一、primer3设计引物
1. Primer3 简介
参考:http://primer3.wi.mit.edu/primer3web_help.htm。
Primer3的主页:http://primer3.sourceforge.net/。主页上能看到对于Primer3的简要介绍和下载通道。
Primer3的网页版:http://primer3.wi.mit.edu/。
Primer3的源代码:http://sourceforge.net/projects/primer3/.
Primer3文献:Steve 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
2. 安装 Primer3(推荐conda安装简易)
$ tar zxf primer3-src-2.3.6.tar.gz -C /opt/biosoft
$ cd primer3-2.3.6/src/
$ make all
$ make test 此项用于检测 primer3 是否正确安装,确保输出中不能有 FAILED。
$ echo 'PATH=$PATH:/opt/biosoft/primer3-2.3.6/src/' >> ~/.bashrc
$ source ~/.bashrc
3. 使用 Primer3
Primer3 的主程序是 primer3_core
3.1 primer3_core 的常用例子和参数
primer3_core --p3_settings_file=$PWD/config/primer3_settings.txt --output=$PWD/Primer3/query/gene-A1BG.primer.out $PWD/Primer3/query/gene-A1BG.input
# -format_output
让 primer3_core 产生人类易读的结果,否则产生机器易读的结果(Boulder-IO 格式结果)。
# -strict_tags
要求 input file 中的标签要全部正确。设置该参数后,如果有标签不能被程序识别,则报错并停止执行。不设置此参数,则软件忽略不识别的参数。
# -p3_settings_file=file_path
指定 primer_core 的配置文件,该配置文件的设定会取代默认设置。当然,input file 的设置也能取代这个文件的设置。此文件的格式为:第 1 行固定为 "Primer3 File - http://primer3.sourceforge.net"; 第 2 行固定为 "P3_FILE_TYPE=settings"; 第 3 行是个空行;从第 4 行开始则是标准的 Boulder-IO 格式内容。(下文会具体讲解配置文件内容)
# -echo_settings_file
打印出 p3_settings_file 中的设置信息。如果没有指定设置文件,或含有 -format_output,则该参数失效。
# -output=file_path
指定输出文件路径,如果不指定,则输出到标准输出。
3.2 Primer3 的输入和输出
默认设置下,primer3 的输入文件为 Boulder-IO 格式的,该格式有利于程序间的相互交流。同时输出文件的格式也为 Boulder-IO。该文件格式是文本形式的,每一个引物设计的记录的结尾以“=\n”进行分隔。每个记录由多个标签和对应的值构成。例如:
# 输入文件
SEQUENCE_ID=gene-A1BG;rna-NM_130786.4
SEQUENCE_TEMPLATE=ATTGCTGCAGACGCTCACCCCAGACACTCACTGCACCGGAGTGAGCGCGACCATCATGTCCATGCTCGTGGTCTTTCTCTTGCTGTGGGGTGTCACCTGGGGCCCAGTGACAGAAGCAGCCATATTTTATGAGACGCAGCCCAGCCTGTGGGCAGAGTCCGAATCACTGCTGAAACCCTTGGCCAATGTGACGCTGACGTGCCAGGCCCACCTGGAGACTCCAGACTTCCAGCTGTTCAAGAATGGGGTGGCCCAGGAGCCTGTGCACCTTGACTCACCTGCCATCAAGCACCAGTTCCTGCTGACGGGTGACACCCAGGGCCGCTACCGCTGCCGCTCGGGCTTGTCCACAGGATGGACCCAGCTGAGCAAGCTCCTGGAGCTGACAGGGCCAAAGTCCTTGCCTGCTCCCTGGCTCTCGATGGCGCCAGTGTCCTGGATCACCCCCGGCCTGAAAACAACAGCAGTGTGCCGAGGTGTGCTGCGGGGTGTGACTTTTCTGCTGAGGCGGGAGGGCGACCATGAGTTTCTGGAGGTGCCTGAGGCCCAGGAGGATGTGGAGGCCACCTTTCCAGTCCATCAGCCTGGCAACTACAGCTGCAGCTACCGGACCGATGGGGAAGGCGCCCTCTCTGAGCCCAGCGCTACTGTGACCATTGAGGAGCTCGCTGCACCACCACCGCCTGTGCTGATGCACCATGGAGAGTCCTCCCAGGTCCTGCACCCTGGCAACAAGGTGACCCTCACCTGCGTGGCTCCCCTGAGTGGAGTGGACTTCCAGCTACGGCGCGGGGAGAAAGAGCTGCTGGTACCCAGGAGCAGCACCAGCCCAGATCGCATCTTCTTTCACCTGAACGCGGTGGCCCTGGGGGATGGAGGTCACTACACCTGCCGCTACCGGCTGCATGACAACCAAAACGGCTGGTCCGGGGACAGCGCGCCGGTCGAGCTGATTCTGAGCGATGAGACGCTGCCCGCGCCGGAGTTCTCCCCGGAGCCGGAGTCCGGCAGGGCCTTGCGGCTGCGGTGCCTGGCGCCCCTGGAGGGCGCGCGCTTCGCCCTGGTGCGCGAGGACAGGGGCGGGCGCCGCGTGCACCGTTTCCAGAGCCCCGCTGGGACCGAGGCGCTCTTCGAGCTGCACAACATTTCCGTGGCTGACTCCGCCAACTACAGCTGCGTCTACGTGGACCTGAAGCCGCCTTTCGGGGGCTCCGCGCCCAGCGAGCGCTTGGAGCTGCACGTGGACGGACCCCCTCCCAGGCCTCAGCTCCGGGCGACGTGGAGTGGGGCGGTCCTGGCGGGCCGAGATGCCGTCCTGCGCTGCGAGGGACCCATCCCCGACGTCACCTTCGAGCTGCTGCGCGAGGGCGAGACGAAGGCCGTGAAGACGGTCCGCACCCCCGGGGCCGCGGCGAACCTCGAGCTGATCTTCGTGGGGCCCCAGCACGCCGGCAACTACAGGTGCCGCTACCGCTCCTGGGTGCCCCACACCTTCGAATCGGAGCTCAGCGACCCTGTGGAGCTCCTGGTGGCAGAAAGCTGATGCAGCCGCGGGCCCAGGGTGCTGTTGGTGTCCTCAGAAGTGCCGGGGATTCTGGACTGGCTCCCTCCCCTCCTGTTGCAGCACAAGGCCGGGGTCTCTGGGGGGCTGGAGAAGCCTCCCTCATTCCTCCCAGGAATTAATAAATGTGAAGAGAGGCTCTGTTTAAAATGTCTTTGGACTCCCAGGGCTGAGTGGGCTGGGATCTCGTGTCCTCAAAGTGGATGGGTTCTGGGGTGGCTCCTGAGGTAGAGGAGTGGAGAACTGGCTCTTAAGAGCACAGTTTTCTTTTCTTTTTTCTTTTTTGAGACAGGGTCTCACACTGTCACCCAGGCTGGAGTGCAGTGTTGCAGTCCGGCTCACTGCAGTTTTGACCTCCCAGGCTCAAGCGATCCTCCTGCCTCAGCCTCCCAAGTAGCTGGGAGCCTGGGCATGCATCGCCACGTCTGGCTAATTATTATTTTTTGTAGACAGGGTCTCACTGTGTTGCCCAAGCTTGTCTTGAACTCCTGGCCTTAAGTGATCCTCCCACCTCAGCCTCCTGAGTAGCTGGGACTACAGGCATGAGCCACCATGCCTGGCCAACTCACATTTTTCTTTCTATTATTTATTTTTTGTAGAGATGAGTCTCACTATGTTGCCCAGGCTGGTCTTGAACTCTTGGGCTCAAGTGATCCTCCCGCCTCAGCCTCCCAAAGTGTTGGGATTACAGGTGTGAGCCACAGCACCTGGCCAACCAACACTTCTCAGGGCCTCTTTCATCTGTGCTCTTCCAGGATGCTGCCTCTTACTCCCTGGGCACCTCGGCCTGGTCCCAGCAGGTATGGGCAGTTGCTTGAGGCTCCAGACATACTCACCTCTACCTCGACCACATCAACCCCATCACCAAGAGGAGGTTCAGGGAAGCTGCATTTTGTGGTCTTGTCCTCCCAGTCCAGGGTGGTAGTGCTGGGCTGTTCCCAGCCTCCCACAGCAGAGCTGGACGCTGTGGAAATGGCTGGATTCCTCTGTGTTCTTTCCCATTATCCCTCAGCTCTGAGTCCTCTTGTGCCATCTGACATCTGATGCCTTCCCAACCACTGCCGAGTTTCTGCCTGGAGCAGGGCCTCAAGGCCCTGGCACACAGAAGATGCGTATCAGTATTATCAACCAATAGTTGATGAATTGTGTTTTTCAACGAATTTGCTAGTGATCTGGTTTACTGCCTTAGTAATATCTAGTTCCTAATATGCCTATGCCTTTTAATGTCTGCACAGTCTATGATGATGTCACTTCTCTCAAGCCTGATGTGTCCTCTCTCTCTTTCTTGCTAGTGGTTTATCAATTTTTAAAACCTTTTCTTGTTTATTTGTTTTTGAGACAGAATTTTGCTCTTGTTGCCCAGGCTGGAGTGCAATGGCGTGATCTTGGCTCACAGCAACCTCTACCTCCTGGGTTCAAGTGATTCTCGTGCCTCAGCCTCCTGAGTAGCTGGGCTGACAGGCACCCACCACCACACCCAGCTAATTTTTATGTTTTTAGTAGAGATGGGGTTTCACCTTGTTGGGTGGCCAGGCTGGTCTCCAACTCCTGAGCTCAAGTGATCCACCCGCCTCAGCCTCCCAAAGTGCTGGGATTACAGGGGTGAGCCACCACGCCCAGCCCCAGCTTAGTTTTTTAAAAAGTTTATTTTAGCCATTCTAATAGGTATGTAGACATATCTAATAGTGGTTTTCCTTGCGTTTCCTTAATGTCTGATGATGTTAAGCATTTTTTCCCCAAGTGCTTAGTTGCCATCTATATAGCATCTTTGATGAAATGTCTGTTTATATATTTTGCACACTTTAAAATATTGGGTTGTTT
=
# 输出文件
SEQUENCE_ID=gene-A1BG;rna-NM_130786.4
SEQUENCE_TEMPLATE=ATTGCTGCAGACGCTCACCCCAGACACTCACTGCACCGGAGTGAGCGCGACCATCATGTCCATGCTCGTGGTCTTTCTCTTGCTGTGGGGTGTCACCTGGGGCCCAGTGACAGAAGCAGCCATATTTTATGAGACGCAGCCCAGCCTGTGGGCAGAGTCCGAATCACTGCTGAAACCCTTGGCCAATGTGACGCTGACGTGCCAGGCCCACCTGGAGACTCCAGACTTCCAGCTGTTCAAGAATGGGGTGGCCCAGGAGCCTGTGCACCTTGACTCACCTGCCATCAAGCACCAGTTCCTGCTGACGGGTGACACCCAGGGCCGCTACCGCTGCCGCTCGGGCTTGTCCACAGGATGGACCCAGCTGAGCAAGCTCCTGGAGCTGACAGGGCCAAAGTCCTTGCCTGCTCCCTGGCTCTCGATGGCGCCAGTGTCCTGGATCACCCCCGGCCTGAAAACAACAGCAGTGTGCCGAGGTGTGCTGCGGGGTGTGACTTTTCTGCTGAGGCGGGAGGGCGACCATGAGTTTCTGGAGGTGCCTGAGGCCCAGGAGGATGTGGAGGCCACCTTTCCAGTCCATCAGCCTGGCAACTACAGCTGCAGCTACCGGACCGATGGGGAAGGCGCCCTCTCTGAGCCCAGCGCTACTGTGACCATTGAGGAGCTCGCTGCACCACCACCGCCTGTGCTGATGCACCATGGAGAGTCCTCCCAGGTCCTGCACCCTGGCAACAAGGTGACCCTCACCTGCGTGGCTCCCCTGAGTGGAGTGGACTTCCAGCTACGGCGCGGGGAGAAAGAGCTGCTGGTACCCAGGAGCAGCACCAGCCCAGATCGCATCTTCTTTCACCTGAACGCGGTGGCCCTGGGGGATGGAGGTCACTACACCTGCCGCTACCGGCTGCATGACAACCAAAACGGCTGGTCCGGGGACAGCGCGCCGGTCGAGCTGATTCTGAGCGATGAGACGCTGCCCGCGCCGGAGTTCTCCCCGGAGCCGGAGTCCGGCAGGGCCTTGCGGCTGCGGTGCCTGGCGCCCCTGGAGGGCGCGCGCTTCGCCCTGGTGCGCGAGGACAGGGGCGGGCGCCGCGTGCACCGTTTCCAGAGCCCCGCTGGGACCGAGGCGCTCTTCGAGCTGCACAACATTTCCGTGGCTGACTCCGCCAACTACAGCTGCGTCTACGTGGACCTGAAGCCGCCTTTCGGGGGCTCCGCGCCCAGCGAGCGCTTGGAGCTGCACGTGGACGGACCCCCTCCCAGGCCTCAGCTCCGGGCGACGTGGAGTGGGGCGGTCCTGGCGGGCCGAGATGCCGTCCTGCGCTGCGAGGGACCCATCCCCGACGTCACCTTCGAGCTGCTGCGCGAGGGCGAGACGAAGGCCGTGAAGACGGTCCGCACCCCCGGGGCCGCGGCGAACCTCGAGCTGATCTTCGTGGGGCCCCAGCACGCCGGCAACTACAGGTGCCGCTACCGCTCCTGGGTGCCCCACACCTTCGAATCGGAGCTCAGCGACCCTGTGGAGCTCCTGGTGGCAGAAAGCTGATGCAGCCGCGGGCCCAGGGTGCTGTTGGTGTCCTCAGAAGTGCCGGGGATTCTGGACTGGCTCCCTCCCCTCCTGTTGCAGCACAAGGCCGGGGTCTCTGGGGGGCTGGAGAAGCCTCCCTCATTCCTCCCAGGAATTAATAAATGTGAAGAGAGGCTCTGTTTAAAATGTCTTTGGACTCCCAGGGCTGAGTGGGCTGGGATCTCGTGTCCTCAAAGTGGATGGGTTCTGGGGTGGCTCCTGAGGTAGAGGAGTGGAGAACTGGCTCTTAAGAGCACAGTTTTCTTTTCTTTTTTCTTTTTTGAGACAGGGTCTCACACTGTCACCCAGGCTGGAGTGCAGTGTTGCAGTCCGGCTCACTGCAGTTTTGACCTCCCAGGCTCAAGCGATCCTCCTGCCTCAGCCTCCCAAGTAGCTGGGAGCCTGGGCATGCATCGCCACGTCTGGCTAATTATTATTTTTTGTAGACAGGGTCTCACTGTGTTGCCCAAGCTTGTCTTGAACTCCTGGCCTTAAGTGATCCTCCCACCTCAGCCTCCTGAGTAGCTGGGACTACAGGCATGAGCCACCATGCCTGGCCAACTCACATTTTTCTTTCTATTATTTATTTTTTGTAGAGATGAGTCTCACTATGTTGCCCAGGCTGGTCTTGAACTCTTGGGCTCAAGTGATCCTCCCGCCTCAGCCTCCCAAAGTGTTGGGATTACAGGTGTGAGCCACAGCACCTGGCCAACCAACACTTCTCAGGGCCTCTTTCATCTGTGCTCTTCCAGGATGCTGCCTCTTACTCCCTGGGCACCTCGGCCTGGTCCCAGCAGGTATGGGCAGTTGCTTGAGGCTCCAGACATACTCACCTCTACCTCGACCACATCAACCCCATCACCAAGAGGAGGTTCAGGGAAGCTGCATTTTGTGGTCTTGTCCTCCCAGTCCAGGGTGGTAGTGCTGGGCTGTTCCCAGCCTCCCACAGCAGAGCTGGACGCTGTGGAAATGGCTGGATTCCTCTGTGTTCTTTCCCATTATCCCTCAGCTCTGAGTCCTCTTGTGCCATCTGACATCTGATGCCTTCCCAACCACTGCCGAGTTTCTGCCTGGAGCAGGGCCTCAAGGCCCTGGCACACAGAAGATGCGTATCAGTATTATCAACCAATAGTTGATGAATTGTGTTTTTCAACGAATTTGCTAGTGATCTGGTTTACTGCCTTAGTAATATCTAGTTCCTAATATGCCTATGCCTTTTAATGTCTGCACAGTCTATGATGATGTCACTTCTCTCAAGCCTGATGTGTCCTCTCTCTCTTTCTTGCTAGTGGTTTATCAATTTTTAAAACCTTTTCTTGTTTATTTGTTTTTGAGACAGAATTTTGCTCTTGTTGCCCAGGCTGGAGTGCAATGGCGTGATCTTGGCTCACAGCAACCTCTACCTCCTGGGTTCAAGTGATTCTCGTGCCTCAGCCTCCTGAGTAGCTGGGCTGACAGGCACCCACCACCACACCCAGCTAATTTTTATGTTTTTAGTAGAGATGGGGTTTCACCTTGTTGGGTGGCCAGGCTGGTCTCCAACTCCTGAGCTCAAGTGATCCACCCGCCTCAGCCTCCCAAAGTGCTGGGATTACAGGGGTGAGCCACCACGCCCAGCCCCAGCTTAGTTTTTTAAAAAGTTTATTTTAGCCATTCTAATAGGTATGTAGACATATCTAATAGTGGTTTTCCTTGCGTTTCCTTAATGTCTGATGATGTTAAGCATTTTTTCCCCAAGTGCTTAGTTGCCATCTATATAGCATCTTTGATGAAATGTCTGTTTATATATTTTGCACACTTTAAAATATTGGGTTGTTT
PRIMER_LEFT_EXPLAIN=considered 37148, GC content failed 9703, low tm 4898, high tm 17097, high any compl 2, high end compl 24, high hairpin stability 2904, long poly-x seq 918, high template mispriming score 12, ok 1496
PRIMER_RIGHT_EXPLAIN=considered 37285, GC content failed 10222, low tm 5010, high tm 16651, high any compl 1, high end compl 10, high hairpin stability 3187, long poly-x seq 905, high template mispriming score 14, ok 1197
PRIMER_PAIR_EXPLAIN=considered 6913, unacceptable product size 6887, tm diff too large 3, high end compl 5, primer in pair overlaps a primer in a better pair 1558, high template mispriming score 4, ok 14
PRIMER_LEFT_NUM_RETURNED=10
PRIMER_RIGHT_NUM_RETURNED=10
PRIMER_INTERNAL_NUM_RETURNED=0
PRIMER_PAIR_NUM_RETURNED=10
PRIMER_PAIR_0_PENALTY=44.751507
PRIMER_LEFT_0_PENALTY=22.480585
PRIMER_RIGHT_0_PENALTY=22.270922
PRIMER_LEFT_0_SEQUENCE=GGCTCCAGACATACTCACCTCTA
PRIMER_RIGHT_0_SEQUENCE=AACACAGAGGAATCCAGCCATTT
PRIMER_LEFT_0=2380,23
PRIMER_RIGHT_0=2554,23
PRIMER_LEFT_0_TM=60.689
PRIMER_RIGHT_0_TM=60.501
PRIMER_LEFT_0_GC_PERCENT=52.174
PRIMER_RIGHT_0_GC_PERCENT=43.478
PRIMER_LEFT_0_SELF_ANY_TH=0.00
PRIMER_RIGHT_0_SELF_ANY_TH=0.61
PRIMER_LEFT_0_SELF_END_TH=0.00
PRIMER_RIGHT_0_SELF_END_TH=0.00
PRIMER_LEFT_0_HAIRPIN_TH=0.00
PRIMER_RIGHT_0_HAIRPIN_TH=0.00
PRIMER_LEFT_0_END_STABILITY=2.4300
PRIMER_RIGHT_0_END_STABILITY=2.3200
PRIMER_LEFT_0_TEMPLATE_MISPRIMING_TH=26.0714
PRIMER_RIGHT_0_TEMPLATE_MISPRIMING_TH=20.2539
PRIMER_PAIR_0_COMPL_ANY_TH=0.00
PRIMER_PAIR_0_COMPL_END_TH=0.00
PRIMER_PAIR_0_PRODUCT_SIZE=175
PRIMER_PAIR_0_TEMPLATE_MISPRIMING_TH=43.65
PRIMER_PAIR_1_PENALTY=45.151534
PRIMER_LEFT_1_PENALTY=22.983399
PRIMER_RIGHT_1_PENALTY=22.168134
# 省略其它primer结果!!!
4. 常用 primer3 配置文件
Primer3 File - http://primer3.org
P3_FILE_TYPE=settings
PRIMER_FIRST_BASE_INDEX=1
PRIMER_TASK=generic
PRIMER_NUM_RETURN=10 #默认输出10条引物对
P3_FILE_ID=Settings for PCR amplification followed by Sanger sequencing on both strands using PCR primers
PRIMER_MIN_THREE_PRIME_DISTANCE=3
PRIMER_EXPLAIN_FLAG=1
PRIMER_MISPRIMING_LIBRARY=
PRIMER_MAX_LIBRARY_MISPRIMING=12.00
PRIMER_PAIR_MAX_LIBRARY_MISPRIMING=20.00
PRIMER_PRODUCT_SIZE_RANGE=100-250 #扩增产物长度在100-250bp之间
# 引物自身进行 3' 端反向互补形成引物二聚体
PRIMER_MAX_SELF_END=3.00
PRIMER_WT_SELF_END=0.0
PRIMER_MAX_SELF_END_TH=35.00
PRIMER_WT_SELF_END_TH=302.4
# left primer 和 right primer 进行 3' 端反向互补形成引物二聚体
PRIMER_PAIR_MAX_COMPL_END=3.00
PRIMER_PAIR_WT_COMPL_END=0.0
PRIMER_PAIR_MAX_COMPL_END_TH=35.00
PRIMER_PAIR_WT_COMPL_END_TH=302.4
# 引物自身进行反向互补
PRIMER_MAX_SELF_ANY=8.00
PRIMER_WT_SELF_ANY=0.0
PRIMER_MAX_SELF_ANY_TH=45.00
PRIMER_WT_SELF_ANY_TH=123.2
# left primer 和 right primer 序列的反向互补
PRIMER_PAIR_MAX_COMPL_ANY=8.00
PRIMER_PAIR_WT_COMPL_ANY=0.0
PRIMER_PAIR_MAX_COMPL_ANY_TH=45.00
PRIMER_PAIR_WT_COMPL_ANY_TH=123.2
# 发夹结构
PRIMER_MAX_HAIRPIN_TH=24.00
PRIMER_WT_HAIRPIN_TH=672
# 3' 端碱基的稳定性
PRIMER_MAX_END_STABILITY=9.0
PRIMER_WT_END_STABILITY=1
# 模板比对到非引物数据库
PRIMER_MAX_TEMPLATE_MISPRIMING_TH=40.00
PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH=70.00
# 引物长度在17-30碱基之间,Tm值在58-62度之间,上下游引物Tm值相差不超过2℃。
PRIMER_MIN_SIZE=17
PRIMER_OPT_SIZE=23
PRIMER_MAX_SIZE=30
PRIMER_WT_SIZE_LT=1.0 # 引物长度每偏离最优值 1 bp, 则罚 1 分
PRIMER_WT_SIZE_GT=1.0
PRIMER_MIN_TM=58.0
PRIMER_OPT_TM=60.0
PRIMER_MAX_TM=62.0
PRIMER_PAIR_MAX_DIFF_TM=2.0
PRIMER_TM_FORMULA=1
PRIMER_WT_TM_LT=1.0
PRIMER_WT_TM_GT=1.0
### PCR 反应体系设定:
PRIMER_SALT_CORRECTIONS=1
PRIMER_SALT_MONOVALENT=50.0
PRIMER_INTERNAL_SALT_MONOVALENT=50.0
PRIMER_SALT_DIVALENT=1.5
PRIMER_INTERNAL_SALT_DIVALENT=1.5
PRIMER_DNTP_CONC=0.6
PRIMER_INTERNAL_DNTP_CONC=0.0
PRIMER_DNA_CONC=50.0
PRIMER_INTERNAL_DNA_CONC=50.0
### 引物的热力学计算: ###
PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1 # 开启热力学计算
PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT=1
#PRIMER_THERMODYNAMIC_PARAMETERS_PATH=/mnt/nas1/wanghw/software/primer3/primer3-2.4.0/src/primer3_config
PRIMER_LOWERCASE_MASKING=0
PRIMER_MAX_POLY_X=3 # 引物序列中不能包含单核苷酸连续长度超过 3 bp
PRIMER_MAX_NS_ACCEPTED=0
PRIMER_WT_NUM_NS=0.0
PRIMER_MAX_END_GC=4
PRIMER_GC_CLAMP=0
PRIMER_MIN_GC=30.0 #引物GC含量在30%~70%之间
PRIMER_MAX_GC=70.0
PRIMER_WT_GC_PERCENT_LT=0.0
PRIMER_WT_GC_PERCENT_GT=0.0
PRIMER_MAX_NS_ACCEPTED=0
PRIMER_OUTSIDE_PENALTY=0
PRIMER_GC_CLAMP=0
PRIMER_LIBERAL_BASE=1
PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=0
PRIMER_PICK_ANYWAY=1
PRIMER_WT_NUM_NS=0.0
PRIMER_WT_LIBRARY_MISPRIMING=0.0
PRIMER_WT_SEQ_QUAL=0.0
PRIMER_WT_END_QUAL=0.0
PRIMER_WT_POS_PENALTY=0.0
PRIMER_WT_TEMPLATE_MISPRIMING_TH=0.0
PRIMER_PAIR_WT_PRODUCT_SIZE_LT=0.0
PRIMER_PAIR_WT_PRODUCT_SIZE_GT=0.0
PRIMER_PAIR_WT_PRODUCT_TM_LT=0.0
PRIMER_PAIR_WT_PRODUCT_TM_GT=0.0
PRIMER_PAIR_WT_DIFF_TM=0.0
PRIMER_PAIR_WT_COMPL_ANY_TH=0.0
PRIMER_PAIR_WT_COMPL_END_TH=0.0
PRIMER_PAIR_WT_LIBRARY_MISPRIMING=0.0
PRIMER_PAIR_WT_PR_PENALTY=1.0
PRIMER_PAIR_WT_IO_PENALTY=0.0
PRIMER_PAIR_WT_TEMPLATE_MISPRIMING=0.0
=
4.2 我用的配置文件(程序运行把“#”删除,否则会报错)
Primer3 File - http://primer3.org
P3_FILE_TYPE=settings
PRIMER_FIRST_BASE_INDEX=1
PRIMER_TASK=generic
PRIMER_NUM_RETURN=10
P3_FILE_ID=Settings for PCR amplification followed by Sanger sequencing on both strands using PCR primers
PRIMER_MIN_THREE_PRIME_DISTANCE=3
PRIMER_EXPLAIN_FLAG=1
PRIMER_MISPRIMING_LIBRARY=
PRIMER_MAX_LIBRARY_MISPRIMING=12.00
PRIMER_PAIR_MAX_LIBRARY_MISPRIMING=20.00
PRIMER_PRODUCT_SIZE_RANGE=100-250
PRIMER_MAX_SELF_END=3.00
PRIMER_WT_SELF_END=0.0
PRIMER_MAX_SELF_END_TH=35.00
PRIMER_WT_SELF_END_TH=302.4
PRIMER_PAIR_MAX_COMPL_END=3.00
PRIMER_PAIR_WT_COMPL_END=0.0
PRIMER_PAIR_MAX_COMPL_END_TH=35.00
PRIMER_PAIR_WT_COMPL_END_TH=302.4
PRIMER_MAX_SELF_ANY=8.00
PRIMER_WT_SELF_ANY=0.0
PRIMER_MAX_SELF_ANY_TH=45.00
PRIMER_WT_SELF_ANY_TH=123.2
PRIMER_PAIR_MAX_COMPL_ANY=8.00
PRIMER_PAIR_WT_COMPL_ANY=0.0
PRIMER_PAIR_MAX_COMPL_ANY_TH=45.00
PRIMER_PAIR_WT_COMPL_ANY_TH=123.2
PRIMER_MAX_HAIRPIN_TH=24.00
PRIMER_WT_HAIRPIN_TH=672
PRIMER_MAX_END_STABILITY=9.0
PRIMER_WT_END_STABILITY=1
PRIMER_MAX_TEMPLATE_MISPRIMING_TH=40.00
PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH=70.00
PRIMER_MIN_SIZE=17
PRIMER_OPT_SIZE=23
PRIMER_MAX_SIZE=30
PRIMER_WT_SIZE_LT=1.0
PRIMER_WT_SIZE_GT=1.0
PRIMER_MIN_TM=58.0
PRIMER_OPT_TM=60.0
PRIMER_MAX_TM=62.0
PRIMER_PAIR_MAX_DIFF_TM=2.0
PRIMER_TM_FORMULA=1
PRIMER_WT_TM_LT=1.0
PRIMER_WT_TM_GT=1.0
PRIMER_SALT_CORRECTIONS=1
PRIMER_SALT_MONOVALENT=50.0
PRIMER_INTERNAL_SALT_MONOVALENT=50.0
PRIMER_SALT_DIVALENT=1.5
PRIMER_INTERNAL_SALT_DIVALENT=1.5
PRIMER_DNTP_CONC=0.6
PRIMER_INTERNAL_DNTP_CONC=0.0
PRIMER_DNA_CONC=50.0
PRIMER_INTERNAL_DNA_CONC=50.0
PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1
PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT=1
#PRIMER_THERMODYNAMIC_PARAMETERS_PATH=/mnt/nas1/wanghw/software/primer3/primer3-2.4.0/src/primer3_config
PRIMER_LOWERCASE_MASKING=0
PRIMER_MAX_POLY_X=3
PRIMER_MAX_NS_ACCEPTED=0
PRIMER_WT_NUM_NS=0.0
PRIMER_MAX_END_GC=4
PRIMER_GC_CLAMP=0
PRIMER_MIN_GC=30.0
PRIMER_MAX_GC=70.0
PRIMER_WT_GC_PERCENT_LT=0.0
PRIMER_WT_GC_PERCENT_GT=0.0
PRIMER_MAX_NS_ACCEPTED=0
PRIMER_OUTSIDE_PENALTY=0
PRIMER_GC_CLAMP=0
PRIMER_LIBERAL_BASE=1
PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=0
PRIMER_PICK_ANYWAY=1
PRIMER_WT_NUM_NS=0.0
PRIMER_WT_LIBRARY_MISPRIMING=0.0
PRIMER_WT_SEQ_QUAL=0.0
PRIMER_WT_END_QUAL=0.0
PRIMER_WT_POS_PENALTY=0.0
PRIMER_WT_TEMPLATE_MISPRIMING_TH=0.0
PRIMER_PAIR_WT_PRODUCT_SIZE_LT=0.0
PRIMER_PAIR_WT_PRODUCT_SIZE_GT=0.0
PRIMER_PAIR_WT_PRODUCT_TM_LT=0.0
PRIMER_PAIR_WT_PRODUCT_TM_GT=0.0
PRIMER_PAIR_WT_DIFF_TM=0.0
PRIMER_PAIR_WT_COMPL_ANY_TH=0.0
PRIMER_PAIR_WT_COMPL_END_TH=0.0
PRIMER_PAIR_WT_LIBRARY_MISPRIMING=0.0
PRIMER_PAIR_WT_PR_PENALTY=1.0
PRIMER_PAIR_WT_IO_PENALTY=0.0
PRIMER_PAIR_WT_TEMPLATE_MISPRIMING=0.0
=
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