457\u4f18\u52bf\u5728\u4e8e\u8fde\u63a5\u65f6\u7684\u8f83\u9ad8\u6e29\u5ea6\uff08\u7ea650-60\u2103\uff09\u53ef\u4ee5\u51cf\u5f31\u4e8c\u7ea7\u7ed3\u6784\u7684\u5f62\u6210\uff0c\u4ece\u800c\u63d0\u5347\u7ec4\u88c5\u6210\u529f\u7387\uff1b\u5e76\u4e14\uff0c\u5be1\u6838\u82f7\u9178\u5728\u9ad8\u6e29\u6761\u4ef6\u4e0b\u7684\u9000\u706b\u53ca\u8fde\u63a5\u53ef\u4ee5\u63d0\u9ad8\u6b63\u786e\u914d\u5bf9\u7684\u6bd4\u4f8b\uff0c\u63d0\u9ad8\u8fde\u63a5\u7684\u51c6\u786e\u5ea6\u30021990\u5e74Smith\u8bfe\u9898\u7ec4\u4f7f\u7528\u642d\u6865\u5f0f\u8fde\u63a5\uff08\u56fe5A\uff09\uff0c\u6784\u5efa\u4e86\u4e00\u6761924 bp\u7684\u7f16\u7801\u8fa3\u6839\u8fc7\u6c27\u5316\u7269\u9176\u540c\u5de5\u9176\u7684\u57fa\u56e0\u30022003\u5e74Smith\u8bfe\u9898\u7ec4\u5e94\u7528LCR-PCR\u601d\u8def\uff0c\u6210\u529f\u5730\u5728\u4e24\u5468\u5185\u5b8c\u6210\u566c\u83cc\u4f53Phi X174 (5386 bp)\u57fa\u56e0\u7ec4\u7684\u4eba\u5de5\u5408\u6210\u5e76\u9a8c\u8bc1\u5176\u529f\u80fd\uff08\u5982\u56fe6\uff09\u3002<\/div>\n\n
\n
\"RmFicvQBAkgDGqJ25wBjOKiaY8dUKgVawQQRgibjIICwMUdPyTg0ic4xibREa12bnJrrrmKY4fsWCYs2BVlLh45uRJA\"\/<\/figure><\/div>\n\n
\u56fe5\uff1a\u57fa\u4e8eLCR-PCA\u7684DNA\u7ec4\u88c5\u65b9\u6cd5\u3002\uff08\u6765\u6e90\uff1a\u4f5c\u8005\uff09<\/div>\n\n
\n
\"RmFicvQBAkgDGqJ25wBjOKiaY8dUKgVawQPlP0e0ksN6XHvs5HgRV0vMmxBQS6tZu4pRXnCvIBt3dNKH6nGmKlTQ\"\/<\/figure><\/div>\n\n
\u56fe6\uff1aphi X174\u566c\u83cc\u4f53\u57fa\u56e0\u7ec4\uff085386 bp\uff09\u7684\u4ece\u5934\u5408\u6210\u6d41\u7a0b\u793a\u610f\u56fe\u3002\uff08\u6765\u6e90\uff1aProc Natl Acad Sci USA\uff09<\/div>\n
2\uff09 \u57fa\u4e8eDNA\u805a\u5408\u9176\u7684\u7ec4\u88c5\u65b9\u6cd5<\/div>\n
\u805a\u5408\u9176\u94fe\u5f0f\u53cd\u5e94\uff08PCR\uff09\u662f\u4e00\u79cd\u6700\u5e38\u7528\u7684\u57fa\u4e8e\u5be1\u805a\u6838\u82f7\u9178\u7684\u57fa\u56e0\u7ec4\u88c5\u65b9\u6848\u3002\u901a\u8fc7\u4e00\u6b65PCR\u7ec4\u88c5\u7684\u65b9\u6cd5\uff0c\u53ef\u4ee5\u5c06\u4e00\u7ec4\u5be1\u6838\u82f7\u9178\u6df7\u5408\u7269\u4e00\u6b65\u7ec4\u88c5\u6210\u4e3a\u57fa\u56e0\u957f\u5ea6DNA\uff0c\u4f46\u8fd9\u79cd\u65b9\u6cd5\u5f97\u5230\u7684PCR\u4ea7\u7269\u901a\u5e38\u662f\u4e00\u7ec4\u957f\u5ea6\u4e0d\u4e00\u7684DNA\uff08\u5982\u56fe7A\uff09\u3002<\/div>\n
\u00a0<\/div>\n
\u53e6\u5916\u4e00\u79cd\u57fa\u4e8ePCR\u7684\u4e24\u6b65\u6cd5\u7ec4\u88c5\u65b9\u6cd5\uff0c\u9996\u5148\u5c06\u57fa\u56e0\u62c6\u5206\u6210\u4e00\u7cfb\u5217\u4e9a\u5e93\uff08subpools\/ subassembly reactions\uff09\uff0c\u5206\u522b\u7ec4\u88c5\u6210\u4e3a\u7247\u6bb5\uff0c\u518d\u5c06\u8fd9\u4e9b\u7247\u6bb5\u6df7\u5408\uff0c\u6700\u7ec8\u7ec4\u88c5\u6210\u4e3a\u5b8c\u6574\u57fa\u56e0\uff08\u5982\u56fe7B\uff09\u3002<\/div>\n\n
\n
\"RmFicvQBAkgDGqJ25wBjOKiaY8dUKgVawQVlwDug7s7O2ywFSecYIfJo8JicsqrA0PUxItQHhqEsXBicnVc5fkEXUg\"\/<\/figure><\/div>\n\n
\u56fe7\uff1a\u57fa\u4e8ePCR\u7684DNA\u7ec4\u88c5\u65b9\u6cd5\u3002\uff08\u6765\u6e90\uff1a\u4f5c\u8005\uff09<\/div>\n
Stemmer\u7b49\u4eba\u57281995\u5e74\u62a5\u9053\u4e86\u57fa\u4e8ePCR\u539f\u7406\u7684\u3001\u7531\u5be1\u6838\u82f7\u9178\u4e00\u6b65\u6cd5\u7ec4\u88c5\u5168\u957f\u57fa\u56e0\u7684\u65b9\u6cd5(Stemmer, Crameri et al. 1995)\u3002\u968f\u7740\u76ee\u6807\u57fa\u56e0\u7ed3\u6784\u590d\u6742\u6027\u7684\u63d0\u5347\u548c\u957f\u5ea6\u589e\u957f\uff0c\u5be1\u6838\u82f7\u9178\u7684\u5408\u6210\u6570\u91cf\u5728\u4e0d\u65ad\u6500\u5347\u3002\u4e3a\u4e86\u63d0\u9ad8\u7ec4\u88c5\u7684\u6210\u529f\u7387\uff0c\u63d0\u9ad8\u901a\u91cf\u5e76\u964d\u4f4e\u51fa\u9519\u7387\uff0cDNA\u7684\u7ec4\u88c5\u6280\u672f\u4e5f\u9700\u8981\u4e0d\u65ad\u6539\u8fdb\u3002Young\u548cDong\u7b49\u4eba\u7ed3\u5408\u4e86\u4e0d\u5bf9\u79f0PCR\uff08Dual asymmetrical PCR, DA-PCR\uff09(Sandhu, Aleff et al. 1992)\u548c\u91cd\u53e0PCR\uff08Overlap extension PCR, OE-PCR\uff09(Horton, Hunt et al. 1989)\u6280\u672f\uff0c\u5f00\u53d1\u4e86\u4e00\u79cd\u65b0\u7684PCR\u7ec4\u88c5\u65b9\u6cd5\uff08\u56fe8\uff09\u3002<\/div>\n
\u00a0<\/div>\n
\u5728\u8bbe\u8ba1\u65f6\uff0c\u7247\u6bb5\u4e4b\u95f4\u7684\u91cd\u53e0\u533a\u57df\u957f\u5ea6\u5927\u4e8e\u5be1\u6838\u82f7\u9178\u4e4b\u95f4\u7684\u91cd\u53e0\u533a\u57df\u3002\u5f53\u5916\u4fa7\u7684\u5f15\u7269\u6d53\u5ea6\u9ad8\u4e8e\u5be1\u6838\u82f7\u9178\u6d53\u5ea6\u65f6\uff0c\u957f\u7684\u4e9a\u7ec4\u88c5PCR\u4ea7\u7269\uff08DA-PCR\u4ea7\u7269\uff09\u4f1a\u88ab\u4f18\u5148\u9009\u62e9\u6269\u589e\u51fa\u6765\u3002\u6bcf\u7ec4DA-PCR\u4ea7\u7269\u53ef\u4ee5\u901a\u8fc7OE-PCR\u65b9\u6cd5\u5ef6\u4f38\u5f97\u5230\u5168\u957f\u4ea7\u7269\u3002\u4f7f\u7528\u591a\u4e2a\u4e9a\u7ec4\u88c5\uff08\u524d\u7ec4\u88c5\uff09\u53cd\u5e94\u53ef\u4ee5\u964d\u4f4e\u5be1\u6838\u82f7\u9178\u6df7\u5408\u7269\u4e4b\u95f4\u7684\u975e\u7279\u5f02\u6027\u9000\u706b\uff0c\u4ece\u800c\u63d0\u9ad8\u7ec4\u88c5\u4ea7\u7269\u7684\u7eaf\u5ea6\u3002\u00a0<\/div>\n
\u00a0<\/div>\n
Gao\u7b49\u4eba\u57282003\u5e74\u53d1\u660e\u4e86\u70ed\u52a8\u529b\u5b66\u5e73\u8861\u6cd5 (thermodynamically balanced inside-out, TBIO)\uff0c\u5f00\u521b\u4e86\u4e00\u79cd\u65b0\u7684\u4f9d\u8d56PCR\u7684DNA\u62fc\u88c5\u6280\u672f(Gao, Yo et al. 2003)\uff0c\u4e3b\u8981\u539f\u7406\u662f\u8c03\u6574\u5be1\u6838\u82f7\u9178\u7ec4\u88c5\u8fc7\u7a0b\u4e2d\u5355\u94fe\u5be1\u6838\u82f7\u9178\u4e4b\u95f4\u53ca\u5b83\u4eec\u548c\u5f15\u7269\u4e4b\u95f4\u7684\u70ed\u529b\u5b66\u5e73\u8861\u3002\u8be5\u65b9\u6cd5\u7684\u8bbe\u8ba1\u53ca\u5408\u6210\u8fc7\u7a0b\u5305\u62ec\u4e94\u4e2a\u4e3b\u8981\u6b65\u9aa4\uff0c\u5c24\u5176\u91cd\u8981\u7684\u662f\u5f15\u7269\u8bbe\u8ba1\u548c\u7ec4\u88c5\u7b56\u7565\u3002\u5728\u8be5\u7b56\u7565\u4e2d\uff0c\u6b63\u4e49\u94fe\u7684\u5be1\u6838\u82f7\u9178\u8986\u76d6\u4e86\u57fa\u56e0N-\u7aef\u7684\u4e00\u534a\u5e8f\u5217\uff0c\u800c\u53cd\u4e49\u94fe\u4e0a\u7684\u5be1\u6838\u82f7\u9178\u5219\u8986\u76d6\u4e86C-\u7aef\u7684\u4e00\u534a\u5e8f\u5217\u3002\u4e24\u7ec4\u5be1\u6838\u82f7\u9178\u4f1a\u5728\u57fa\u56e0\u6216\u8005\u7247\u6bb5\u7684\u4e2d\u5fc3\u76f8\u9047\uff08\u56fe9\uff09\u3002\u5229\u7528TBIO\u65b9\u6cd5\uff0c4-6\u5bf9\u7ea660 nt\u957f\u7684\u5be1\u6838\u82f7\u9178\u53ef\u4ee5\u7528\u6765\u62fc\u88c5\u4e00\u6bb5\u7ea6400-500 bp\u957f\u7684\u57fa\u56e0\u7247\u6bb5\u3002TBIO\u62fc\u88c5\u8fc7\u7a0b\u53ef\u4ee5\u91cd\u590d\u8fdb\u884c\uff0c\u76f4\u5230\u5f97\u5230\u6240\u9700\u7684\u5168\u957fDNA\u3002Gao\u7b49\u4eba\u4f7f\u7528TBIO-PCR\u62fc\u88c5\u65b9\u6cd5\u5f97\u5230\u768415\u6761\u57fa\u56e0\u5e8f\u5217\u7684\u9519\u8bef\u7387\u662f0%-0.3% (Gao, Yo et al. 2003)\u3002<\/div>\n\n
\n
\"RmFicvQBAkgDGqJ25wBjOKiaY8dUKgVawQCJ8qtUPR8vH5iauBIG1GshPYHna8jtzSdqMjicuDNB7JQglPbFZgZanA\"\/<\/figure><\/div>\n\n
\u56fe8\uff1a\u57fa\u4e8eDA-PCR\u548cOE-PCR\u7684DNA\u7ec4\u88c5\u65b9\u6cd5\u3002\uff08\u6765\u6e90\uff1aNucleic Acids Research\uff09<\/div>\n\n
\n
\"RmFicvQBAkgDGqJ25wBjOKiaY8dUKgVawQ5JDPpWkIBoNLGuHVEGeIa3F91ic2lzPmxXibLZ9orWebtqhNguoTOobg\"\/<\/figure><\/div>\n\n
\u56fe9\uff1aTBIO-PCR\u65b9\u6cd5\u62fc\u88c5DNA\u7684\u539f\u7406\u793a\u610f\u56fe\u3002\uff08\u6765\u6e90\uff1a\u4f5c\u8005\uff09<\/div>\n
Xiong\u7b49\u4eba\u57282004\u5e74\u63cf\u8ff0\u4e86\u53e6\u5916\u4e00\u79cd\u57fa\u4e8ePCR\u7684\u7ec4\u88c5\u6280\u672f\u2014\u2014\u4e24\u6b65\u6cd5DNA\u5408\u6210\u65b9\u6cd5\uff08PCR-based, two-step DNA synthesis, PTDS\uff09\uff0c\u6210\u529f\u5408\u6210\u4e861,230 bp\u957f\u7684Peniophora lycii phytase\u57fa\u56e0(Xiong, Yao et al. 2006)\u548c5,367 bp\u957f\u7684CrtEBWY\u57fa\u56e0(Xiong, Yao et al. 2004)\u3002\u800c\u8be5\u65b9\u6cd5\u7684\u6539\u8fdb\u7248\u672c\uff0c\u5f15\u8fdb\u4e86\u5bf9\u5be1\u6838\u82f7\u9178\u7684\u80f6\u56de\u6536\u6b65\u9aa4\u548c\u57fa\u4e8eOE-PCR\u7684\u9519\u8bef\u7ea0\u6b63\u6b65\u9aa4\uff0c\u53ef\u4ee5\u63d0\u9ad8\u5408\u6210\u4ea7\u7269\u7684\u6574\u4f53\u4fdd\u771f\u6027(Xiong, Yao et al. 2006)\u3002<\/div>\n
\u00a0<\/div>\n
3\uff09 \u7ec4\u88c5\u65b9\u6cd5\u7684\u9009\u62e9<\/div>\n
\u8fde\u63a5\u4ecb\u5bfc\u7684DNA\u62fc\u88c5\u65b9\u6cd5\u548cPCR\u4ecb\u5bfc\u7684\u62fc\u88c5\u65b9\u6cd5\u5404\u6709\u5229\u5f0a\uff0c\u5bf9\u5927\u591a\u6570DNA\u5e8f\u5217\u6765\u8bf4\uff0c\u4e24\u79cd\u65b9\u6cd5\u90fd\u53ef\u4ee5\u5b9e\u73b0\u4ece\u5be1\u6838\u82f7\u9178\u5230\u5168\u957f\u57fa\u56e0\u7684\u62fc\u88c5\u3002\u5728PCR\u4ecb\u5bfc\u7684\u7ec4\u88c5\u65b9\u6cd5\u4e2d\uff0c\u53cc\u94fe\u4e2d\u4efb\u610f\u4e00\u6761\u94fe\u4e0a\u7684\u76f8\u90bb\u5be1\u6838\u82f7\u9178\uff08\u5c5e\u4e8e\u4e24\u6761\u94fe\u6216\u8005\u540c\u4e00\u6761\u94fe\uff09\u4e4b\u95f4\u90fd\u53ef\u80fd\u5b58\u5728\u6838\u82f7\u9178\u7684\u7f3a\u5931\uff0c\u8fd9\u5728\u8fde\u63a5\u4ecb\u5bfc\u7684\u7ec4\u88c5\u8fc7\u7a0b\u4e2d\u5219\u4e0d\u4f1a\u51fa\u73b0\u3002\u8fd9\u4e00\u7279\u70b9\u4f7f\u5f97PCR\u4ecb\u5bfc\u7684\u7ec4\u88c5\u65b9\u6cd5\u5728\u5408\u6210\u5927\u91cfDNA\u65f6\u7a0d\u5177\u6210\u672c\u4f18\u52bf\u3002\u00a0<\/div>\n
\u00a0<\/div>\n
PCR\u4ecb\u5bfc\u7684\u4e00\u6b65\u7ec4\u88c5\u65b9\u6cd5\u7684\u4f18\u52bf\u662f\u5feb\u901f\u3001\u65b9\u4fbf\u3002\u4f46\u662f\uff0c\u5e76\u4e0d\u662f\u6240\u6709\u5e8f\u5217\u90fd\u53ef\u4ee5\u901a\u8fc7PCR\u7684\u65b9\u6cd5\u7ec4\u88c5\u5f97\u5230\u3002\u5728\u5408\u6210\u4e00\u4e9b\u5177\u6709\u7279\u6b8a\u7ed3\u6784\uff0c\u5982\u91cd\u590d\u5e8f\u5217\u6216\u590d\u6742\u4e8c\u7ea7\u7ed3\u6784\u7684DNA\u5e8f\u5217\u65f6\uff0c\u70ed\u7a33\u5b9a\u6027\u8fde\u63a5\u9176\u4ecb\u5bfc\u7684\u57fa\u56e0\u62fc\u88c5\u65b9\u6cd5\u662f\u66f4\u597d\u7684\u9009\u62e9\u3002\u53e6\u5916\uff0c\u76f8\u6bd4\u8fde\u63a5\u53cd\u5e94\uff0c\u5168\u90e8\u7531PCR\u8fc7\u7a0b\u6784\u6210\u7684\u62fc\u88c5\u65b9\u6cd5\u53ef\u80fd\u4f1a\u5f15\u5165\u8f83\u591a\u989d\u5916\u7684\u9519\u8bef\u3002\u5316\u5b66\u5408\u6210\u7684\u5be1\u6838\u82f7\u9178\u7684\u9519\u8bef\u7387\u4e00\u822c\u662f1-3 errors\/kb (Hoover and Lubkowski 2002, Binkowski, Richmond et al. 2005)\uff0c\u57fa\u4e8e\u8be5\u4fdd\u771f\u6c34\u5e73\u7684\u5be1\u6838\u82f7\u9178\u4e3a\u6750\u6599\uff0c\u901a\u8fc7PCR\u65b9\u6cd5\u62fc\u88c5\u5f97\u5230\u7684DNA\u5e8f\u5217\u7684\u9519\u8bef\u7387\u4f1a\u63d0\u9ad8\u4e00\u4e2a\u6570\u91cf\u7ea7(Binkowski, Richmond et al. 2005)\u3002\u56e0\u6b64\uff0c\u6240\u6709PCR\u62fc\u88c5\u7b56\u7565\u90fd\u9700\u8981\u9ad8\u8d28\u91cf\u7684\u5be1\u6838\u82f7\u9178\u3002\u5728\u8bb8\u591a\u4f8b\u5b50\u4e2d\uff0c\u62fc\u88c5\u957f\u94feDNA\u4f7f\u7528\u7684\u5be1\u6838\u82f7\u9178\u90fd\u662f\u5546\u4e1a\u63d0\u4f9b\u5e76\u4e14\u7ecf\u8fc7\u80f6\u7eaf\u5316\u540e\u7684\u4ea7\u7269(Wu, de Kievit et al. 2004, Bi, Zhou et al. 2005)\u3002<\/div>\n
\u00a0<\/div>\n
\u00a0\u4e94\u00a0\u00a0\u00a0\u57fa\u56e0\u7ec4\u5408\u6210\u548c\u57fa\u56e0\u7ec4\u7f16\u8f91\u6280\u672f<\/div>\n
\u968f\u7740\u957f\u7247\u6bb5DNA\u7ec4\u88c5\u6280\u672f\u7684\u4e0d\u65ad\u8fdb\u6b65\uff0c\u51fa\u73b0\u4e86\u6784\u5efa\u5927\u578b\u9176\u590d\u5408\u7269(Kodumal et al. 2004)\u3001\u6574\u4e2a\u4ee3\u8c22\u901a\u8def(Temme et al. 2012)\u751a\u81f3\u6574\u4e2a\u57fa\u56e0\u7ec4\u7684\u65b9\u6cd5(Smith et al. 2003;Gibson et al. 2008a)\u3002\u4f53\u5916\u7ec4\u88c5\u548c\u4f53\u5185\u7ec4\u88c5\u6cd5\u5171\u540c\u63a8\u8fdb\u66f4\u5927\u7684DNA\u7ed3\u6784\u7684\u4eba\u5de5\u5408\u6210\u3002<\/div>\n
\u00a0<\/div>\n
\u4f53\u5916\u7ec4\u88c5\u5305\u62ec\u4f20\u7edf\u9176\u5207\u8fde\u63a5\u3001BioBrickTM\u7ec4\u88c5\u6cd5\uff08\u56fe10\uff09\u3001Gibson\u7ec4\u88c5\uff08\u56fe11\uff09\u7b49\u3002\u5176\u4e2d\uff0cGibson\u7ec4\u88c5\u53ef\u80fd\u662f\u5c06\u591a\u4e2aDNA\u7247\u6bb5\u7ec4\u88c5\u6210\u66f4\u5927\u7684\u7ed3\u6784\u6700\u5e38\u7528\u7684\u7684\u65b9\u6cd5(Gibson et al. 2010)\u3002\u8be5\u65b9\u6cd5\u91c7\u7528\u4e00\u9505\u7b49\u6e29\u6280\u672f\uff0c\u5229\u7528\u542b\u6709\u70ed\u7a33\u5b9aDNA\u805a\u5408\u9176\u3001DNA\u8fde\u63a5\u9176\u548c\u5916\u5207\u9176\u7684\u9176\u6df7\u5408\u7269\uff0c\u53d8\u6027\u3001\u9000\u706b\u548c\u4fee\u590d\u76f8\u90bb\u91cd\u53e0\u7684DNA\u5e8f\u5217\uff0c\u7ec4\u88c5\u6240\u9700\u7684\u7ed3\u6784\u3002\u8be5\u65b9\u6cd5\u88ab\u7528\u6765\u76f4\u63a5\u4ee560 mer\u957f\u5ea6\u5be1\u6838\u82f7\u9178\u7ec4\u88c5\u4e8616.3 kb\u7684\u5c0f\u9f20\u7ebf\u7c92\u4f53\u57fa\u56e0\u7ec4(Gibson et al. 2010)\u3002<\/div>\n
\u00a0<\/div>\n
\u5229\u7528\u917f\u9152\u9175\u6bcd\u7684\u540c\u6e90\u91cd\u7ec4\u80fd\u529b\uff0c\u8fd8\u53ef\u4ee5\u5728\u4f53\u5185\u9ad8\u6548\u7ec4\u88c5\u66f4\u5927\u7684\u5408\u6210DNA\u7247\u6bb5\uff08\u56fe12\uff09\u3002\u4f8b\u5982\uff0c\u9175\u6bcd\u53ef\u4ee5\u5c06\u5916\u6e90DNA\u4ece\u76f8\u4e92\u91cd\u53e0\u7684\u7247\u6bb5\u7ec4\u88c5\u6210\u66f4\u5927\u7684DNA\u7ed3\u6784\uff0cJ. Craig Venter Institute\u7684\u7814\u7a76\u4eba\u5458\u5df2\u7ecf\u6210\u529f\u5730\u4f7f\u7528\u9175\u6bcd\u7ec4\u88c5\u4e86\u591a\u4e2a0.5-1 Mb\u7684\u7ec6\u83cc\u57fa\u56e0\u7ec4(Gibson et al. 2008a)\uff0c\u751a\u81f3\u76f4\u63a5\u4ee5\u76f8\u4e92\u91cd\u53e0\u7684\u5be1\u6838\u82f7\u9178\u8fdb\u884c\u7ec4\u88c5 (Gibson 2009, 2011b, 2012)\u3002<\/div>\n\n
\n
\"640?wx_fmt=png\"\/<\/figure><\/div>\n\n
\u56fe10\uff1aBioBrickTM\u62fc\u88c5\u957f\u94feDNA\u7684\u539f\u7406\u793a\u610f\u56fe\u3002\uff08\u6765\u6e90\uff1aTrends in Biotechnology\uff09<\/div>\n\n
\n
\"RmFicvQBAkgDGqJ25wBjOKiaY8dUKgVawQd6sT8IpHoblm8p5ReeGm0ItrYwo0Omx8lhwgQeeCzLAicYZpy23OIjw\"\/<\/figure><\/div>\n\n
\u56fe11\uff1a\u5916\u5207\u9176\u4ecb\u5bfc\u7684\u957f\u94feDNA\u62fc\u88c5\u65b9\u6cd5\uff08Gibson\u6cd5\uff09\u7684\u539f\u7406\u793a\u610f\u56fe\u3002\uff08\u6765\u6e90\uff1a\u4f5c\u8005\uff09<\/div>\n\n
\n
\"RmFicvQBAkgDGqJ25wBjOKiaY8dUKgVawQOibNNDqhBiany8zl8HCCngHGwjLPOh8S4L4RtmZI76eRcygO4VhBYo7w\"\/<\/figure><\/div>\n\n
\u56fe12\uff1a\u57fa\u4e8e\u9175\u6bcd\u4f53\u5185\u91cd\u7ec4\u539f\u7406\u7684\u4e00\u6b65\u6cd5\u62fc\u63a5\u4ee3\u8c22\u901a\u8def\u3002\uff08\u6765\u6e90\uff1aNucleic Acids Research\uff09<\/div>\n\n
\n
\"RmFicvQBAkgDGqJ25wBjOKiaY8dUKgVawQicIAibsiaPdQRTTulv1CMNrnDWLp8CWBYMhBdGJBdt37Y1bjmxI5oI4uw\"\/<\/figure><\/div>\n\n
\u88681\uff1a\u91cd\u53e0\u533a\u57df\u957f\u5ea6\u5bf9\u9175\u6bcd\u4f53\u5185\u91cd\u7ec4\u6709\u6548\u7387\u7684\u5f71\u54cd\uff08\u6765\u6e90\uff1aNucleic Acids\uff09<\/div>\n
\u57fa\u56e0\u7ec4\u5b8c\u6210\u7ec4\u88c5\u540e\uff0c\u9700\u8981\u4f9d\u6258\u57fa\u56e0\u7ec4\u7f16\u8f91\u6280\u672f\u8fdb\u884c\u9a8c\u8bc1\u5e76\u8fdb\u884c\u90e8\u5206\u6821\u6b63\u3002<\/div>\n\n
\n
\"640?wx_fmt=png\"\/<\/figure><\/div>\n\n
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\u53c2\u8003\u8d44\u6599\uff1a<\/div>\n
1. Bi, L. J., Y. F. Zhou\u00a0et. al.,\u00a0 (2005). “Construction and characterization of different MutS fusion proteins as recognition elements of DNA chip for detection of DNA mutations.” Biosens Bioelectron 21(1): 135-144.\u00a0<\/div>\n
2. Binkowski, B. F., K. E. Richmond\u00a0et. al.,\u00a0(2005). “Correcting errors in synthetic DNA through consensus shuffling.” Nucleic Acids Research 33(6)<\/div>\n
3. Gao, X. X., P. Yo\u00a0et. al.,\u00a0(2003).”Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences.” Nucleic Acids Research 31(22)<\/div>\n
4. Gibson, D. G., J. I. Glass\u00a0et. al.,\u00a0(2010).\u00a0“Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome.” Science 329(5987): 52-56.\u00a0<\/div>\n
5. Hoover, D. M. and J. Lubkowski (2002). “DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis.” Nucleic Acids Research 30(10).\u00a0<\/div>\n
6. Horton, R. M., H. D. Hunt\u00a0et. al.,\u00a0(1989).”Engineering Hybrid Genes without the Use of Restriction Enzymes – Gene-Splicing by Overlap Extension.” Gene 77(1): 61-68.\u00a0<\/div>\n
7. Sandhu, G. S., R. A. Aleff and B. C. Kline (1992). “Dual Asymmetric Pcr – One-Step Construction of Synthetic Genes.” Biotechniques 12(1): 14-16.<\/div>\n
8. Stemmer, W. P. C., A. Crameri\u00a0et. al.,\u00a0 (1995). “Single-Step Assembly of a Gene and Entire Plasmid from Large Numbers of Oligodeoxyribonucleotides.” Gene 164(1): 49-53.\u00a0<\/div>\n
9. Wu, Y., P. de Kievit, L. Vahlkamp et. al.,\u00a0(2004).\u00a0“Quantitative assessment of a novel flow-through porous microarray for the rapid analysis of gene expression profiles.” Nucleic Acids Research 32(15).\u00a0<\/div>\n
10. Xiong, A. S., Q. H. Yao, R. H. Peng et. al.,\u00a0(2004). “A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences.” Nucleic Acids Research 32(12)<\/div>\n
11. Xiong, A. S., Q. H. Yao, R. H. Penget. al.,\u00a0 (2006).\u00a0“High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris.” Applied Microbiology and Biotechnology 72(5): 1039-1047.\u00a0<\/div>\n
12. Hughes, R.A. and A.D. Ellington, Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology. Cold Spring Harb Perspect Biol, 2017. 9(1)\u00a0<\/div>\n
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17. Quan, J., et al., Parallel on-chip gene synthesis and application to optimization of protein expression. Nat Biotechnol, 2011. 29(5): p. 449-52.\u00a0<\/div>\n
18. Kosuri, S. and G.M. Church, Large-scale de novo DNA synthesis: technologies and applications. Nature Methods, 2014. 11: p. 499.\u00a0<\/div>\n
19. Richmond, K.E., Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis. Nucleic Acids Res., 2004. 32: p. 5011-5018.\u00a0<\/div>\n
20. Lubock, N.B., et al., A systematic comparison of error correction enzymes by next-generation sequencing. Nucleic acids research, 2017. 45(15): p. 9206-9217.\u00a0<\/div>\n
21. Fodor, S.P., et al., Light-directed, spatially addressable parallel chemical synthesis. Science, 1991. 251(4995): p. 767-73.\u00a0<\/div>\n
22. Pease, A.C., et al., Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc Natl Acad Sci U S A, 1994. 91(11): p. 5022-6.\u00a0<\/div>\n
23. Egeland, R.D., F. Marken, and E.M. Southern, An electrochemical redox couple activitated by microelectrodes for confined chemical patterning of surfaces. Anal Chem, 2002. 74(7): p. 1590-6.\u00a0<\/div>\n
24. Ghindilis, A.L., et al., CombiMatrix oligonucleotide arrays: genotyping and gene expression assays employing electrochemical detection. Biosens Bioelectron, 2007. 22(9-10): p. 1853-60.\u00a0<\/div>\n
25. Recht, M.I., et al., RNA sequence determinants for aminoglycoside binding to an A-site rRNA model oligonucleotide. J Mol Biol, 1996. 262(4): p. 421-36.\u00a0<\/div>\n
26. Hughes, T.R., et al., Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat Biotechnol, 2001. 19(4): p. 342-7.\u00a0<\/div>\n
27. \u674e\u8bd7\u6e0a\u3001\u8d75\u56fd\u5c4f\u3001\u738b\u91d1.\uff082017\uff09\u5408\u6210\u751f\u7269\u5b66\u6280\u672f\u7684\u7814\u7a76\u8fdb\u5c55\u2014\u2014DNA\u5408\u6210\u3001\u7ec4\u88c5\u4e0e\u57fa\u56e0\u7ec4\u7f16\u8f91\uff0c\u751f\u7269\u5de5\u7a0b\u5b66\u62a5 33 (3):343-360<\/div>\n
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