\u53c2\u8003\u81ea\uff1ahttp:\/\/primer3.wi.mit.edu\/primer3web_help.htm\u3002<\/a> Primer3 \u7684\u4e3b\u7a0b\u5e8f\u662f primer3_core .<\/p>\n \u5e38\u7528\u4f8b\u5b50\uff1a<\/p>\n \u5e38\u7528\u53c2\u6570\uff1a<\/p>\n \u9ed8\u8ba4\u8bbe\u7f6e\u4e0b\uff0cprimer3 \u7684\u8f93\u5165\u6587\u4ef6\u4e3a Boulder-IO \u683c\u5f0f\u7684\uff0c\u8be5\u683c\u5f0f\u6709\u5229\u4e8e\u7a0b\u5e8f\u95f4\u7684\u76f8\u4e92\u4ea4\u6d41\u3002\u540c\u65f6\u8f93\u51fa\u6587\u4ef6\u7684\u683c\u5f0f\u4e5f\u4e3a Boulder-IO\u3002\u8be5\u6587\u4ef6\u683c\u5f0f\u662f\u6587\u672c\u5f62\u5f0f\u7684\uff0c\u6bcf\u4e00\u4e2a\u5f15\u7269\u8bbe\u8ba1\u7684\u8bb0\u5f55\u7684\u7ed3\u5c3e\u4ee5\u201c=\\n\u201d\u8fdb\u884c\u5206\u9694\u3002\u6bcf\u4e2a\u8bb0\u5f55\u7531\u591a\u4e2a\u6807\u7b7e\u548c\u5bf9\u5e94\u7684\u503c\u6784\u6210\u3002\u4f8b\u5982\uff1a<\/p>\n primer3\u53d1\u5e032.0\u540e\uff0c\u8bb8\u591a\u7684Boulder-IO\u7684\u6807\u7b7e\u6709\u4e86\u53d8\u52a8\uff0c\u800c\u4e14\u65b0\u589e\u4e86\u8bb8\u591a\u6807\u7b7e\u3002\u6807\u7b7e\u5206\u4e09\u7c7b\uff1a\u5206\u522b\u4ee5SEQUENCE_, PRIMER_ \u548c P3_\u5f00\u5934\u3002\u5173\u4e8e\u6807\u7b7e\u7684\u53d8\u52a8\uff0c\u5177\u4f53\u53c2\u8003\u672c\u6587\u5f00\u5934\u7684\u53c2\u8003\u7f51\u5740\u3002<\/p>\n \u9996\u5148\uff0c\u901a\u8fc7\u4e00\u4e9b\u9650\u5b9a\u9608\u503c\uff0c\u7a0b\u5e8f\u627e\u51fa\u6240\u6709\u7684\u5f15\u7269\u5bf9\u3002\u7136\u540e\u518d\u5bf9\u5176\u8fdb\u884c\u6253\u5206\u3002 1. Primer3 \u7b80\u4ecb \u53c2\u8003\u81ea\uff1ahttp:\/\/primer3.wi.mit. … \u7ee7\u7eed\u9605\u8bfb
\nPrimer3\u7684\u4e3b\u9875\uff1ahttp:\/\/primer3.sourceforge.net\/<\/a>\u3002\u4e3b\u9875\u4e0a\u80fd\u770b\u5230\u5bf9\u4e8ePrimer3\u7684\u7b80\u8981\u4ecb\u7ecd\u548c\u4e0b\u8f7d\u901a\u9053\u3002
\nPrimer3\u7684\u7f51\u9875\u7248\uff1ahttp:\/\/primer3.wi.mit.edu\/<\/a>\u3002
\nPrimer3\u7684\u6e90\u4ee3\u7801\uff1ahttp:\/\/sourceforge.net\/projects\/primer3\/<\/a>.
\nPrimer3\u6587\u732e\uff1aSteve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386<\/a><\/p>\n2. \u5b89\u88c5 Primer3<\/h1>\n
$ wget http:\/\/downloads.sourceforge.net\/project\/primer3\/primer3\/2.3.6\/primer3-src-2.3.6.tar.gz\r\n$ tar zxf primer3-src-2.3.6.tar.gz -C \/opt\/biosoft\r\n$ cd primer3-2.3.6\/src\/\r\n$ make all\r\n$ make test \u6b64\u9879\u7528\u4e8e\u68c0\u6d4b primer3 \u662f\u5426\u6b63\u786e\u5b89\u88c5\uff0c\u786e\u4fdd\u8f93\u51fa\u4e2d\u4e0d\u80fd\u6709 FAILED\u3002\r\n$ echo 'PATH=$PATH:\/opt\/biosoft\/primer3-2.3.6\/src\/' >> ~\/.bashrc\r\n$ source ~\/.bashrc\r\n<\/pre>\n
3. \u4f7f\u7528 Primer3<\/h1>\n
3.1 primer3_core \u7684\u5e38\u7528\u4f8b\u5b50\u548c\u53c2\u6570<\/h2>\n
\r\n$ perl -n -e 's\/\\s*#.*\/\/;print' \/opt\/biosoft\/primer3-2.3.6\/my_default_settings.txt > p3_settings_file \r\n$ primer3_core -p3_settings_file p3_settings_file -strict_tags input_file > result.p3.out\r\n\u5982\u679c\u6ca1\u6709input file\uff0c\u5219 primer3_core \u4ece\u6807\u51c6\u8f93\u5165\u8bfb\u53d6\u6570\u636e\u3002\r\n<\/pre>\n
-about\r\n\u663e\u793aprimer3\u7248\u672c\u53f7\u5e76\u9000\u51fa\r\n\r\n-default_version=n\r\n\u9ed8\u8ba4\u8bbe\u7f6e\u4e3a n=2\uff0c\u8ba9primer3\u4f7f\u7528\u6700\u65b0\u7684\u9ed8\u8ba4\u8bbe\u7f6e\uff1bn=1 \u5219\u8ba9\u7a0b\u5e8f\u4f7f\u7528 2.23 \u6216\u4e4b\u524d\u7248\u672c\u7684\u8bbe\u7f6e\r\n\r\n-format_output\r\n\u8ba9 primer3_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\r\n\r\n-strict_tags\r\n\u8981\u6c42 input file \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\r\n\r\n-p3_settings_file=file_path\r\n\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\uff0cinput file \u7684\u8bbe\u7f6e\u4e5f\u80fd\u53d6\u4ee3\u8fd9\u4e2a\u6587\u4ef6\u7684\u8bbe\u7f6e\u3002\u6b64\u6587\u4ef6\u7684\u683c\u5f0f\u4e3a\uff1a\u7b2c 1 \u884c\u56fa\u5b9a\u4e3a \"Primer3 File - http:\/\/primer3.sourceforge.net\"; \u7b2c 2 \u884c\u56fa\u5b9a\u4e3a \"P3_FILE_TYPE=settings\"; \u7b2c 3 \u884c\u662f\u4e2a\u7a7a\u884c\uff1b\u4ece\u7b2c 4 \u884c\u5f00\u59cb\u5219\u662f\u6807\u51c6\u7684 Boulder-IO \u683c\u5f0f\u5185\u5bb9\u3002\r\n\r\n-echo_settings_file\r\n\u6253\u5370\u51fa p3_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\r\n\r\n-io_version=n \r\n\u6b64\u53c2\u6570\u73b0\u5728\u4ec5\u80fd\u8bbe\u7f6e\u4e3a\u9ed8\u8ba4\u503c 4 \u3002\u8868\u793a\u6b64\u53c2\u6570\u7b97\u662f\u53d6\u6d88\u4e86\u3002\r\n\r\n-output=file_path\r\n\u6307\u5b9a\u8f93\u51fa\u6587\u4ef6\u8def\u5f84\uff0c\u5982\u679c\u4e0d\u6307\u5b9a\uff0c\u5219\u8f93\u51fa\u5230\u6807\u51c6\u8f93\u51fa\u3002\r\n\r\n-error=file_path\r\n\u6307\u5b9a\u9519\u8bef\u4fe1\u606f\u8f93\u51fa\u8def\u5f84\uff0c\u5982\u679c\u4e0d\u6307\u5b9a\uff0c\u5219\u8f93\u51fa\u5230stderr\u4e2d\u3002\r\n<\/pre>\n
3.2 Primer3 \u7684\u8f93\u5165\u548c\u8f93\u51fa<\/h2>\n
SEQUENCE_ID=example\r\nSEQUENCE_TEMPLATE=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG\r\nSEQUENCE_TARGET=37,21\r\nPRIMER_TASK=generic\r\nPRIMER_PICK_LEFT_PRIMER=1\r\nPRIMER_PICK_INTERNAL_OLIGO=1\r\nPRIMER_PICK_RIGHT_PRIMER=1\r\nPRIMER_OPT_SIZE=18\r\nPRIMER_MIN_SIZE=15\r\nPRIMER_MAX_SIZE=21\r\nPRIMER_MAX_NS_ACCEPTED=1\r\nPRIMER_PRODUCT_SIZE_RANGE=75-100\r\nP3_FILE_FLAG=1\r\nSEQUENCE_INTERNAL_EXCLUDED_REGION=37,21\r\nPRIMER_EXPLAIN_FLAG=1\r\n=\r\n<\/pre>\n
3.3 \u5e38\u7528 primer3 \u6807\u7b7e<\/h2>\n
SEQUENCE \u6807\u7b7e \uff08\u4ee5 SEQUENCE_ \u5f00\u5934\uff09\uff1a<\/h3>\n
SEQUENCE_ID (string; default empty)\r\n\u5e8f\u5217\u7684\u540d\u79f0\uff0c\u4e3a\u5e8f\u5217\u7684\u7b80\u8981\u63cf\u8ff0\u3002\r\n\r\nSEQUENCE_TEMPLATE (nucleotide sequence; default empty)\r\n\u8fdb\u884c\u5f15\u7269\u8bbe\u8ba1\u7684\u6a21\u677f\u740f\u5e8f\u5217\uff0c\u65b9\u5411\u5fc5\u987b\u662f\u4ece5'\u52303'\u3002\u78b1\u57fa\u5e8f\u5217\u6700\u597d\u7528\u5927\u5199\u5b57\u6bcd\uff0c\u5e76\u4e14\u5e8f\u5217\u4e2d\u7684\u6240\u6709\u78b1\u57fa\u90fd\u5728\u4e00\u884c\u4e0a\u4e66\u5199\u3002\u5982\u679c\u7528\u5c0f\u5199\u5b57\u6bcd\uff0c\u5219\u80fd\u88ab\u6807\u7b7e PRIMER_LOWERCASE_MASKING \u8fdb\u884c\u5c4f\u853d\u3002\r\n\r\nSEQUENCE_INCLUDED_REGION (interval list; default empty)\r\n\u8be5\u6807\u7b7e\u7684\u503c\u4e3a\uff1a<start>,<length>\u3002\u5373\u5728\u4ece\u5e8f\u5217\u7684\u7b2c<start>\u4f4d\u70b9\u7684\u78b1\u57fa\u5f00\u59cb\uff0c\u5230\u5f80\u540e<length>\u957f\u5ea6\u7684\u5b50\u5e8f\u5217\u4e2d\u8bbe\u8ba1\u5f15\u7269\u3002\r\n\r\nSEQUENCE_TARGET (interval list; default empty)\r\n\u8be5\u6807\u7b7e\u7684\u503c\u4e3a\uff1a<start>,<length>\u3002\u5373\u4ece\u5e8f\u5217\u7684\u7b2c<start>\u4f4d\u70b9\u7684\u78b1\u57fa\u5f00\u59cb\uff0c\u5230\u5f80\u540e<length>\u957f\u5ea6\u7684\u533a\u57df\u662f\u8981\u6269\u589e\u7684\u76ee\u7684\u7247\u6bb5\uff0c\u8bbe\u8ba1\u7684\u5f15\u7269\u5728\u6b64\u533a\u57df\u7684\u4fa7\u7ffc\u624d\u80fd\u53bb\u5f97\u8f83\u597d\u7684\u5206\u503c\u3002\u53ef\u4ee5\u5728\u591a\u884c\u4e0a\u8bbe\u7f6e\u591a\u6b21\u8be5\u6807\u7b7e\uff0c\u4ece\u800c\u8bbe\u5b9a\u591a\u4e2a\u76ee\u6807\u533a\u95f4\u3002\r\n\r\nSEQUENCE_EXCLUDED_REGION (interval list; default empty)\r\n\u8be5\u6807\u7b7e\u7684\u503c\u4e3a\uff1a<start><length>\u3002\u8bbe\u8ba1\u5f15\u7269\u65f6\u8981\u907f\u5f00\u6b64\u6bb5\u5b50\u5e8f\u5217\u533a\u57df\u3002\u6bd4\u5982\u6b64\u533a\u57df\u542b\u6709\u8bf8\u5982ALUs\u6216\u8005LINEs\u7b49\u91cd\u590d\u5e8f\u5217\u65f6\u3002\r\n\r\nSEQUENCE_PRIMER_PAIR_OK_REGION_LIST (semicolon separated list of integer \"quadruples\"; default empty)\r\n\u8be5\u6807\u7b7e\u7684\u503c\u4e3a\u591a\u4e2a\uff0c\u503c\u548c\u503c\u4e4b\u95f4\u7528\u201c;\u201c\u9694\u5f00\uff0c\u5355\u4e2a\u503c\u4e3a\uff1a<left_start>,<left_length>,<right_start>,<right_length>\u3002\r\n\u6bd4\u5982\uff1a\r\nSEQUENCE_PRIMER_PAIR_OK_REGION_LIST=100,50,300,50 ; 900,60,, ; ,,930,100\r\n\u8868\u660e\u6709\u5f15\u7269\u8bbe\u8ba1\u6709\u4e09\u79cd\u9009\u62e9\uff1a\r\n\u5de6\u5f15\u7269\u5728100\uff5e150bp\u533a\u95f4\u8fdb\u884c\u8bbe\u8ba1\uff0c\u53f3\u5f15\u7269\u5728300\uff5e350bp\u7684\u533a\u95f4\u8fdb\u884c\u8bbe\u8ba1\uff1b\r\n\u5de6\u5f15\u7269\u5728900\uff5e960bp\u533a\u95f4\u8fdb\u884c\u8bbe\u8ba1\uff0c\u53f3\u5f15\u7269\u968f\u610f\uff1b\r\n\u53f3\u5f15\u7269\u5728930\uff5e1030bp\u533a\u95f4\u8fdb\u884c\u8bbe\u8ba1\uff0c\u5de6\u5f15\u7269\u968f\u610f\u3002\r\n\r\nSEQUENCE_OVERLAP_JUNCTION_LIST (space separated integers; default empty)\r\n\u6307\u5b9a\u5f15\u7269\u5728\u6b64\u4f4d\u7f6e\u8981\u6709\u91cd\u53e0\u3002\u4e0e\u6807\u7b7e PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION \u6216 PRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION \u642d\u914d\u4f7f\u7528\u3002\u4f8b\u5982\uff1a\u6b64\u503c\u8bbe\u4e3a 20\uff0c \u9ed8\u8ba4\u4e0b\uff0c\u8bbe\u8ba1\u7684\u5f15\u7269\u7684 3' \u7aef\u5fc5\u987b\u8981\u5305\u542b\u6709 20~23 \u533a\u6bb5\u7684\u78b1\u57fa\uff0c\u6216\u5f15\u7269\u7684 5' \u7aef\u5fc5\u987b\u5305\u542b\u6709 20~26 \u533a\u6bb5\u7684\u78b1\u57fa\u3002\r\n\r\nSEQUENCE_INTERNAL_EXCLUDED_REGION (interval list; default empty)\r\n\u8be5\u6807\u7b7e\u7684\u503c\u4e3a\uff1a<start>,<length>\u3002\u4e2d\u95f4oligos\u7684\u8bbe\u8ba1\u907f\u5f00\u6b64\u6bb5\u5b50\u5e8f\u5217\u3002\r\n\r\nSEQUENCE_PRIMER (nucleotide sequence; default empty)\r\n\u8be5\u6807\u7b7e\u7684\u503c\u4e3a\u5de6\u5f15\u7269\u5e8f\u5217\uff0c\u8be5\u5e8f\u5217\u5fc5\u987b\u662f SEQUENCE_TEMPLATE \u7684\u4e00\u6bb5\u5b50\u5e8f\u5217\uff1b\u7a0b\u5e8f\u8fd0\u884c\u65f6\u4f1a\u68c0\u67e5\u8be5\u5b50\u5e8f\u5217\u5e76\u5728\u5176\u9644\u8fd1\u8bbe\u8ba1\u53f3\u5f15\u7269\u6216\u4e2d\u95f4oligos.\r\n\r\nSEQUENCE_INTERNAL_OLIGO (nucleotide sequence; default empty)\r\n\u8be5\u6807\u7b7e\u7684\u503c\u4e3a\u4e2d\u95f4oligo\u5e8f\u5217\uff0c\u8be5\u5e8f\u5217\u5fc5\u987b\u662fSEQUENCE_TEMPLATE\u7684\u4e00\u6bb5\u5b50\u5e8f\u5217\uff1b\u7a0b\u5e8f\u8fd0\u884c\u65f6\u4f1a\u68c0\u67e5\u8be5\u5b50\u5e8f\u5217\u5e76\u5728\u5176\u9644\u8fd1\u8bbe\u8ba1\u5de6\u53f3\u5f15\u7269\u3002\r\n\r\nSEQUENCE_PRIMER_REVCOMP (nucleotide sequence; default empty)\r\n\u8be5\u6807\u7b7e\u7684\u503c\u4e3a\u53f3\u5f15\u7269\u5e8f\u5217\uff0c\u8be5\u5e8f\u5217\u5fc5\u987b\u662fSEQUENCE_TEMPLATE\u7684\u4e00\u6bb5\u53cd\u5411\u4e92\u8865\u5b50\u5e8f\u5217\uff1b\u7a0b\u5e8f\u8fd0\u884c\u65f6\u4f1a\u68c0\u67e5\u8be5\u5b50\u5e8f\u5217\u5e76\u5728\u5176\u9644\u8fd1\u8bbe\u8ba1\u5de6\u5f15\u7269\u3002\r\n\r\nSEQUENCE_START_CODON_POSITION (int; default -2000000)\r\n\u6807\u8bb0\u8d77\u59cb\u5bc6\u7801\u5b50\u7684\u7b2c\u4e00\u4e2a\u78b1\u57fa\u4f4d\u70b9\uff0c\u4ece\u800c\u8ba9primer3\u5728\u8d77\u59cb\u5bc6\u7801\u5b50\u7684\u5de6\u8fb9\u8bbe\u8ba1\u5de6\u5f15\u7269\uff0c\u53ea\u6709\u5728\u5fc5\u8981\u65f6\u624d\u5728\u8d77\u59cb\u5bc6\u7801\u5b50\u7684\u53f3\u8fb9\u8bbe\u8ba1\u5de6\u5f15\u7269\u3002\u53ef\u4ee5\u8bbe\u7f6e\u4e3a\u8d1f\u6570\uff0c\u8868\u793a\u8d77\u59cb\u5bc6\u7801\u5b50\u5728\u6574\u6761\u5e8f\u5217\u4e0d\u53ef\u89c1\u7684\u5de6\u8fb9\u3002\u5982\u679c\u8be5\u503c\u4e3a\u975e\u8d1f\u6570\u4e14\u8be5\u4f4d\u70b9\u5904\u7684\u78b1\u57fa\u4e0d\u662fATG\uff0c\u5219primer3\u4f1a\u53d1\u51fa\u4e00\u4e2a\u9519\u8bef\u4fe1\u53f7\u3002\u5982\u679c\u503c\u5c0f\u4e8e\u6216\u7b49\u4e8e-10^6,\u5219\u5ffd\u7565\u8be5\u53c2\u6570\u3002primer3\u4ece\u8d77\u59cb\u5bc6\u7801\u5b50\u5f00\u59cb\u626b\u63cf\uff0c\u76f4\u5230\u83b7\u5f97\u4e00\u4e2a\u7ec8\u6b62\u5bc6\u7801\u5b50\uff0c\u7406\u60f3\u72b6\u51b5\u4e0b\u8bbe\u8ba1\u7684\u53f3\u5f15\u7269\u7ed3\u675f\u4e8e\u8be5\u7ec8\u6b62\u5bc6\u7801\u5b50\u6216\u5176\u540e\u7684\u78b1\u57fa\u3002\r\n\r\nSEQUENCE_QUALITY (space separated integers; default empty)\r\n\u8be5\u6807\u7b7e\u7684\u503c\u662f\u4e00\u7cfb\u5217\u7684\u4ee5\u7a7a\u683c\u9694\u5f00\u7684\u78b1\u57fa\u8d28\u91cf\u503c\uff0c\u6bcf\u4e00\u4e2a\u78b1\u57fa\u8d28\u91cf\u503c\u5bf9\u5e94\u7740\u6a21\u677f\u740f\u4e0a\u7684\u78b1\u57fa\u3002\r\n\r\nSEQUENCE_FORCE_LEFT_START (int; default -1000000)\r\n\u5f3a\u5236\u5de6\u5f15\u7269\u76845'\u7aef\u4ece\u6307\u5b9a\u7684\u4f4d\u70b9\u5f00\u59cb\uff0c\u4e0d\u7ba1\u662f\u5426\u8fdd\u80cc\u4e86\u4e00\u4e9b\u5176\u5b83\u7684\u6761\u4ef6\u3002\u9ed8\u8ba4\u503c\u8868\u793a\u5de6\u5f15\u7269\u7684\u8d77\u59cb\u4f4d\u70b9\u662f\u4efb\u610f\u4f4d\u7f6e\u3002\r\n\r\nSEQUENCE_FORCE_LEFT_END (int; default -1000000)\r\n\u5f3a\u5236\u5de6\u5f15\u7269\u76843'\u7aef\u5230\u6307\u5b9a\u4f4d\u70b9\u7ed3\u675f\uff0c\u4e0d\u7ba1\u662f\u5426\u8fdd\u80cc\u4e86\u4e00\u4e9b\u5176\u5b83\u7684\u6761\u4ef6\u3002\u9ed8\u8ba4\u503c\u8868\u793a\u5de6\u5f15\u7269\u7684\u7ed3\u675f\u4f4d\u70b9\u662f\u4efb\u610f\u4f4d\u7f6e\u3002\r\n\r\nSEQUENCE_FORCE_RIGHT_START (int; default -1000000)\r\n\u5f3a\u5236\u53f3\u5f15\u7269\u76845'\u7aef\u4ece\u6307\u5b9a\u7684\u4f4d\u70b9\u5f00\u59cb\uff0c\u4e0d\u7ba1\u662f\u5426\u8fdd\u80cc\u4e86\u4e00\u4e9b\u5176\u5b83\u7684\u6761\u4ef6\u3002\u9ed8\u8ba4\u503c\u8868\u793a\u5de6\u5f15\u7269\u7684\u8d77\u59cb\u4f4d\u70b9\u662f\u4efb\u610f\u4f4d\u7f6e\u3002\r\n\r\nSEQUENCE_FORCE_RIGHT_END (int; default -1000000)\r\n\u5f3a\u5236\u53f3\u5f15\u7269\u76843'\u7aef\u5230\u6307\u5b9a\u4f4d\u70b9\u7ed3\u675f\uff0c\u4e0d\u7ba1\u662f\u5426\u8fdd\u80cc\u4e86\u4e00\u4e9b\u5176\u5b83\u7684\u6761\u4ef6\u3002\u9ed8\u8ba4\u503c\u8868\u793a\u5de6\u5f15\u7269\u7684\u7ed3\u675f\u4f4d\u70b9\u662f\u4efb\u610f\u4f4d\u7f6e\u3002\r\n<\/pre>\n
\u5168\u5c40\u8f93\u5165\u6807\u7b7e \uff08\u4ee5 PRIMER_ \u5f00\u5934\uff09\uff1a<\/h3>\n
PRIMER_TASK (string; default generic)\r\n\u8be5\u6807\u7b7e\u786e\u5b9a primer3 \u8bbe\u8ba1\u4ec0\u4e48\u5f15\u7269\uff0c\u5176\u6709\u6548\u7684\u503c\u6709\uff1a\r\n1. generic\r\n\u6700\u5e38\u7528\u7684\u503c\uff0c\u5373\u8bbe\u8ba1\u5f15\u7269\u5bf9\u3002\r\n2. pick_detection_primers\r\n\u8be5\u503c\u5df2\u53d6\u6d88\u3002\u548c generic \u7b49\u540c\u3002\r\n3. check_primers\r\n\u5bf9\u5f15\u7269\u8fdb\u884c\u68c0\u6d4b\uff0c\u5fc5\u987b\u8981\u5728 SEQUENCE_PRIMER, SEQUENCE_INTERNAL_OLIGO \u548c SEQUENCE_PRIMER_REVCOMP \u6807\u7b7e\u4e2d\u63d0\u4f9b\u5f15\u7269\u5e8f\u5217\u3002\r\n4. pick_primer_list\r\n\u4ec5\u4ec5\u72ec\u7acb\u5730\u8fdb\u884c left primers, right primers \u548c internal oligos \u7684\u63d0\u53d6\u3002\r\n5. pick_sequencing_primers\r\n\u8bbe\u8ba1\u5f15\u7269\u9002\u5408\u4e8e\u5bf9\u76ee\u6807\u533a\u57df\u8fdb\u884c\u6d4b\u5e8f\u3002\u53ef\u4ee5\u4f7f\u7528 SEQUENCE_TARGET \u6765\u5236\u5b9a\u591a\u4e2a\u76ee\u6807\u533a\u95f4\u3002\u4e3a\u4e86\u83b7\u5f97\u6700\u4f73\u7684\u6d4b\u5e8f\u7ed3\u679c\uff0c\u6765\u8ba1\u7b97 primer \u7684\u4f4d\u7f6e\u3002\u6b64\u65f6\u4f1a\u5ffd\u7565 PRIMER_PRODUCT_SIZE_RANGE \u6240\u8bbe\u5b9a\u7684\u503c\u3002\r\n6. pick_cloning_primers\r\n\u8bbe\u8ba1\u5f15\u7269\u7528\u4e8e\u57fa\u56e0\u7684\u514b\u9686\u3002\u5fc5\u987b\u4f7f\u7528 SEQUENCE_INCLUDED_REGION \u6765\u6307\u5b9a\u6a21\u677f\u5e8f\u5217\u4e2d\u8d77\u59cb\u548c\u7ec8\u6b62\u7684\u6838\u82f7\u9178\u4f4d\u7f6e\uff08ORF\u7684\u8d77\u59cb\u4e0e\u7ec8\u6b62\uff09\u3002\u4ece\u800c\uff0c\u7a0b\u5e8f\u4ec5\u80fd\u8c03\u8282\u5f15\u7269\u5e8f\u5217\u7684\u957f\u5ea6\u3002\u540c\u65f6\u518d\u8fdb\u884c PRIMER_PICK_ANYWAY=1 \u800c\u4e0d\u8003\u8651\u548c\u5176\u5b83\u5f15\u7269\u8bbe\u8ba1\u6761\u4f8b\u76f8\u51b2\u7a81\uff0c\u6765\u5f3a\u5236\u8bbe\u8ba1\u5f15\u7269\u3002\r\n7. pick_discriminative_primers\r\n\u8bbe\u8ba1\u591a\u6001\u6027\u6269\u589e\u5f15\u7269\uff0c\u7528\u4e8e\u5c06\u4e00\u4e2a\u5f15\u7269\u7684\u5e8f\u5217\u5f3a\u5236\u8bbe\u8ba1\u5230\u4e00\u4e2a\u591a\u6001\u6027\u4f4d\u70b9\u4e0a\u3002\u548c\u4e0a\u4e00\u4e2a\u65b9\u6cd5\u539f\u7406\u4e00\u81f4\uff0c\u9700\u8981\u8bbe\u7f6e SEQUENCE_INCLUDED_REGION \u548c PRIMER_PICK_ANYWAY=1 \u6807\u7b7e\u3002\r\n8. pick_pcr_primers\r\n\u8be5\u503c\u5df2\u53d6\u6d88\u3002\u7b49\u540c\u4e8e\uff1a\r\nPRIMER_TASK=generic\r\nPRIMER_PICK_LEFT_PRIMER=1\r\nPRIMER_PICK_INTERNAL_OLIGO=1\r\nPRIMER_PICK_RIGHT_PRIMER=1\r\n9. pick_pcr_primers_and_hyb_probe\r\n\u8be5\u503c\u5df2\u53d6\u6d88\u3002 \u548c pick_pcr_primers \u5b8c\u5168\u4e00\u81f4\u3002\r\n10. pick_left_only\r\n\u8be5\u503c\u5df2\u53d6\u6d88\u3002\u4ec5\u8bbe\u8ba1 left primer.\r\n11. pick_right_only\r\n\u8be5\u503c\u5df2\u53d6\u6d88\u3002\u4ec5\u8bbe\u8ba1 right primer.\r\n12. pick_hyb_probe_only\r\n\u8be5\u503c\u5df2\u53d6\u6d88\u3002\u4ec5\u8bbe\u8ba1 interanl oligo.\r\n\r\nPRIMER_PICK_LEFT_PRIMER (boolean; default 1)\r\n\u662f\u5426\u8bbe\u8ba1 left primer.\r\n\r\nPRIMER_PICK_INTERNAL_OLIGO (boolean; default 0)\r\n\u662f\u5426\u8bbe\u8ba1 internal oligo.\r\n\r\nPRIMER_PICK_RIGHT_PRIMER (boolean; default 1)\r\n\u662f\u5426\u8bbe\u8ba1 right primer.\r\n\r\nPRIMER_NUM_RETURN (int; default 5)\r\n\u6700\u5927\u8fd4\u56de\u7684\u5f15\u7269\uff08\u5bf9\uff09\u6570\u76ee\u3002\u8fd9\u4e9b\u5f15\u7269\uff08\u5bf9\uff09\u6309\u5176\u8d28\u91cf\u8fdb\u884c\u6392\u5e8f\u3002\u589e\u5927\u8be5\u503c\u5c06\u589e\u52a0\u8fd0\u7b97\u65f6\u95f4\u3002\r\n\r\nPRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION (int; default 4)\r\n\u9ed8\u8ba4\u8bbe\u7f6e\u4e0b\uff0c\u5f15\u7269\u7684 3' \u7aef\u5e8f\u5217\u548c\u6a21\u677f\u6307\u5b9a\u4f4d\u7f6e\u5fc5\u987b\u8981\u6709 4 \u4e2a\u78b1\u57fa\u7684\u91cd\u53e0\u3002\r\n\r\nPRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION (int; default 7)\r\n\u9ed8\u8ba4\u8bbe\u7f6e\u4e0b\uff0c\u5f15\u7269\u7684 5' \u7aef\u5e8f\u5217\u548c\u6a21\u677f\u6307\u5b9a\u4f4d\u7f6e\u5fc5\u987b\u8981\u6709 4 \u4e2a\u78b1\u57fa\u7684\u91cd\u53e0\u3002\r\n\r\nPRIMER_MUST_MATCH_FIVE_PRIME (ambiguous nucleotide sequence; default empty)\r\n\u8be5\u503c\u662f\u4e2a 5bp \u957f\u5ea6\u7684\u78b1\u57fa\u5e8f\u5217\uff08\u53ef\u4ee5\u4f7f\u7528\u5404\u79cd\u6a21\u7cca\u78b1\u57fa\u5b57\u7b26\uff09\uff0c\u7528\u4e8e\u6307\u5b9a\u5f15\u7269 5' \u7aef\u7684\u5e8f\u5217\u3002\r\n\r\nPRIMER_INTERNAL_MUST_MATCH_FIVE_PRIME (ambiguous nucleotide sequence; default empty)\r\n\u548c\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u81f4\uff0c\u7528\u4e8e\u6307\u5b9a internal oligo \u7684 5' \u7aef\u5e8f\u5217\u3002\r\n\r\nPRIMER_MUST_MATCH_THREE_PRIME (ambiguous nucleotide sequence; default empty)\r\n\u8be5\u503c\u662f\u4e2a 5bp \u957f\u5ea6\u7684\u78b1\u57fa\u5e8f\u5217\uff08\u53ef\u4ee5\u4f7f\u7528\u5404\u79cd\u6a21\u7cca\u78b1\u57fa\u5b57\u7b26\uff09\uff0c\u7528\u4e8e\u6307\u5b9a\u5f15\u7269 3' \u7aef\u7684\u5e8f\u5217\u3002\r\n\r\nPRIMER_INTERNAL_MUST_MATCH_THREE_PRIME (ambiguous nucleotide sequence; default empty)\r\n\u548c\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u81f4\uff0c\u7528\u4e8e\u6307\u5b9a internal oligo \u7684 3' \u7aef\u7684\u5e8f\u5217\u3002\r\n\r\nPRIMER_PRODUCT_SIZE_RANGE (size range list; default 100-300)\r\n\u6307\u5b9a\u5f15\u7269\u4ea7\u7269\u7684\u957f\u5ea6\u8303\u56f4\u3002\r\n\r\nPRIMER_PRODUCT_OPT_SIZE (int; default 0)\r\n\u6307\u5b9a\u5f15\u7269\u4ea7\u7269\u6700\u4f73\u7684\u957f\u5ea6\u503c\u3002\r\n\r\nPRIMER_PAIR_WT_PRODUCT_SIZE_LT (float; default 0.0)\r\n\u5982\u679c\u4ea7\u7269\u957f\u5ea6\u4f4e\u4e8e PRIMER_PRODUCT_OPT_SIZE \u7684\u503c\uff0c\u6240\u5f97\u5230\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_PAIR_WT_PRODUCT_SIZE_GT (float; default 0.0)\r\n\u5982\u679c\u4ea7\u7269\u957f\u5ea6\u9ad8\u4e8e PRIMER_PRODUCT_OPT_SIZE \u7684\u503c\uff0c\u6240\u5f97\u5230\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_MIN_SIZE (int; default 18)\r\n\u6240\u5141\u8bb8\u7684\u6700\u5c0f\u7684 primer \u957f\u5ea6\u3002\r\n\r\nPRIMER_INTERNAL_MIN_SIZE (int; default 18)\r\n\u6240\u5141\u8bb8\u7684\u6700\u5c0f\u7684 internal oligo \u957f\u5ea6\u3002\r\n\r\nPRIMER_OPT_SIZE (int; default 20)\r\n\u6700\u4f73\u7684 primer \u957f\u5ea6\u3002\r\n\r\nPRIMER_INTERNAL_OPT_SIZE (int; default 20)\r\n\u6700\u4f73\u7684 internal oligo \u957f\u5ea6\u3002\r\n\r\nPRIMER_MAX_SIZE (int; default 27)\r\n\u6240\u5141\u8bb8\u7684\u6700\u5927\u7684 primer \u957f\u5ea6\u3002\r\n\r\nPRIMER_INTERNAL_MAX_SIZE (int; default 27)\r\n\u6240\u5141\u8bb8\u7684\u6700\u5927\u7684 internal oligo \u957f\u5ea6\u3002\r\n\r\nPRIMER_WT_SIZE_LT (float; default 1.0)\r\nprimer \u6bd4\u6700\u4f73\u957f\u5ea6\u5c0f\u6240\u83b7\u5f97\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_INTERNAL_WT_SIZE_LT (float; default 1.0)\r\ninternal oligo \u6bd4\u6700\u4f73\u957f\u5ea6\u5c0f\u6240\u83b7\u5f97\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_WT_SIZE_GT (float; default 1.0)\r\nprimer \u6bd4\u6700\u4f73\u957f\u5ea6\u5927\u6240\u83b7\u5f97\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_INTERNAL_WT_SIZE_GT (float; default 1.0)\r\ninternal oligo \u6bd4\u6700\u4f73\u957f\u5ea6\u5927\u6240\u83b7\u5f97\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_MIN_GC (float; default 20.0)\r\nprimer \u4e2d\u6240\u5141\u8bb8\u7684\u6700\u5c0f\u7684 GC \u542b\u91cf\u7684\u767e\u5206\u6bd4\u3002\r\n\r\nPRIMER_INTERNAL_MIN_GC (float; default 20.0)\r\ninternal oligo \u4e2d\u6240\u5141\u8bb8\u7684\u6700\u5c0f\u7684 GC \u542b\u91cf\u7684\u767e\u5206\u6bd4\u3002\r\n\r\nPRIMER_OPT_GC_PERCENT (float; default 50.0)\r\nprimer \u4e2d\u6700\u4f73\u7684 GC \u542b\u91cf\u503c\u3002\r\n\r\nPRIMER_INTERNAL_OPT_GC_PERCENT (float; default 50.0)\r\ninternal oligo \u4e2d\u6700\u4f73\u7684 GC \u542b\u91cf\u503c\u3002\r\n\r\nPRIMER_MAX_GC (float; default 80.0)\r\nprimer \u4e2d\u6240\u5141\u8bb8\u7684\u6700\u5927\u7684 GC \u542b\u91cf\u7684\u767e\u5206\u6bd4\u3002\r\n\r\nPRIMER_INTERNAL_MAX_GC (float; default 80.0)\r\ninternal oligo \u4e2d\u6240\u5141\u8bb8\u7684\u6700\u5927\u7684 GC \u542b\u91cf\u7684\u767e\u5206\u6bd4\u3002\r\n\r\nPRIMER_WT_GC_PERCENT_LT (float; default 0.0)\r\nprimer \u6bd4\u6700\u4f73 GC \u542b\u91cf\u503c\u4f4e\u6240\u83b7\u5f97\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_INTERNAL_WT_GC_PERCENT_LT (float; default 0.0)\r\ninternal oligo \u6bd4\u6700\u4f73 GC \u542b\u91cf\u503c\u4f4e\u6240\u83b7\u5f97\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_WT_GC_PERCENT_GT (float; default 0.0)\r\nprimer \u6bd4\u6700\u4f73 GC \u542b\u91cf\u503c\u9ad8\u6240\u83b7\u5f97\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_INTERNAL_WT_GC_PERCENT_GT (float; default 0.0)\r\ninternal oligo \u6bd4\u6700\u4f73 GC \u542b\u91cf\u503c\u9ad8\u6240\u83b7\u5f97\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_GC_CLAMP (int; default 0)\r\n\u8981\u6c42 left primer \u548c right primer \u7684 3' \u672b\u7aef\u5e8f\u5217\u4e2d\u6709\u8fde\u7eed\u6307\u5b9a\u6570\u76ee\u7684 Gs \u6216 Cs \u78b1\u57fa\u3002\r\n\r\nPRIMER_MAX_END_GC (int; default 5)\r\nleft primer \u548c right primer \u7684 3' \u672b\u7aef\u7684 5bp \u78b1\u57fa\u4e2d\u6700\u591a\u5141\u8bb8\u6709\u6307\u5b9a\u6570\u76ee\u7684 Gs \u6216 Cs \u78b1\u57fa\u3002\r\n\r\nPRIMER_MIN_TM (float; default 57.0)\r\nprimer \u6240\u5141\u8bb8\u7684\u6700\u5c0f\u7684 TM \u503c\u3002\r\n\r\nPRIMER_INTERNAL_MIN_TM (float; default 57.0)\r\ninternal oligo \u6240\u5141\u8bb8\u7684\u6700\u5c0f\u7684 TM \u503c\u3002\r\n\r\nPRIMER_OPT_TM (float; default 60.0)\r\nprimer \u6700\u4f18\u7684 TM \u503c\u3002\r\n\r\nPRIMER_INTERNAL_OPT_TM (float; default 60.0)\r\ninternal oligo \u6700\u4f18\u7684 TM \u503c\u3002\r\n\r\nPRIMER_MAX_TM (float; default 63.0)\r\nprimer \u6240\u5141\u8bb8\u7684\u6700\u5927\u7684 TM \u503c\u3002\r\n\r\nPRIMER_INTERNAL_MAX_TM (float; default 63.0)\r\ninternal oligo \u6240\u5141\u8bb8\u7684\u6700\u5927\u7684 TM \u503c\u3002\r\n\r\nPRIMER_WT_TM_LT (float; default 1.0)\r\nprimer \u4f4e\u4e8e\u6700\u4f18 TM \u503c\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_INTERNAL_WT_TM_LT (float; default 1.0)\r\ninternal oligo \u4f4e\u4e8e\u6700\u4f18 TM \u503c\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_WT_TM_GT (float; default 1.0)\r\nprimer \u9ad8\u4e8e\u6700\u4f18 TM \u503c\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_INTERNAL_WT_TM_GT (float; default 1.0)\r\ninternal oligo \u9ad8\u4e8e\u6700\u4f18 TM \u503c\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_PAIR_WT_DIFF_TM (float; default 0.0)\r\nleft primer \u548c right primer \u7684 TM \u503c\u4e0d\u7b49\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_PRODUCT_MIN_TM (float; default -1000000.0)\r\n\u6269\u589e\u4ea7\u7269\u6240\u5141\u8bb8\u6700\u5c0f\u7684 TM \u503c\u3002\r\n\r\nPRIMER_PRODUCT_OPT_TM (float; default 0.0)\r\n\u6269\u589e\u4ea7\u7269\u6700\u4f73\u7684 TM \u503c\u3002 0 \u8868\u793a\u6ca1\u6709\u6700\u4f73\u7684 TM \u503c\u3002\r\n\r\nPRIMER_PRODUCT_MAX_TM (float; default 1000000.0)\r\n\u6269\u589e\u4ea7\u7269\u6240\u5141\u8bb8\u6700\u5927\u7684 TM \u503c\u3002\u8ba1\u7b97\u5176 TM \u503c\u7684\u516c\u5f0f\uff1aTm = 81.5 + 16.6(log10([Na+])) + 0.41*(%GC) - 600\/length\u3002\u5176\u4e2d[Na+]\u662f\u94a0\u76d0\u7684\u6469\u5c14\u6d53\u5ea6\uff1blength \u662f\u5e8f\u5217\u7684\u957f\u5ea6\u3002\u6b64\u516c\u5f0f\u4e5f\u7528\u4e8e\u8ba1\u7b97 primer and internal oligo \u7684 Tm \u503c\u3002\r\n\r\nPRIMER_PAIR_WT_PRODUCT_TM_LT (float; default 0.0)\r\n\u6269\u589e\u4ea7\u7269\u4f4e\u4e8e\u6700\u4f73 TM \u503c\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_PAIR_WT_PRODUCT_TM_GT (float; default 0.0)\r\n\u6269\u589e\u4ea7\u7269\u9ad8\u4e8e\u6700\u4f73 TM \u503c\u7684\u7f5a\u5206\u3002\r\n\r\nPRIMER_TM_FORMULA (int; default 1)\r\n\u6307\u5b9a TM \u8be6\u7ec6\u8ba1\u7b97\u65b9\u6cd5\u3002 0 \u8868\u793a\u4f7f\u7528\u4ee5\u524d\u7248\u672c\u7684\u517c\u5bb9\u7684\u7b97\u6cd5\u3002\r\n\r\nPRIMER_SALT_MONOVALENT (float; default 50.0)\r\n\u6307\u5b9a PCR \u53cd\u5e94\u4f53\u7cfb\u4e2d\u4e00\u4ef7\u76d0\u7684\u6d53\u5ea6\u3002\u9ed8\u8ba4\u662f 50 mM\u3002\r\n\r\nPRIMER_INTERNAL_SALT_MONOVALENT (float; default 50.0)\r\n\u548c\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u81f4\u3002\u4f46\u662f for internal oligo.\r\n\r\nPRIMER_SALT_DIVALENT (float; default 1.5)\r\n\u6307\u5b9a PCR \u53cd\u5e94\u4f53\u7cfb\u4e2d\u4e8c\u4ef7\u76d0\uff08\u4e00\u822c\u662f Mg2+\uff09\u7684\u6d53\u5ea6\u3002\u9ed8\u8ba4\u662f 1.6 mM\u3002\r\n\r\nPRIMER_INTERNAL_SALT_DIVALENT (float; default 0.0)\r\n\u548c\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u81f4\uff0c\u4f46\u662f for internal oligo.\r\n\r\nPRIMER_DNTP_CONC (float; default 0.6)\r\nDNTP \u7684\u603b\u6d53\u5ea6\u3002\r\n\r\nPRIMER_INTERNAL_DNTP_CONC (float; default 0.0)\r\n\u548c\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u81f4\uff0c\u4f46\u662f for internal oligo.\r\n\r\nPRIMER_SALT_CORRECTIONS (int; default 1)\r\n\u5bf9\u76d0\u6d53\u5ea6\u7684\u6821\u6b63\u65b9\u6cd5\u3002\u6709 0,1,2 \u4e09\u79cd\u65b9\u6cd5\u3002\r\n\r\nPRIMER_DNA_CONC (float; default 50.0)\r\nPCR \u5b8c\u6bd5\u540e\u7684 annealing oligo \u7684\u6d53\u5ea6\u3002\r\n\r\nPRIMER_INTERNAL_DNA_CONC (float; default 50.0)\r\n\u548c\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u81f4\uff0c\u4f46\u662f for internal oligo.\r\n\r\nPRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT (boolean; default 1)\r\n\u8be5\u503c\u8bbe\u4e3a 1\uff0c \u5219\u7a0b\u5e8f\u4f1a\u4f7f\u7528\u70ed\u529b\u5b66\u6a21\u578b\u6765\u8ba1\u7b97 oligos \u5f62\u6210\u53d1\u5939\u7ed3\u6784\u548c\u4e8c\u805a\u4f53\u7684\u53ef\u80fd\u6027\u3002\r\n\r\nPRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT (boolean; default 0)\r\n\u8be5\u503c\u8bbe\u4e3a 1\uff0c \u5219\u7a0b\u5e8f\u4f1a\u4f7f\u7528\u70ed\u529b\u5b66\u6a21\u578b\u6765\u8ba1\u7b97 oligos \u9000\u706b\u65f6\u7ed3\u5408\u5230\u6a21\u677f\u94fe\u4e0a\u975e\u7279\u5f02\u6027\u4f4d\u70b9\u7684\u53ef\u80fd\u6027\u3002\r\n\r\nPRIMER_THERMODYNAMIC_PARAMETERS_PATH (string; default .\/primer3_config)\r\n\u6b64\u6807\u7b7e\u6307\u5b9a\u8fdb\u884c\u70ed\u52a8\u529b\u5b66\u65b9\u6cd5\u8fdb\u884c\u5206\u6790\u6240\u9700\u8981\u7684\u76f8\u5173\u53c2\u6570\u6587\u4ef6\u7684\u8def\u5f84\u3002\u5728 Linux \u4e0b\u6709 2 \u4e2a\u9ed8\u8ba4\u7684\u8def\u5f84\uff1a.\/primer3_config\/ and \/opt\/primer3_config\/ \uff1b \u5728 Windows \u4e0b\uff0c\u53ea\u6709 1 \u4e2a\u9ed8\u8ba4\u8def\u5f84\uff1a.\\primer3_config\\ \u3002\u6b64\u53c2\u6570\u5728 Linux \u4e0b\u9700\u8981\u8bbe\u7f6e\u4e3a \/opt\/biosoft\/primer3-2.3.6\/src\/primer3_config\/ \u3002\r\n\r\nPRIMER_MAX_SELF_ANY (decimal, 9999.99; default 8.00)\r\n\u5f15\u7269\u8fdb\u884c\u81ea\u6211\u7ed3\u5408\u7684\u53ef\u80fd\u6027\u3002\u81ea\u6211\u5339\u914d\u4f4d\u70b9\u5219\u8bb0 1 \u5206\uff1b\u5339\u914d\u5230 N \u5219\u8bb0 -0.25 \u5206\uff1b\u4e0d\u5339\u914d\u8bb0 -1 \u5206\uff1b\u6253\u5f00\u4e00\u4e2a gap \u8bb0 -2 \u5206\u3002\u4f8b\u5982\uff1a\r\n 5' ATCGNA 3'\r\n || | |\r\n 3' TA-CGT 5'\r\n\u8fd9\u4e2a\u4ea7\u751f\u7684\u5f97\u5206\u4e3a 1.75 \u3002\u5f97\u5206\u8d8a\u4f4e\uff0c\u5219\u8868\u793a\u8d8a\u4e0d\u53ef\u80fd\u53d1\u751f\u5f15\u7269\u5185\u78b1\u57fa\u5339\u914d\u3002\r\n\r\nPRIMER_MAX_SELF_ANY_TH (decimal, 9999,99; default 47.00)\r\n\u548c PRIMER_MAX_SELF_ANY \u4e00\u6837\uff0c\u4f46\u662f\u4f7f\u7528\u70ed\u529b\u5b66\u65b9\u6cd5\u8fdb\u884c\u8ba1\u7b97\u3002\u6b64\u6807\u7b7e\u5fc5\u987b\u5728 PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1 \u65f6\u751f\u6548\u3002\u8be5\u6807\u7b7e\u7684\u9ed8\u8ba4\u503c\u6bd4 PRIMER_MIN_TM \u6807\u7b7e\u7684\u503c\u4f4e 10 \u5ea6\u3002\r\n\r\nPRIMER_INTERNAL_MAX_SELF_ANY (decimal, 9999.99; default 12.00)\r\n\u548c\u4e0a\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u81f4\uff0c\u4f46\u662f for internal oligo.\r\n\r\nPRIMER_INTERNAL_MAX_SELF_ANY_TH (decimal, 9999.99; default 47.00)\r\n\u548c\u4e0a\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u81f4\uff0c\u4f46\u662f for internal oligo.\r\n\r\nPRIMER_PAIR_MAX_COMPL_ANY (decimal, 9999.99; default 8.00)\r\n\u548c PRIMER_MAX_SELF_ANY \u4e00\u81f4\uff0c\u4f46\u662f\u9488\u5bf9 left primer \u7ed3\u5408\u5230 right primer \u4e0a\u7684\u53ef\u80fd\u6027\u3002\r\n\r\nPRIMER_PAIR_MAX_COMPL_ANY_TH (decimal, 9999.99; default 47.00)\r\n\u548c\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u6837\uff0c\u4f46\u662f\u4f7f\u7528\u70ed\u529b\u5b66\u7684\u65b9\u6cd5\u8fdb\u884c\u8ba1\u7b97\u3002\r\n\r\nPRIMER_WT_SELF_ANY (float; default 0.0)\r\n\u7f5a\u5206\u6743\u91cd for PRIMER_MAX_SELF_ANY \u7684\u503c\u3002\r\n\r\nPRIMER_WT_SELF_ANY_TH (float; default 0.0)\r\n\u7f5a\u5206\u6743\u91cd for PRIMER_MAX_SELF_ANY_TH \u7684\u503c\u3002\r\n\r\nPRIMER_INTERNAL_WT_SELF_ANY (float; default 0.0)\r\n\u548c\u4e0a\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u81f4\uff0c\u4f46\u662f for internal oligo.\r\n\r\nPRIMER_INTERNAL_WT_SELF_ANY_TH (float; default 0.0)\r\n\u548c\u4e0a\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u81f4\uff0c\u4f46\u662f for internal oligo.\r\n\r\nPRIMER_PAIR_WT_COMPL_ANY (float; default 0.0)\r\n\u7f5a\u5206\u6743\u91cd for PRIMER_MAX_SELF_ANY \u7684\u503c\u3002\r\n\r\nPRIMER_PAIR_WT_COMPL_ANY_TH (float; default 0.0)\r\n\u7f5a\u5206\u6743\u91cd for PRIMER_MAX_SELF_ANY_TH \u7684\u503c\u3002\r\n\r\nPRIMER_MAX_SELF_END (decimal, 9999.99; default 3.00)\r\n\u5f15\u7269\u7684 3' \u7aef\u53ef\u80fd\u7ed3\u5408\u5230\u4e00\u8d77\uff0c\u4ece\u800c\u5f62\u6210\u4e00\u6761\u5e8f\u5217\uff0c\u7136\u540e\u4ee5\u4e4b\u4e3a\u6a21\u677f\u8fdb\u884c\u6269\u589e\uff0c\u5f62\u6210\u5f15\u7269\u4e8c\u805a\u4f53\uff0c\u4ece\u800c\u5bfc\u81f4\u76ee\u6807\u5e8f\u5217\u7684\u6269\u589e\u5931\u8d25\u3002\u8be5\u53c2\u6570\u8bbe\u7f6e\u6700\u591a\u5141\u8bb8\u78b1\u57fa\u5728 3' \u7aef\u8fdb\u884c\u4e92\u8865\u914d\u5bf9\u7684\u5f97\u5206\u4e3a 3\u3002\u4f8b\u5982\uff1a\r\n 5' ATGCCCTAGCTTCCGGATG 3'\r\n ||| |||||\r\n 3' AAGTCCTACATTTAGCCTAGT 5'\r\n\r\nor 5` AGGCTATGGGCCTCGCGA 3'\r\n ||||||\r\n 3' AGCGCTCCGGGTATCGGA 5'\r\n\u524d\u8005\u5f97\u5206\u4e3a 7\uff0c \u540e\u8005\u5f97\u5206\u4e3a 6 \u3002\r\n\r\nPRIMER_MAX_SELF_END_TH (decimal 9999.99; default 47.00)\r\n\u548c\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u6837\uff0c\u4e0d\u8fc7\u4f7f\u7528\u70ed\u529b\u5b66\u65b9\u6cd5\u8fdb\u884c\u8ba1\u7b97\u3002\r\n\r\nPRIMER_INTERNAL_MAX_SELF_END (decimal 9999.99; default 12.00)\r\n\u548c\u4e0a\u4e0a\u4e2a\u6807\u7b7e\u4e00\u6837\uff0c\u4f46\u662f for internal oligo.\r\n\r\nPRIMER_INTERNAL_MAX_SELF_END_TH (decimal 9999.99; default 47.00\r\n\u548c\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u6837\uff0c\u4e0d\u8fc7\u4f7f\u7528\u70ed\u529b\u5b66\u65b9\u6cd5\u8fdb\u884c\u8ba1\u7b97\u3002\r\n\r\nPRIMER_PAIR_MAX_COMPL_END (decimal, 9999.99; default 3.00)\r\n\u548c PRIMER_MAX_SELF_END \u6807\u7b7e\u4e00\u6837\uff0c\u4f46\u662f\u9488\u5bf9 left primer \u548c right primer \u7684 3' \u7aef\u4e92\u8865\u914d\u5bf9\u3002\r\n\r\nPRIMER_PAIR_MAX_COMPL_END_TH (decimal, 9999.99; default 47.00)\r\n\u548c\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u6837\uff0c\u4e0d\u8fc7\u4f7f\u7528\u70ed\u529b\u5b66\u65b9\u6cd5\u8fdb\u884c\u8ba1\u7b97\u3002\r\n\r\nPRIMER_WT_SELF_END (float; default 0.0)\r\n\u7f5a\u5206\u6743\u91cd for PRIMER_MAX_SELF_END \u7684\u503c\u3002\r\n\r\nPRIMER_WT_SELF_END_TH (float; default 0.0)\r\n\u7f5a\u5206\u6743\u91cd for PRIMER_MAX_SELF_END_TH \u7684\u503c\u3002\r\n\r\nPRIMER_INTERNAL_WT_SELF_END (float; default 0.0)\r\n\u548c\u4e0a\u4e0a\u4e2a\u6807\u7b7e\u4e00\u6837\uff0c\u4f46\u662f for internal oligo.\r\n\r\nPRIMER_INTERNAL_WT_SELF_END_TH (float; default 0.0)\r\n\u548c\u4e0a\u4e0a\u4e2a\u6807\u7b7e\u4e00\u6837\uff0c\u4f46\u662f for internal oligo.\r\n\r\nPRIMER_PAIR_WT_COMPL_END (float; default 0.0)\r\n\u7f5a\u5206\u6743\u91cd for PRIMER_PAIR_MAX_COMPL_END \u7684\u503c\u3002\r\n\r\nPRIMER_PAIR_WT_COMPL_END_TH (float; default 0.0)\r\n\u7f5a\u5206\u6743\u91cd for PRIMER_PAIR_MAX_COMPL_END_TH \u7684\u503c\u3002\r\n\r\nPRIMER_MAX_HAIRPIN_TH (float; default 47.0)\r\n\u548c PRIMER_MAX_SELF_ANY_TH \u7c7b\u4f3c\uff0c\u8be5\u6807\u7b7e\u5229\u7528\u70ed\u529b\u5b66\u65b9\u6cd5\u8ba1\u7b97\u5f62\u6210\u53d1\u5939\u7ed3\u6784\u540e\u6240\u5141\u8bb8\u6700\u5927\u7684 TM \u503c\u3002\u8be5\u6807\u7b7e\u7684\u9ed8\u8ba4\u503c\u6bd4 PRIMER_MIN_TM \u6807\u7b7e\u7684\u503c\u4f4e 10 \u5ea6\u3002\r\n\r\nPRIMER_INTERNAL_MAX_HAIRPIN_TH (float; default 47.0)\r\n\u548c\u4e0a\u4e2a\u6807\u7b7e\u4e00\u6837\uff0c\u4f46\u662f for internal oligo.\r\n\r\nPRIMER_WT_HAIRPIN_TH (float; default 0.0)\r\n\u7f5a\u5206\u6743\u91cd for PRIMER_MAX_HAIRPIN_TH \u7684\u503c\u3002\r\n\r\nPRIMER_INTERNAL_WT_HAIRPIN_TH (float; default 0.0)\r\n\u7f5a\u5206\u6743\u91cd for PRIMER_INTERNAL_MAX_HAIRPIN_TH \u7684\u503c\u3002\r\n\r\nPRIMER_MAX_END_STABILITY (float, 999.9999; default 100.0)\r\nleft or right primer \u7684 5bp 3' \u78b1\u57fa\u6700\u5927\u7684\u7a33\u5b9a\u6027\u3002\u8be5\u503c\u9009\u53d6 left or right primer \u5206\u522b\u8ba1\u7b97\u51fa\u7684\u503c\u4e2d\u7684\u8f83\u5927\u503c\u3002\u8ba1\u7b97\u65b9\u6cd5\u4e3a PRIMER_TM_FORMULA \u6807\u7b7e\u4e2d\u6307\u5b9a\u7684\u65b9\u6cd5\u3002\u9ed8\u8ba4\u65b9\u6cd5\u4e0b\uff0c GCGCG \u78b1\u57fa\u5e8f\u5217\u7684\u503c\u4e3a6.86; TATAT \u78b1\u57fa\u5e8f\u5217\u7684\u503c\u4e3a 0.86 \u3002\u5982\u679c\u662f\u4f7f\u7528\u65e7\u7248\u672c\u7684\u65b9\u6cd5\uff0c\u6b64\u503c\u5219\u4f1a\u53d8\u5927\uff0c\u76f8\u5e94\u5e94\u8be5\u8bbe\u7f6e\u6b64\u503c\u5927\u4e9b\u3002\r\n\r\nPRIMER_WT_END_STABILITY (float; default 0.0)\r\n\u7f5a\u5206\u56e0\u5b50 for PRIMER_MAX_END_STABILITY \u7684\u503c\u3002\r\n\r\nPRIMER_MAX_NS_ACCEPTED (int; default 0)\r\n\u6240\u5141\u8bb8\u7684 N \u78b1\u57fa\u7684\u6570\u76ee\u3002\r\n\r\nPRIMER_WT_NUM_NS (float; default 0.0)\r\n\u7f5a\u5206\u6743\u91cd for PRIMER_MAX_NS_ACCEPTED \u7684\u503c\u3002\r\n\r\nPRIMER_MAX_POLY_X (int; default 5)\r\n\u6240\u5141\u8bb8\u7684\u5355\u6838\u82f7\u9178\u91cd\u590d\u7684\u6b21\u6570\uff0c\u4f8b\u5982\uff0c \u9ed8\u8ba4\u4e0b AAAAAA \u662f\u4e0d\u5141\u8bb8\u7684\u3002\r\n\r\nPRIMER_INTERNAL_MAX_POLY_X (int; default 5)\r\n\u548c\u4e0a\u4e2a\u6807\u7b7e\u4e00\u81f4\uff0c\u4f46\u662f for internal oligo.\r\n\r\nPRIMER_MIN_LEFT_THREE_PRIME_DISTANCE (int; default -1)\r\n\u5728\u7ed9\u51fa\u5f15\u7269\u5bf9\u7684\u65f6\u5019\uff0c\u4efb\u610f\u4e24\u4e2a left primers \u7684 3' \u672b\u7aef\u5728\u6a21\u677f\u94fe\u4e0a\u7684\u4f4d\u70b9\u76f8\u5dee\u7684\u78b1\u57fa\u6570\u4e0d\u8d85\u8fc7\u6b64\u503c\u3002 -1 \u8868\u793a\u4e0d\u4f5c\u9650\u5236\uff0c 0 \u5219\u8868\u793a\u4e24\u5bf9\u5f15\u7269\u7684 left primers \u7684 3' \u672b\u7aef\u5728\u6a21\u677f\u94fe\u4e0a\u7684\u4f4d\u70b9\u662f\u76f8\u540c\u7684\u3002\r\n\r\nPRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE (int; default -1)\r\n\u548c\u4e0a\u4e00\u4e2a\u6807\u7b7e\u4e00\u6837\uff0c\u4f46\u662f for right primers\u3002\r\n\r\nPRIMER_MIN_THREE_PRIME_DISTANCE (int; default -1)\r\n\u662f\u4e0a\u4e24\u4e2a\u6807\u7b7e\u7684\u7efc\u5408\u4f53\uff0c\u7528\u6b64\u6807\u7b7e\u76f8\u5f53\u4e8e\u7ed9\u4e0a\u4e24\u4e2a\u6807\u7b7e\u8d4b\u4e88\u540c\u6837\u7684\u503c\u3002\u4e0d\u80fd\u548c\u4e0a\u4e24\u4e2a\u6807\u7b7e\u540c\u65f6\u4f7f\u7528\u3002\r\n\r\nPRIMER_PICK_ANYWAY (boolean; default 0)\r\n\u4e0d\u7ba1\u5982\u4f55\u4e5f\u8981\u8bbe\u8ba1\u5f15\u7269\u3002\r\n\r\nPRIMER_LOWERCASE_MASKING (int; default 0)\r\n\u5982\u679c\u8bbe\u7f6e\u6b64\u6807\u7b7e\u503c\u4e3a 1\uff0c \u5219\u8bbe\u8ba1\u7684\u5f15\u7269\u7684 3' \u7aef\u5c06\u4e0d\u4f1a\u843d\u5728\u5c0f\u5199\u5b57\u7b26\u7684\u78b1\u57fa\u533a\u95f4\u3002\r\n\r\n\u540e\u9762\u7684\u8fd8\u6709\u4e00\u5c0f\u90e8\u5206\uff0c\u4e0d\u60f3\u5199\u4e86... \u4e0d\u662f\u7279\u522b\u91cd\u8981\uff0c\u800c\u4e14\u7528\u5230\u7684\u65f6\u5019\u5f88\u5c11\u3002\r\n<\/pre>\n
\u7a0b\u5e8f\u8f93\u5165\u6807\u7b7e \uff08\u4ee5 P3_ \u5f00\u5934\uff09\uff1a<\/h3>\n
P3_FILE_ID (string; default empty)\r\n\u8be5\u6807\u7b7e\u7528\u4e8e\u6307\u5b9a primer3 \u7684\u914d\u7f6e\u6587\u4ef6\u8def\u5f84\u3002\u548c\u4e3b\u7a0b\u5e8f\u7684 -p3_setting_file \u7684\u6548\u679c\u4e00\u81f4\u3002\r\nP3_FILE_FLAG (boolean; default 0)\r\n\u5982\u679c\u8bbe\u7f6e\u8be5\u503c\u4e3a 1\uff0c \u5219\u7a0b\u5e8f\u4f1a\u989d\u5916\u751f\u6210\u51e0\u4e2a\u6587\u4ef6\uff0c\u6587\u4ef6\u540d\u4ee5 SEQUENCE_ID \u6807\u7b7e\u7684\u503c\u4e3a\u524d\u7f00\uff0c\u5206\u522b\u4ee5 .for, .rev \u6216 .int \u4e3a\u540e\u7f00\uff0c\u5bf9\u5e94\u7740\u72ec\u7acb\u8bbe\u8ba1\u7684 left primer, right primer \u548c internal oligos \u7684\u5e8f\u5217\u3002\r\nP3_COMMENT (string; default empty)\r\n\u8be5\u6807\u7b7e\u7684\u503c\u662f\u65e0\u6548\u7684\u3002\u53ef\u4ee5\u7528\u4e8e\u8f93\u5165\u4fe1\u606f\u7684\u63cf\u8ff0\u3002\r\n<\/pre>\n
3.4 PRIMER3 \u6253\u5206<\/h2>\n
\n\u9ed8\u8ba4\u60c5\u51b5\u4e0b\uff0c\u5f15\u7269\u7684 TM \u6bcf\u504f\u79bb\u6700\u4f18 TM 1 \u6444\u6c0f\u5ea6\uff0c\u5219\u7f5a 1 \u5206\uff1b \u5f15\u7269\u7684\u957f\u5ea6\u6bcf\u504f\u79bb\u6700\u4f18\u957f\u5ea6 1 bp\uff0c \u5219\u7f5a 1 \u5206\u3002\u800c\u6ca1\u6709\u901a\u8fc7 GC \u542b\u91cf\u3001\u53d1\u5939\u7ed3\u6784\u30013′ \u7aef\u4e92\u8865\u3001\u81ea\u8eab\u4e92\u8865\u914d\u5bf9\u3001\u6a21\u677f\u662f\u91cd\u590d\u5e8f\u5217 \u7b49\u65b9\u5f0f\u8fdb\u884c\u7f5a\u5206\u3002\u8981\u6839\u636e\u81ea\u8eab\u9700\u6c42\u6765\u8fdb\u884c\u7f5a\u5206\u5e38\u6570\u7684\u8bbe\u5b9a\u3002<\/p>\n4. \/opt\/biosoft\/primer3-2.3.6\/my_default_settings.txt<\/h1>\n
\r\nPrimer3 File - http:\/\/primer3.sourceforge.net\r\nP3_FILE_TYPE=settings\r\n\r\nP3_FILE_ID=Settings for Batch task of searching SSR Primers\r\nPRIMER_FIRST_BASE_INDEX=1\r\nPRIMER_TASK=generic\r\nPRIMER_NUM_RETURN=5\r\nPRIMER_PICK_LEFT_PRIMER=1\r\nPRIMER_PICK_INTERNAL_OLIGO=0\r\nPRIMER_PICK_RIGHT_PRIMER=1\r\nPRIMER_PICK_ANYWAY=1\r\nPRIMER_THERMODYNAMIC_PARAMETERS_PATH=\/opt\/biosoft\/primer3-2.3.6\/src\/primer3_config\/\r\n\r\n\r\n### \u5f15\u7269 TM \u503c\u8bbe\u5b9a: ###\r\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)\r\nPRIMER_MIN_TM=55.0\r\nPRIMER_OPT_TM=60.0\r\nPRIMER_MAX_TM=65.0\r\nPRIMER_PAIR_MAX_DIFF_TM=5.0 # \u4e24\u4e2a\u5f15\u7269\u4e4b\u95f4\u7684 TM \u503c\u6700\u591a\u76f8\u5dee 5 \u6444\u6c0f\u5ea6\r\nPRIMER_WT_TM_LT=1.0\r\nPRIMER_WT_TM_GT=1.0 # TM \u6bcf\u504f\u79bb\u6700\u4f18 TM \u503c 1 \u6444\u6c0f\u5ea6\uff0c\u5219\u7f5a 1\u5206\u3002\r\nPRIMER_PAIR_WT_DIFF_TM=0.0\r\n\r\n\r\n### \u5f15\u7269\u957f\u5ea6\u8bbe\u5b9a: ###\r\nPRIMER_MIN_SIZE=18\r\nPRIMER_OPT_SIZE=20\r\nPRIMER_MAX_SIZE=22\r\nPRIMER_WT_SIZE_LT=1.0\r\nPRIMER_WT_SIZE_GT=1.0 # \u5f15\u7269\u957f\u5ea6\u6bcf\u504f\u79bb\u6700\u4f18\u503c 1 bp\uff0c \u5219\u7f5a 1 \u5206\u3002\r\n\r\n\r\n### \u5f15\u7269 GC \u542b\u91cf\u8bbe\u5b9a\uff1a###\r\nPRIMER_MIN_GC=30.0\r\nPRIMER_MAX_GC=70.0\r\nPRIMER_WT_GC_PERCENT_LT=0.0\r\nPRIMER_WT_GC_PERCENT_GT=0.0\r\n\r\n\r\n### \u5f15\u7269\u7684\u70ed\u529b\u5b66\u8ba1\u7b97: ###\r\n# \u5f00\u542f\u70ed\u529b\u5b66\u8ba1\u7b97\r\nPRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1\r\n\r\n# \u5f15\u7269\u81ea\u8eab\u8fdb\u884c\u53cd\u5411\u4e92\u8865\r\nPRIMER_MAX_SELF_ANY=8.00\r\nPRIMER_WT_SELF_ANY=0.0\r\nPRIMER_MAX_SELF_ANY_TH=45.00\r\nPRIMER_WT_SELF_ANY_TH=0.0\r\n\r\n# \u5f15\u7269\u81ea\u8eab\u8fdb\u884c 3' \u7aef\u53cd\u5411\u4e92\u8865\u5f62\u6210\u5f15\u7269\u4e8c\u805a\u4f53\r\nPRIMER_MAX_SELF_END=3.00\r\nPRIMER_WT_SELF_END=0.0\r\nPRIMER_MAX_SELF_END_TH=35.00\r\nPRIMER_WT_SELF_END_TH=0.0\r\n\r\n# left primer \u548c right primer \u5e8f\u5217\u7684\u53cd\u5411\u4e92\u8865\r\nPRIMER_PAIR_MAX_COMPL_ANY=8.00\r\nPRIMER_PAIR_WT_COMPL_ANY=0.0\r\nPRIMER_PAIR_MAX_COMPL_ANY_TH=45.00\r\nPRIMER_PAIR_WT_COMPL_ANY_TH=0.0\r\n\r\n# left primer \u548c right primer \u8fdb\u884c 3' \u7aef\u53cd\u5411\u4e92\u8865\u5f62\u6210\u5f15\u7269\u4e8c\u805a\u4f53\r\nPRIMER_PAIR_MAX_COMPL_END=3.00\r\nPRIMER_PAIR_WT_COMPL_END=0.0\r\nPRIMER_PAIR_MAX_COMPL_END_TH=35.00\r\nPRIMER_PAIR_WT_COMPL_END_TH=0.0\r\n\r\n# \u53d1\u5939\u7ed3\u6784\r\nPRIMER_MAX_HAIRPIN_TH=24.00\r\nPRIMER_WT_HAIRPIN_TH=0.0\r\n\r\n# 3' \u7aef\u78b1\u57fa\u7684\u7a33\u5b9a\u6027\r\nPRIMER_MAX_END_STABILITY=9.0\r\nPRIMER_WT_END_STABILITY=0.0\r\n\r\n\r\n### \u78b1\u57fa\u5e8f\u5217\u8bbe\u5b9a\uff1a ###\r\nPRIMER_LOWERCASE_MASKING=0 # \u6a21\u677f\u5e8f\u5217\u4e2d\u5305\u542b\u5c0f\u5199\u5b57\u7b26\u4e0d\u5f71\u54cd\u5f15\u7269\u8bbe\u8ba1\r\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\r\nPRIMER_MAX_NS_ACCEPTED=0 # \u5f15\u7269\u4e2d\u5141\u8bb8\u7684 N \u7684\u6570\u76ee\r\nPRIMER_WT_NUM_NS=0.0 # \u6bcf\u4e2a N \u7684\u7f5a\u5206\r\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\r\nPRIMER_GC_CLAMP=0 # \u5f15\u7269\u4e2d 3' \u7aef\u78b1\u57fa\u4e2d\u4e0d\u80fd\u51fa\u73b0\u8fde\u7eed\u7684 Gs \u548c Cs \u5e8f\u5217\r\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\r\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\r\n\r\n\r\n\r\n### \u78b1\u57fa\u8d28\u91cf\u8bbe\u5b9a\uff1a ###\r\nPRIMER_MIN_QUALITY=0 # primer \u5e8f\u5217\u5141\u8bb8\u6700\u5c0f\u7684\u78b1\u57fa\u8d28\u91cf\r\nPRIMER_MIN_END_QUALITY=0\r\nPRIMER_QUALITY_RANGE_MIN=0\r\nPRIMER_QUALITY_RANGE_MAX=100\r\nPRIMER_WT_SEQ_QUAL=0.0\r\nPRIMER_WT_END_QUAL=0.0\r\n\r\n\r\n## \u5f15\u7269\u4f4d\u7f6e\u8bbe\u7f6e\uff1a ###\r\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\r\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.\r\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\r\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.\r\nPRIMER_OUTSIDE_PENALTY=0\r\nPRIMER_INSIDE_PENALTY=-1.0\r\nPRIMER_WT_POS_PENALTY=0.0\r\n\r\n\r\n### \u975e\u5f15\u7269\u6570\u636e\u5e93\u8bbe\u5b9a\uff1a###\r\n# \u542f\u7528\u975e\u5f15\u7269\u6570\u636e\u5e93\r\n#PRIMER_MISPRIMING_LIBRARY=\r\n\r\n# \u5f15\u7269\u6bd4\u5bf9\u5230\u975e\u5f15\u7269\u6570\u636e\u5e93\r\n#PRIMER_MAX_LIBRARY_MISPRIMING=12.00\r\n#PRIMER_WT_LIBRARY_MISPRIMING=0.0\r\n#PRIMER_PAIR_MAX_LIBRARY_MISPRIMING=20.00\r\n#PRIMER_PAIR_WT_LIBRARY_MISPRIMING=0.0\r\n\r\n# \u6a21\u677f\u6bd4\u5bf9\u5230\u975e\u5f15\u7269\u6570\u636e\u5e93\r\n#PRIMER_MAX_TEMPLATE_MISPRIMING=12.00\r\n#PRIMER_WT_TEMPLATE_MISPRIMING=0.0\r\n#PRIMER_MAX_TEMPLATE_MISPRIMING_TH=40.00\r\n#PRIMER_WT_TEMPLATE_MISPRIMING_TH=0.0\r\n#PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING=24.00\r\n#PRIMER_PAIR_WT_TEMPLATE_MISPRIMING=0.0\r\n#PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH=70.00\r\n#PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH=0.0\r\n\r\n### PCR \u53cd\u5e94\u4f53\u7cfb\u8bbe\u5b9a: ###\r\nPRIMER_SALT_MONOVALENT=50.0 # \u5355\u4ef7\u76d0\u79bb\u5b50\u6d53\u5ea6(mM)\r\nPRIMER_SALT_CORRECTIONS=1 # PRIMER_SALT_CORRECTIONS=1 means use the salt correction in SantaLucia et al 1998\r\nPRIMER_SALT_DIVALENT=1.5 # \u4e8c\u4ef7\u9541\u79bb\u5b50\u6d53\u5ea6(mM)\r\nPRIMER_DNTP_CONC=0.6 # \u603bdNTPs\u6d53\u5ea6(mM)\r\nPRIMER_DNA_CONC=50.0 # DNA\u4ea7\u7269\u6d53\u5ea6(mM)\r\n\r\n\r\n### PCR \u4ea7\u7269\u7684\u8bbe\u5b9a\uff1a ###\r\nPRIMER_PRODUCT_MIN_TM=-1000000.0\r\nPRIMER_PRODUCT_OPT_TM=0.0\r\nPRIMER_PRODUCT_MAX_TM=1000000.0\r\nPRIMER_PAIR_WT_PRODUCT_TM_LT=0.0\r\nPRIMER_PAIR_WT_PRODUCT_TM_GT=0.0\r\nPRIMER_PRODUCT_OPT_SIZE=0\r\nPRIMER_PAIR_WT_PRODUCT_SIZE_LT=0.0\r\nPRIMER_PAIR_WT_PRODUCT_SIZE_GT=0.0\r\n\r\n\r\n## \u7f5a\u5206\u56e0\u5b50\uff1a ###\r\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\r\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\r\n\r\n\r\n### internal oligo \u7684\u8bbe\u5b9a\uff1a###\r\n# TM \u503c\r\nPRIMER_INTERNAL_MIN_TM=57.0\r\nPRIMER_INTERNAL_OPT_TM=60.0\r\nPRIMER_INTERNAL_MAX_TM=63.0\r\nPRIMER_INTERNAL_WT_TM_LT=1.0\r\nPRIMER_INTERNAL_WT_TM_GT=1.0\r\n\r\n# \u957f\u5ea6\r\nPRIMER_INTERNAL_MIN_SIZE=18\r\nPRIMER_INTERNAL_OPT_SIZE=20\r\nPRIMER_INTERNAL_MAX_SIZE=27\r\nPRIMER_INTERNAL_WT_SIZE_LT=1.0\r\nPRIMER_INTERNAL_WT_SIZE_GT=1.0\r\n\r\n# GC \u542b\u91cf\r\nPRIMER_INTERNAL_MIN_GC=20.0\r\nPRIMER_INTERNAL_MAX_GC=80.0\r\nPRIMER_INTERNAL_OPT_GC_PERCENT=50.0\r\nPRIMER_INTERNAL_WT_GC_PERCENT_LT=0.0\r\nPRIMER_INTERNAL_WT_GC_PERCENT_GT=0.0\r\n\r\n# \u70ed\u529b\u5b66\r\nPRIMER_INTERNAL_MAX_SELF_ANY=12.00\r\nPRIMER_INTERNAL_WT_SELF_ANY=0.0\r\nPRIMER_INTERNAL_MAX_SELF_END=12.00\r\nPRIMER_INTERNAL_WT_SELF_END=0.0\r\n\r\n# \u78b1\u57fa\u5e8f\u5217\r\nPRIMER_INTERNAL_MAX_POLY_X=5\r\nPRIMER_INTERNAL_MAX_NS_ACCEPTED=0\r\n\r\n# \u78b1\u57fa\u8d28\u91cf\r\nPRIMER_INTERNAL_MIN_QUALITY=0\r\nPRIMER_INTERNAL_WT_END_QUAL=0.0\r\nPRIMER_INTERNAL_WT_SEQ_QUAL=0.0\r\n\r\n# \u975e\u5f15\u7269\u6570\u636e\u5e93\r\n#PRIMER_INTERNAL_MAX_LIBRARY_MISHYB=12.00\r\n#PRIMER_INTERNAL_WT_LIBRARY_MISHYB=0.0\r\n\r\n# PCR \u53cd\u5e94\u4f53\u7cfb\r\nPRIMER_INTERNAL_SALT_MONOVALENT=50.0\r\nPRIMER_INTERNAL_SALT_DIVALENT=1.5\r\nPRIMER_INTERNAL_DNA_CONC=50.0\r\nPRIMER_INTERNAL_DNTP_CONC=0.0\r\n=\r\n<\/pre>\n","protected":false},"excerpt":{"rendered":"