{"id":2741,"date":"2019-04-10T22:33:56","date_gmt":"2019-04-10T14:33:56","guid":{"rendered":"http:\/\/www.chenlianfu.com\/?p=2741"},"modified":"2019-04-11T01:45:24","modified_gmt":"2019-04-10T17:45:24","slug":"%e4%bd%bf%e7%94%a8ncbi-epcr%e5%92%8cprimer3%e8%bf%9b%e8%a1%8c%e5%bc%95%e7%89%a9%e6%89%b9%e9%87%8f%e5%8c%96%e8%ae%be%e8%ae%a1","status":"publish","type":"post","link":"http:\/\/www.chenlianfu.com\/?p=2741","title":{"rendered":"\u4f7f\u7528NCBI-ePCR\u548cPrimer3\u8fdb\u884c\u5f15\u7269\u6279\u91cf\u5316\u8bbe\u8ba1"},"content":{"rendered":"\n<p>NCBI\u5df2\u7ecf\u4e0d\u518d\u7ef4\u62a4\u5e76\u4e0b\u67b6\u4e86ePCR\u8f6f\u4ef6\uff0c\u8f6c\u800c\u63a8\u8350\u4f7f\u7528\u5176Primer-BLAST\u7f51\u9875\u5de5\u5177\u3002\u8fd9\u5bf9\u4e8e\u5355\u4e2a\u5f15\u7269\u8bbe\u8ba1\u4efb\u52a1\u6bd4\u8f83\u65b9\u4fbf\uff0c\u4f46\u4e0d\u5229\u4e8e\u57fa\u56e0\u7ec4\u8f83\u5927\u7684\u975e\u6a21\u5f0f\u7269\u79cd\u7684\u7279\u5f02\u6027\u5f15\u7269\u8bbe\u8ba1\u6216\u6279\u91cf\u5316\u7684\u5f15\u7269\u8bbe\u8ba1\u3002<\/p>\n\n\n\n<p>\u672c\u6559\u7a0b\u8bb2\u89e3\u4f7f\u7528NCBI-ePCR\u548cPrimer3\u8fdb\u884c\u5f15\u7269\u6279\u91cf\u5316\u8bbe\u8ba1\u3002<\/p>\n\n\n\n<h2>1.\u4e0b\u8f7d\u5e76\u5b89\u88c5NCBI-ePCR\u548cPrimer3\u8f6f\u4ef6<\/h2>\n\n\n\n<pre class=\"wp-block-code\"><code>$ wget http:\/\/ftp.debian.org\/debian\/pool\/main\/e\/epcr\/epcr_2.3.12-1.orig.tar.gz -P ~\/software\n$ tar zxf ~\/software\/epcr_2.3.12-1.orig.tar.gz -C \/opt\/biosoft\/\n$ make -j 4\n$ echo 'PATH=$PATH:\/opt\/biosoft\/e-PCR-2.3.12\/' >> ~\/.bashrc\n$ source ~\/.bashrc<\/code><\/pre>\n\n\n\n<pre class=\"wp-block-code\"><code>$ wget https:\/\/sourceforge.net\/projects\/primer3\/files\/primer3\/2.4.0\/primer3-2.4.0.tar.gz -P ~\/software\/\n$ tar zxf ~\/software\/primer3-2.4.0.tar.gz -C \/opt\/biosoft\/\n$ cd \/opt\/biosoft\/primer3-2.4.0\/src\/\n$ make all\n$ echo 'PATH=$PATH:\/opt\/biosoft\/primer3-2.4.0\/src\/' >> ~\/.bashrc\n$ source ~\/.bashrc<\/code><\/pre>\n\n\n\n<h2>2. \u4f7f\u7528ePCR\u8fdb\u884c\u5f15\u7269\u9a8c\u8bc1<\/h2>\n\n\n\n<p>\u9996\u5148\uff0c\u4f7f\u7528famap\u547d\u4ee4\u548cfahash\u547d\u4ee4\u5206\u4e24\u6b65\u5c06\u57fa\u56e0\u7ec4\u5e8f\u5217\u8f6c\u6362\u4e3a\u54c8\u5e0c\u6570\u636e\u5e93\u3002<\/p>\n\n\n\n<pre class=\"wp-block-preformatted\">$ famap -t N -b genome.famap genome.fasta<br>     \u7a0b\u5e8f\u5c06FASTA\u683c\u5f0f\u7684\u5e8f\u5217\u8f6c\u6362\u4e3afamap\u6570\u636e\u5e93\u6587\u4ef6\u3002<br><br><strong>\u5e38\u7528\u53c2\u6570\uff1a<br><\/strong>-t &lt;string&gt;<br><string>    \u8bbe\u7f6e\u78b1\u57fa\u7c7b\u578b\u3002\u53ef\u4ee5\u8bbe\u7f6e4\u79cd\u503c\uff1an\uff0c\u5141\u8bb8\u542b\u6709\u5c0f\u5199\u78b1\u57faatcgn\uff0c\u5176\u5b83\u5b57\u7b26\u8f6c\u6362\u4e3an\u6216N\uff1bnx\uff0c\u5141\u8bb8\u542b\u6709\u5c0f\u5199\u78b1\u57fa\u548c\u517c\u5e76\u78b1\u57fa\u5b57\u7b26\uff0c\u5176\u5b83\u5b57\u7b26\u8f6c\u6362\u4e3an\u6216N\uff1bN\uff0c\u4ec5\u5141\u8bb8\u5927\u5199\u78b1\u57fa\uff0catcgn\u81ea\u52a8\u8f6c\u6362\u4e3a\u5927\u5199\uff0c\u5176\u5b83\u5b57\u7b26\u8f6c\u6362\u4e3aN\uff1bNX\uff0c\u5141\u8bb8\u5927\u5199\u78b1\u57fa\u548c\u517c\u5e76\u78b1\u57fa\u5b57\u7b26\uff0c\u5176\u5b83\u5b57\u7b26\u8f6c\u6362\u4e3aN\u3002 -b &lt;string&gt;<\/string><br><string><string>    \u8bbe\u7f6e\u8f93\u51fa\u7684famap\u6570\u636e\u5e93\u6587\u4ef6\u8def\u5f84\u3002<\/string><\/string><br><br><string><string>$ fahash -b genome.hash -w 12 -f 3 genome.famap<\/string><\/string><br><string><string>\u7a0b\u5e8f\u8fdb\u4e00\u6b65\u5c06famap\u6587\u4ef6\u8f6c\u6362\u4e3ahash\u6570\u636e\u5e93\u6587\u4ef6\u3002<\/string><\/string><br><br><string><string><strong>\u5e38\u7528\u53c2\u6570\uff1a<\/strong><\/string><\/string><strong><br><\/strong><string><string>-b &lt;string&gt;<\/string><\/string><br><string><string><stirng>    \u8f93\u51fahash\u6570\u636e\u5e93\u6587\u4ef6\u3002<\/stirng><\/string><\/string><br><string><string><stirng>-w &lt;int&gt;<\/stirng><\/string><\/string><br><string><string><stirng><int>    \u8bbe\u7f6ewordsize\u957f\u5ea6\u3002<\/int><\/stirng><\/string><\/string><br><string><string><stirng><int>-f &lt;int&gt;<\/int><\/stirng><\/string><\/string><br><string><string><stirng><int><int>    \u8bbe\u7f6ewordcnt\u957f\u5ea6\u3002<\/int><\/int><\/stirng><\/string><\/string><\/pre>\n\n\n\n<p>\u7136\u540e\uff0c\u4f7f\u7528re-PCR\u5c06\u5f15\u7269\u548c\u6570\u636e\u5e93\u8fdb\u884c\u6bd4\u5bf9\uff0c\u5bfb\u627e\u5f15\u7269\u6bd4\u5bf9\u7ed3\u679c\u3002<\/p>\n\n\n\n<pre class=\"wp-block-preformatted\">\u5206\u522b\u5bf9\u591a\u4e2a\u5f15\u7269\u8fdb\u884c\u6bd4\u5bf9\uff0c\u5f97\u5230\u5404\u4e2a\u5f15\u7269\u7684\u5339\u914d\u7ed3\u679c\uff1a<br>$ re-PCR -p genome.hash -n 1 -g 1 ACTATTGATGATGA AGGTAGATGTTTTT \u2026<br><br>\u8f93\u5165\u4e00\u5bf9\u5f15\u7269\uff0c\u5e76\u8bbe\u7f6e\u4ea7\u7269\u957f\u5ea6\u671f\u671b\u503c\uff0c\u5f97\u5230\u4e00\u5bf9\u5f15\u7269\u7684\u5339\u914d\u7ed3\u679c\uff1a<br>$ re-PCR -s genome.hash -n 1 -g 1 ACTATTGATGATGA AGGTAGATGTTTTT 50-1000<br>\u5e38\u7528\u53c2\u6570\uff1a<br>-p <br>     \u8f93\u5165hash\u6570\u636e\u5e93\uff0c\u5728\u547d\u4ee4\u884c\u4e2d\u76f4\u63a5\u8f93\u5165\u5f15\u7269\u5e8f\u5217\uff0c\u5c06\u5f15\u7269\u548c\u6570\u636e\u5e93\u8fdb\u884c\u6bd4\u5bf9\u3002<br>-s <br>     \u8f93\u5165hash\u6570\u636e\u5e93\uff0c\u5728\u547d\u4ee4\u884c\u4e2d\u5fc5\u987b\u8f93\u5165\u4e00\u5bf9\u5f15\u7269\u548c\u4ea7\u7269\u957f\u5ea6\u671f\u671b\u8303\u56f4\uff0c\u5f97\u5230\u4e00\u5bf9\u5f15\u7269\u7684\u5339\u914d\u7ed3\u679c\u3002<br>-n <br>     \u8bbe\u7f6e\u5141\u8bb8\u7684\u9519\u914d\u78b1\u57fa\u6570\u3002<br>-g <br>     \u8bbe\u7f6e\u5141\u8bb8\u7684gap\u6570\u3002<\/pre>\n\n\n\n<h2>3. Primer3\u8f6f\u4ef6\u7684\u4f7f\u7528<\/h2>\n\n\n\n<p><a rel=\"noreferrer noopener\" aria-label=\"Primer3\uff08\u5728\u65b0\u7a97\u53e3\u6253\u5f00\uff09\" href=\"http:\/\/primer3.ut.ee\/\" target=\"_blank\">Primer3<\/a>\u662f\u547d\u4ee4\u884c\u5f62\u5f0f\u7684\u5f15\u7269\u8bbe\u8ba1\u8f6f\u4ef6\uff0c\u80fd\u5f88\u597d\u5730\u7528\u4e8e\u5f15\u7269\u7684\u6279\u91cf\u8bbe\u8ba1\u3002 \u5176\u4e3b\u7a0b\u5e8f\u662fprimer3_core\u3002 \u8be5\u547d\u4ee4\u7684\u8f93\u5165\u662fBoulder-IO\u683c\u5f0f\uff0c\u9002\u5408\u4e8e\u8f6f\u4ef6\u8bfb\u5165\u6570\u636e\uff1b\u8f93\u51fa\u6587\u4ef6\u9ed8\u8ba4\u4e0b\u662f\u9002\u5408\u4eba\u7c7b\u9605\u8bfb\u7684\u683c\u5f0f\uff0c\u4e5f\u53ef\u4ee5\u662fBoulder-IO\u683c\u5f0f\uff0c\u6709\u52a9\u4e8e\u4e0b\u4e00\u6b65\u5f15\u7269\u7ed3\u679c\u7684\u6279\u91cf\u64cd\u4f5c\u3002 Boulder-IO\u683c\u5f0f\u4ee5\u6587\u672c\u5f62\u5f0f\u8bb0\u5f55\u7740\u5f15\u7269\u8bbe\u8ba1\u4fe1\u606f\uff0c\u4e14\u6bcf\u4e2a\u5f15\u7269\u4fe1\u606f\u7684\u7ed3\u5c3e\u4f7f\u7528\u201c\u7b49\u4e8e\u6362\u884c\u7b26\u201d\u5206\u9694\u3002\u6bcf\u4e2a\u8bb0\u5f55\u7531\u591a\u4e2a\u6807\u7b7e\u548c\u5bf9\u5e94\u7684\u503c\u6784\u6210\uff0c\u7528\u4e8e\u6307\u5b9a\u8f93\u5165\u4fe1\u606f\u6216\u8f93\u51fa\u7ed3\u679c\u3002\u4f8b\u5982\uff1a<\/p>\n\n\n\n<pre class=\"wp-block-preformatted\">SEQUENCE_ID=example<br>SEQUENCE_TEMPLATE=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG<br>SEQUENCE_TARGET=37,21<br>PRIMER_TASK=generic<br>PRIMER_PICK_LEFT_PRIMER=1<br>PRIMER_PICK_INTERNAL_OLIGO=1<br>PRIMER_PICK_RIGHT_PRIMER=1<br>PRIMER_OPT_SIZE=18<br>PRIMER_MIN_SIZE=15<br>PRIMER_MAX_SIZE=21<br>PRIMER_MAX_NS_ACCEPTED=1<br>PRIMER_PRODUCT_SIZE_RANGE=75-100<br>P3_FILE_FLAG=1<br>SEQUENCE_INTERNAL_EXCLUDED_REGION=37,21<br>PRIMER_EXPLAIN_FLAG=1<\/pre>\n\n\n\n<p>Primer3\u4f7f\u7528\u793a\u4f8b\u548c\u53c2\u6570\uff1a<\/p>\n\n\n\n<pre class=\"wp-block-preformatted\">$ primer3_core -p3_settings_file p3_settings_file -strict_tags -format_output input_file result.p3.out<br>     \u7a0b\u5e8f\u7684\u8f93\u5165\u6587\u4ef6\u8981\u6c42\u662fBoulder-IO\u683c\u5f0f\uff0c\u5982\u679c\u6ca1\u6709\u8f93\u5165\u6587\u4ef6\uff0c\u5219\u4f1a\u4ece\u6807\u51c6\u8f93\u5165\u8bfb\u53d6\u6570\u636e\u3002 <br><br><strong>\u5e38\u7528\u53c2\u6570\uff1a<\/strong><br>-p3_settings_file<br>    \u7528\u4e8e\u8f93\u5165primer3\u7684\u914d\u7f6e\u6587\u4ef6\u3002\u8fd9\u662f\u56e0\u4e3aprimer3\u53ef\u8bbe\u7f6e\u7684\u53c2\u6570\u592a\u591a\u4e86\uff0c\u5c06\u5927\u90e8\u5206\u53c2\u6570\u653e\u5165\u5230\u8be5\u914d\u7f6e\u6587\u4ef6\uff0c\u6709\u5229\u4e8e\u53c2\u6570\u7684\u8f93\u5165\u3002primer3\u7684\u9ed8\u8ba4\u53c2\u6570\u4e0d\u597d\uff0c\u7279\u522b\u662f\u9ed8\u8ba4\u53c2\u6570\u4e0b\u6ca1\u6709\u5f00\u542f\u70ed\u529b\u5b66\u8ba1\u7b97\u3002\u63a8\u8350\u4f7f\u7528\u914d\u7f6e\u6587\u4ef6\u6765\u6839\u636e\u81ea\u5df1\u7684\u9700\u6c42\u6765\u8bbe\u5b9a\u76f8\u5173\u53c2\u6570\u3002\u6b64\u5916\uff0c\u8f93\u5165\u6587\u4ef6\u4e2d\u7684\u53c2\u6570\u8bbe\u7f6e\u80fd\u53d6\u4ee3\u6b64\u6587\u4ef6\u4e2d\u7684\u8bbe\u5b9a\u503c\u3002<br>    \u6b64\u6587\u4ef6\u683c\u5f0f\uff1a\u7b2c1\u884c\u56fa\u5b9a\u4e3a\"Primer3 File - http:\/\/primer3.sourceforge.net\"\uff1b\u7b2c2\u884c\u56fa\u5b9a\u4e3a\"P3_FILE_TYPE=settings\"\uff1b\u7b2c3\u884c\u662f\u4e2a\u7a7a\u884c\uff1b\u4ece\u7b2c4\u884c\u5f00\u59cb\u5219\u662f\u6807\u51c6\u7684Boulder-IO\u683c\u5f0f\u5185\u5bb9\u3002<br>-format_output<br>    \u8ba9primer3_core\u4ea7\u751f\u4eba\u7c7b\u6613\u8bfb\u7684\u7ed3\u679c\uff0c\u5426\u5219\u4ea7\u751f\u673a\u5668\u6613\u8bfb\u7684\u7ed3\u679c\uff08Boulder-IO\u683c\u5f0f\u7ed3\u679c\uff09\u3002<br>-strict_tags<br>    \u8981\u6c42\u8f93\u5165\u6587\u4ef6\u4e2d\u7684\u6807\u7b7e\u8981\u5168\u90e8\u6b63\u786e\u3002\u8bbe\u7f6e\u8be5\u53c2\u6570\u540e\uff0c\u5982\u679c\u6709\u6807\u7b7e\u4e0d\u80fd\u88ab\u7a0b\u5e8f\u8bc6\u522b\uff0c\u5219\u62a5\u9519\u5e76\u505c\u6b62\u6267\u884c\u3002\u4e0d\u8bbe\u7f6e\u6b64\u53c2\u6570\uff0c\u5219\u8f6f\u4ef6\u5ffd\u7565\u4e0d\u8bc6\u522b\u7684\u53c2\u6570\u3002<br>-p3_settings_file=file_path<br>    \u6307\u5b9a primer_core \u7684\u914d\u7f6e\u6587\u4ef6\uff0c\u8be5\u914d\u7f6e\u6587\u4ef6\u7684\u8bbe\u5b9a\u4f1a\u53d6\u4ee3\u9ed8\u8ba4\u8bbe\u7f6e\u3002\u5f53\u7136\uff0c<br>-echo_settings_file<br>    \u6253\u5370\u51fap3_settings_file\u4e2d\u7684\u8bbe\u7f6e\u4fe1\u606f\u3002\u5982\u679c\u6ca1\u6709\u6307\u5b9a\u8bbe\u7f6e\u6587\u4ef6\uff0c\u6216\u542b\u6709-format_output\uff0c\u5219\u8be5\u53c2\u6570\u5931\u6548\u3002<br>-output=file_path<br>    \u6307\u5b9a\u8f93\u51fa\u6587\u4ef6\u8def\u5f84\uff0c\u5982\u679c\u4e0d\u6307\u5b9a\uff0c\u5219\u8f93\u51fa\u5230\u6807\u51c6\u8f93\u51fa\u3002<br>-error=file_path<br>   \u6307\u5b9a\u9519\u8bef\u4fe1\u606f\u8f93\u51fa\u8def\u5f84\uff0c\u5982\u679c\u4e0d\u6307\u5b9a\uff0c\u5219\u8f93\u51fa\u5230stderr\u4e2d\u3002<\/pre>\n\n\n\n<p>Primer3\u4f7f\u7528\u7684\u96be\u70b9\u662f\u6839\u636e\u81ea\u8eab\u9700\u6c42\u51c6\u5907p3_settings_file\u6587\u4ef6\u3002\u6211\u7684\u4e00\u4e2a\u793a\u4f8b\u5982\u4e0b\uff08\u4f7f\u7528\u65f6\u9700\u8981\u53bb\u6389#\u6ce8\u91ca\u90e8\u5206\u548c\u7a7a\u884c\uff0c\u4e14\u5fc5\u987b\u7b2c\u4e09\u884c\u7559\u7a7a\u884c\uff09\uff1a<\/p>\n\n\n\n<pre class=\"wp-block-code\"><code>Primer3 File - http:\/\/primer3.sourceforge.net\nP3_FILE_TYPE=settings\n\nP3_FILE_ID=P3 Settings from Lianfu Chen\nPRIMER_FIRST_BASE_INDEX=1\nPRIMER_TASK=generic\nPRIMER_NUM_RETURN=5\nPRIMER_PICK_LEFT_PRIMER=1\nPRIMER_PICK_INTERNAL_OLIGO=0\nPRIMER_PICK_RIGHT_PRIMER=1\nPRIMER_PICK_ANYWAY=1\nPRIMER_THERMODYNAMIC_PARAMETERS_PATH=\/opt\/biosoft\/primer3-2.4.0\/src\/primer3_config\/\n\n### \u5f15\u7269 TM \u503c\u8bbe\u5b9a: ###\nPRIMER_TM_FORMULA=1                 # TM \u7684\u8ba1\u7b97\u65b9\u6cd5\uff0c 1 \u8868\u793a\u4f7f\u7528 the SantaLucia parameters (Proc Natl Acad Sci 95:1460-65)\nPRIMER_MIN_TM=55.0  \nPRIMER_OPT_TM=60.0\nPRIMER_MAX_TM=65.0\nPRIMER_PAIR_MAX_DIFF_TM=5.0         # \u4e24\u4e2a\u5f15\u7269\u4e4b\u95f4\u7684 TM \u503c\u6700\u591a\u76f8\u5dee 5 \u6444\u6c0f\u5ea6\nPRIMER_WT_TM_LT=0\nPRIMER_WT_TM_GT=0\nPRIMER_PAIR_WT_DIFF_TM=0.0\n\n### \u5f15\u7269\u957f\u5ea6\u8bbe\u5b9a: ###\nPRIMER_MIN_SIZE=18\nPRIMER_OPT_SIZE=20\nPRIMER_MAX_SIZE=22\nPRIMER_WT_SIZE_LT=0\nPRIMER_WT_SIZE_GT=0\n\n### \u5f15\u7269 GC \u542b\u91cf\u8bbe\u5b9a\uff1a###\nPRIMER_MIN_GC=30.0\nPRIMER_MAX_GC=70.0\nPRIMER_WT_GC_PERCENT_LT=0.0\nPRIMER_WT_GC_PERCENT_GT=0.0\n\n### \u5f15\u7269\u7684\u70ed\u529b\u5b66\u8ba1\u7b97: ###\n# \u5f00\u542f\u70ed\u529b\u5b66\u8ba1\u7b97\uff0c\u5f00\u542f\u540e\uff0c\u6839\u636e\u70ed\u529b\u5b66\u7684\u503c TH \u503c\u6765\u8ba1\u7b97\u7f5a\u5206\u3002 TH \u7684\u7f5a\u5206\u65b9\u6cd5\u4e3a\uff1a\u7f5a\u5206\u7cfb\u6570 * (1 \/ (\u5f15\u7269TM - 4 - TH\u503c))\u3002\n# \u8be5\u65b9\u6cd5\u597d\u5904\u662f\uff0cTH \u503c\u8d8a\u5927\uff0c\u7f5a\u5206\u529b\u5ea6\u8d8a\u91cd\u3002\nPRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1\n\n# \u5f15\u7269\u81ea\u8eab\u8fdb\u884c\u53cd\u5411\u4e92\u8865\uff0c\u7f5a\u5206\u7cfb\u6570\u8ba1\u7b97\u4e3a 9 \/ ( 1\/(60-4-45) - 1\/(60-4-0) ) = 123.2\n# \u8fd9\u6837\u8ba1\u7b97\u7f5a\u5206\u7cfb\u6570\u7684\u539f\u5219\u662f\uff0c\u82e5 TM \u4e3a\u6700\u4f73 60 \u6444\u6c0f\u5ea6\u7684\u65f6\u5019\uff0c\u6240\u5141\u8bb8\u7684\u6700\u5927\u7f5a\u5206\u503c\u51cf\u53bb\u6700\u5c0f\u7f5a\u5206\u503c\u4e3a 9\uff0c\u548c 3' \u7aef\u78b1\u57fa\u7684\u7a33\u5b9a\u6027\u7684\u7f5a\u5206\u989d\u5ea6\u4e00\u81f4\u3002\nPRIMER_MAX_SELF_ANY=8.00\nPRIMER_WT_SELF_ANY=0.0\nPRIMER_MAX_SELF_ANY_TH=45.00\nPRIMER_WT_SELF_ANY_TH=123.2\n\n# \u5f15\u7269\u81ea\u8eab\u8fdb\u884c 3' \u7aef\u53cd\u5411\u4e92\u8865\u5f62\u6210\u5f15\u7269\u4e8c\u805a\u4f53\uff0c\u7f5a\u5206\u7cfb\u6570\u8ba1\u7b97\u4e3a 9 \/ ( 1\/(60-4-35) - 1\/(60-4-0) ) = 302.4\nPRIMER_MAX_SELF_END=3.00\nPRIMER_WT_SELF_END=0.0\nPRIMER_MAX_SELF_END_TH=35.00\nPRIMER_WT_SELF_END_TH=302.4\n\n# left primer \u548c right primer \u5e8f\u5217\u7684\u53cd\u5411\u4e92\u8865\nPRIMER_PAIR_MAX_COMPL_ANY=8.00\nPRIMER_PAIR_WT_COMPL_ANY=0.0\nPRIMER_PAIR_MAX_COMPL_ANY_TH=45.00\nPRIMER_PAIR_WT_COMPL_ANY_TH=123.2\n\n# left primer \u548c right primer \u8fdb\u884c 3' \u7aef\u53cd\u5411\u4e92\u8865\u5f62\u6210\u5f15\u7269\u4e8c\u805a\u4f53\nPRIMER_PAIR_MAX_COMPL_END=3.00\nPRIMER_PAIR_WT_COMPL_END=0.0\nPRIMER_PAIR_MAX_COMPL_END_TH=35.00\nPRIMER_PAIR_WT_COMPL_END_TH=302.4\n\n# \u53d1\u5939\u7ed3\u6784\uff0c\u7f5a\u5206\u7cfb\u6570\u8ba1\u7b97\u4e3a 9 \/ ( 1\/(60-4-24) - 1\/(60-4-0) ) = 672\nPRIMER_MAX_HAIRPIN_TH=24.00\nPRIMER_WT_HAIRPIN_TH=672\n\n# 3' \u7aef\u78b1\u57fa\u7684\u7a33\u5b9a\u6027\nPRIMER_MAX_END_STABILITY=9.0\nPRIMER_WT_END_STABILITY=1\n\n### \u78b1\u57fa\u5e8f\u5217\u8bbe\u5b9a\uff1a ###\nPRIMER_LOWERCASE_MASKING=0              # \u6a21\u677f\u5e8f\u5217\u4e2d\u5305\u542b\u5c0f\u5199\u5b57\u7b26\u4e0d\u5f71\u54cd\u5f15\u7269\u8bbe\u8ba1\nPRIMER_MAX_POLY_X=4                     # \u5f15\u7269\u5e8f\u5217\u4e2d\u4e0d\u80fd\u5305\u542b\u5355\u6838\u82f7\u9178\u8fde\u7eed\u957f\u5ea6\u8d85\u8fc7 4 bp\nPRIMER_MAX_NS_ACCEPTED=0                # \u5f15\u7269\u4e2d\u5141\u8bb8\u7684 N \u7684\u6570\u76ee\nPRIMER_WT_NUM_NS=0.0                    # \u6bcf\u4e2a N \u7684\u7f5a\u5206\nPRIMER_MAX_END_GC=5                     # \u5f15\u7269\u4e2d 3' \u7aef 5bp \u78b1\u57fa\u4e2d\u5141\u8bb8\u7684\u6700\u5927 Gs \u6216 Cs \u7684\u6570\u76ee\nPRIMER_GC_CLAMP=0                       # \u5f15\u7269\u4e2d 3' \u7aef\u78b1\u57fa\u4e2d\u4e0d\u80fd\u51fa\u73b0\u8fde\u7eed\u7684 Gs \u548c Cs \u5e8f\u5217\nPRIMER_LIBERAL_BASE=1                   # \u662f\u5426\u5141\u8bb8\u6709\u8bf8\u5982 N A R Y \u7b49\u7c7b\u578b\u7684\u78b1\u57fa\u3002\u5fc5\u987b\u5728\u8bbe\u5b9aPRIMER_MAX_NS_ACCEPTED \u4e0d\u4e3a 0 \u540e\u65b9\u6709\u6548\u3002\nPRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=0  # \u5982\u679c\u8bbe\u7f6e\u4e3a 1\uff0c\u5219 C \u80fd\u4e0e S \u5b8c\u7f8e\u5339\u914d\uff0c\u4efb\u610f\u78b1\u57fa\u80fd\u548c N \u5b8c\u7f8e\u5339\u914d\u3002\n\n### \u78b1\u57fa\u8d28\u91cf\u8bbe\u5b9a\uff1a ###\nPRIMER_MIN_QUALITY=0                    # primer \u5e8f\u5217\u5141\u8bb8\u6700\u5c0f\u7684\u78b1\u57fa\u8d28\u91cf\nPRIMER_MIN_END_QUALITY=0\nPRIMER_QUALITY_RANGE_MIN=0\nPRIMER_QUALITY_RANGE_MAX=100\nPRIMER_WT_SEQ_QUAL=0.0\nPRIMER_WT_END_QUAL=0.0\n\n## \u5f15\u7269\u4f4d\u7f6e\u8bbe\u7f6e\uff1a ###\nPRIMER_SEQUENCING_LEAD=50               # \u8be5\u53c2\u6570\u4ec5\u5728 PRIMER_TASK=pick_sequencing_primers \u65f6\u6709\u6548. \u8868\u660e\u5f15\u7269\u7684 3' \u7aef\u8ddd\u76ee\u6807\u533a\u57df\u6709 50bp\u3002\nPRIMER_SEQUENCING_SPACING=500           # \u8be5\u53c2\u6570\u4ec5\u5728 PRIMER_TASK=pick_sequencing_primers \u65f6\u6709\u6548. \u8be5\u503c\u51b3\u5b9a\u4e86\u5728\u540c\u4e00\u6761\u94fe\u4e0a\u7684\u4e24\u4e2a\u5f15\u7269\u7684\u8ddd\u79bb.\nPRIMER_SEQUENCING_INTERVAL=250          # \u8be5\u53c2\u6570\u4ec5\u5728 PRIMER_TASK=pick_sequencing_primers \u65f6\u6709\u6548. \u8be5\u503c\u51b3\u5b9a\u4e86\u5728\u4e0d\u540c\u94fe\u4e0a\u7684\u4e24\u4e2a\u5f15\u7269\u7684\u8ddd\u79bb\u3002\nPRIMER_SEQUENCING_ACCURACY=20           # \u8be5\u53c2\u6570\u4ec5\u5728 PRIMER_TASK=pick_sequencing_primers \u65f6\u6709\u6548. \u8be5\u503c\u51b3\u5b9a\u4e86\u5f15\u7269\u8bbe\u8ba1\u7684\u533a\u95f4.\nPRIMER_OUTSIDE_PENALTY=0\nPRIMER_INSIDE_PENALTY=-1.0\nPRIMER_WT_POS_PENALTY=0.0\n\n### PCR \u53cd\u5e94\u4f53\u7cfb\u8bbe\u5b9a: ###\nPRIMER_SALT_MONOVALENT=50.0                 # \u5355\u4ef7\u76d0\u79bb\u5b50\u6d53\u5ea6(mM)\nPRIMER_SALT_CORRECTIONS=1                   # PRIMER_SALT_CORRECTIONS=1 means use the salt correction in SantaLucia et al 1998\nPRIMER_SALT_DIVALENT=1.5                    # \u4e8c\u4ef7\u9541\u79bb\u5b50\u6d53\u5ea6(mM)\nPRIMER_DNTP_CONC=0.6                        # \u603bdNTPs\u6d53\u5ea6(mM)\nPRIMER_DNA_CONC=50.0                        # DNA\u4ea7\u7269\u6d53\u5ea6(mM)\n\n### PCR \u4ea7\u7269\u7684\u8bbe\u5b9a\uff1a ###\nPRIMER_PRODUCT_MIN_TM=-1000000.0\nPRIMER_PRODUCT_OPT_TM=0.0\nPRIMER_PRODUCT_MAX_TM=1000000.0\nPRIMER_PAIR_WT_PRODUCT_TM_LT=0.0\nPRIMER_PAIR_WT_PRODUCT_TM_GT=0.0\nPRIMER_PRODUCT_OPT_SIZE=0\nPRIMER_PAIR_WT_PRODUCT_SIZE_LT=0.0\nPRIMER_PAIR_WT_PRODUCT_SIZE_GT=0.0\n\n## \u7f5a\u5206\u56e0\u5b50\uff1a ###\nPRIMER_PAIR_WT_PR_PENALTY=1.0                # left primer\u548cright primer\u7684\u7f5a\u5206\u4e4b\u548c\u3002\u6b64\u548c\u4e58\u4ee5\u6b64\u7cfb\u6570\uff0c\u518d\u52a0\u5176\u5b83\u7f5a\u5206\u4f5c\u4e3a\u6700\u7ec8\u7f5a\u5206\u3002\nPRIMER_PAIR_WT_IO_PENALTY=0.0                # \u5c06 internal oligo \u7684\u7f5a\u5206\u4e58\u4ee5\u6b64\u7cfb\u6570\uff0c\u52a0\u5165\u5230\u5f15\u7269\u7684\u6700\u7ec8\u7f5a\u5206\u4e2d\u3002\n\n### internal oligo \u7684\u8bbe\u5b9a\uff1a###\n# TM \u503c\nPRIMER_INTERNAL_MIN_TM=57.0\nPRIMER_INTERNAL_OPT_TM=60.0\nPRIMER_INTERNAL_MAX_TM=63.0\nPRIMER_INTERNAL_WT_TM_LT=1.0\nPRIMER_INTERNAL_WT_TM_GT=1.0\n\n# \u957f\u5ea6\nPRIMER_INTERNAL_MIN_SIZE=18\nPRIMER_INTERNAL_OPT_SIZE=20\nPRIMER_INTERNAL_MAX_SIZE=27\nPRIMER_INTERNAL_WT_SIZE_LT=1.0\nPRIMER_INTERNAL_WT_SIZE_GT=1.0\n\n# GC \u542b\u91cf\nPRIMER_INTERNAL_MIN_GC=20.0\nPRIMER_INTERNAL_MAX_GC=80.0\nPRIMER_INTERNAL_OPT_GC_PERCENT=50.0\nPRIMER_INTERNAL_WT_GC_PERCENT_LT=0.0\nPRIMER_INTERNAL_WT_GC_PERCENT_GT=0.0\n\n# \u70ed\u529b\u5b66\nPRIMER_INTERNAL_MAX_SELF_ANY=12.00\nPRIMER_INTERNAL_WT_SELF_ANY=0.0\nPRIMER_INTERNAL_MAX_SELF_END=12.00\nPRIMER_INTERNAL_WT_SELF_END=0.0\n\n# \u78b1\u57fa\u5e8f\u5217\nPRIMER_INTERNAL_MAX_POLY_X=5\nPRIMER_INTERNAL_MAX_NS_ACCEPTED=0\n\n# \u78b1\u57fa\u8d28\u91cf\nPRIMER_INTERNAL_MIN_QUALITY=0\nPRIMER_INTERNAL_WT_END_QUAL=0.0\nPRIMER_INTERNAL_WT_SEQ_QUAL=0.0\n\n# \u975e\u5f15\u7269\u6570\u636e\u5e93\n#PRIMER_INTERNAL_MAX_LIBRARY_MISHYB=12.00\n#PRIMER_INTERNAL_WT_LIBRARY_MISHYB=0.0\n\n# PCR \u53cd\u5e94\u4f53\u7cfb\nPRIMER_INTERNAL_SALT_MONOVALENT=50.0\nPRIMER_INTERNAL_SALT_DIVALENT=1.5\nPRIMER_INTERNAL_DNA_CONC=50.0\nPRIMER_INTERNAL_DNTP_CONC=0.0\n=<\/code><\/pre>\n\n\n\n<h2>4. \u7f16\u5199\u7a0b\u5e8f\u8c03\u7528primer3\u8fdb\u884c\u5f15\u7269\u8bbe\u8ba1\u518d\u8c03\u7528e-PCR\u8f6f\u4ef6\u8fdb\u884c\u7279\u5f02\u6027\u9a8c\u8bc1<\/h2>\n\n\n\n<p>\u7f16\u5199\u7a0b\u5e8fprimer3_with_ePCR_validation.pl\uff0c\u8f93\u5165\u6a21\u677f\u5e8f\u5217\u7684FASTA\u6587\u4ef6\uff0c\u5373\u53ef\u5bf9\u6240\u6709\u7684\u6a21\u677f\u5e8f\u5217\u6279\u91cf\u5316\u8fdb\u884c\u5f15\u7269\u8bbe\u8ba1\u3002\u82e5\u540c\u65f6\u8f93\u5165\u5168\u57fa\u56e0\u7ec4\u7684\u5e8f\u5217\uff0c\u8fdb\u4e00\u6b65\u53ef\u4ee5\u5bf9\u8bbe\u8ba1\u51fa\u7684\u5f15\u7269\u8fdb\u884c\u7279\u5f02\u6027\u7b5b\u9009\u3002<\/p>\n\n\n\n<pre class=\"wp-block-code\"><code>#!\/usr\/bin\/perl\nuse strict;\nuse Getopt::Long;\n\nmy $usage = &lt;&lt;USAGE;\nUsage:\n    $0 [options] seq.fasta > primer3.out\n\n    \u7a0b\u5e8f\u5bf9seq.fasta\u6587\u4ef6\u4e2d\u7684\u5e8f\u5217\u8c03\u7528primer3\u8fdb\u884c\u5f15\u7269\u6279\u91cf\u5316\u8bbe\u8ba1\u3002\u5e76\u5c06\u7ed3\u679c\u8f93\u51fa\u5230\u6807\u51c6\u8f93\u51fa\u3002\n\n    --min_product_length &lt;INT>  default: 80\n    \u8bbe\u7f6e\u5f15\u7269\u5f97\u5230\u7684\u6700\u5c0f\u4ea7\u7269\u957f\u5ea6\u3002\n\n    --max_product_length &lt;INT>  default: 150\n    \u8bbe\u7f6e\u5f15\u7269\u5f97\u5230\u7684\u6700\u5927\u4ea7\u7269\u957f\u5ea6\u3002\n\n    --primer-num &lt;int>    default: 5\n    \u8bbe\u7f6e\u4f7f\u7528primer3\u8f6f\u4ef6\u8bbe\u8ba1\u7684\u5f15\u7269\u5bf9\u6570\u91cf\u3002\n\n    --p3_setting_file &lt;STRING>\n    \u7a0b\u5e8f\u4f7f\u7528Primer3\u8fdb\u884c\u5f15\u7269\u6279\u91cf\u8bbe\u8ba1\uff0c\u8be5\u53c2\u6570\u8bbe\u7f6e\u6240\u4f7f\u7528\u7684Primer3\u914d\u7f6e\u6587\u4ef6\u8def\u5f84\u3002\u8be5\u53c2\u6570\u662f\u5fc5\u987b\u7684\uff0c\u4ee5\u5229\u4e8eprimer3\u8bbe\u7f6e\u6b63\u786e\u6709\u6548\u7684\u5f15\u7269\u3002\n\n    --CPU &lt;int>    default: 1\n    \u8bbe\u7f6e\u5e76\u884c\u5316\u8fd0\u884cprimer3\u7684\u4efb\u52a1\u6570\u3002\u7a0b\u5e8f\u8c03\u7528ParaFly\u547d\u4ee4\u8fdb\u884c\u5e76\u884c\u5316\u8fd0\u7b97\uff0c\u9700\u8981\u80fd\u76f4\u63a5\u5728terminal\u4e2d\u76f4\u63a5\u8fd0\u884cParaFly\u547d\u4ee4\u3002\n\n    --db &lt;string>\n    \u82e5\u4f7f\u7528\u8be5\u53c2\u6570\u8f93\u5165FASTA\u683c\u5f0f\u7684\u6570\u636e\u5e93\u5e8f\u5217\uff0c\u7a0b\u5e8f\u80fd\u989d\u5916\u5bf9primer3\u5f97\u5230\u7684\u5f15\u7269\u8fdb\u884ce-PCR\u9a8c\u8bc1\u3002\n\n    --max-mismatch &lt;int>    default: 1\n    --max-gap &lt;int>    default: 1\n    \u8bbe\u7f6ee-PCR\u8fc7\u7a0b\u4e2d\u5141\u8bb8\u5f15\u7269\u548c\u6570\u636e\u5e93\u5e8f\u5217\u6700\u5927\u7684\u9519\u914d\u548cgap\u6570\u91cf\u3002\u8bbe\u7f6e\u66f4\u9ad8\u80fd\u5f97\u5230\u66f4\u597d\u7684\u7279\u5f02\u6027\u3002\n\n    --max-hit &lt;int>    default: 1\n    \u8bbe\u7f6e\u5141\u8bb8e-PCR\u5bf9\u6570\u636e\u5e93\u641c\u7d22\u5f97\u5230\u7684\u6700\u5927\u7ed3\u679c\u6570\u91cf\u3002\u82e5\u6a21\u677f\u5e8f\u5217\u5b58\u5728\u6570\u636e\u5e93\u4e2d\uff0c\u63a8\u8350\u8bbe\u7f6e\u4e3a1\uff0c\u82e5\u6a21\u677f\u5e8f\u5217\u4e0d\u5b58\u5728\u6570\u636e\u5e93\u4e2d\uff0c\u63a8\u8350\u8bbe\u7f6e\u4e3a0\u3002\n\n    --no-primer-seq &lt;string>\n    \u82e5\u8bbe\u7f6e\u8be5\u53c2\u6570\u7684\u503c\uff0c\u5219\u751f\u6210\u76f8\u5e94\u7684\u6587\u4ef6\uff0c\u8f93\u51fa\u6ca1\u6709\u5f15\u7269\u7ed3\u679c\u7684\u5e8f\u5217\u3002\n\n    --tmp-dir &lt;string>    default: primer3_with_ePCR_validation.tmp\n    \u8bbe\u7f6e\u4e34\u65f6\u6587\u4ef6\u5939\uff0c\u7a0b\u5e8f\u7684\u4e2d\u95f4\u6587\u4ef6\u653e\u5165\u8be5\u6587\u4ef6\u5939\u4e2d\u3002\n\nUSAGE\nif (@ARGV==0){die $usage}\n\n...<\/code><\/pre>\n","protected":false},"excerpt":{"rendered":"<p>NCBI\u5df2\u7ecf\u4e0d\u518d\u7ef4\u62a4\u5e76\u4e0b\u67b6\u4e86ePCR\u8f6f\u4ef6\uff0c\u8f6c\u800c\u63a8\u8350\u4f7f\u7528\u5176Primer-BLAST &hellip; <a href=\"http:\/\/www.chenlianfu.com\/?p=2741\">\u7ee7\u7eed\u9605\u8bfb <span class=\"meta-nav\">&rarr;<\/span><\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":[],"categories":[1],"tags":[],"_links":{"self":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/2741"}],"collection":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=2741"}],"version-history":[{"count":9,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/2741\/revisions"}],"predecessor-version":[{"id":2780,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/2741\/revisions\/2780"}],"wp:attachment":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=2741"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=2741"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=2741"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}