一种基于接头连接的DNAPCR-Free标签文库构建方法 |
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申请号 | CN201010299261.X | 申请日 | 2010-09-21 | 公开(公告)号 | CN101967476A | 公开(公告)日 | 2011-02-09 |
申请人 | 深圳华大基因科技有限公司; 深圳华大基因研究院; | 发明人 | 章文蔚; 田方; 陈海燕; 于竞; 龚梅花; 张艳艳; 周妍; | ||||
摘要 | 本 发明 设计了长度为8bp独特的161个标签序列,并将标签嵌入DNA接头中从而形成DNA PCR-Free标签接头。本发明还提供了通过连接DNA PCR-Free标签接头来导入标签序列,不需要经过PCR反应而成功构建DNA标签文库,并应用于solexa DNA测序。这样将建库方法优化以后,不需要通过PCR反应来构建DNA标签文库。 | ||||||
权利要求 | 1.一组DNA标签,所述DNA标签包括如下或由如下组成:表2所示的161个DNA标签或与之相差1个碱基的DNA标签中的至少10个,或至少20个,或至少30个,或至少40个,至少50个,或至少60个,或至少70个,或至少80个,或90个,或至少100个,或至少110个,或至少120个,或至少130个,或至少140个,或至少150个,或全部161个,所述DNA标签优选地至少包括表2所示的161个DNA标签的DNA Index1~DNA Index10,或 DNA Index11~ DNA Index20,或 DNA Index21 ~ DNA Index30,或 DNA Index31~DNA Index40,或DNA Index41~DNA Index50,或DNA Index51~DNA Index60,或DNA Index61~DNA Index70,或DNA Index71~DNA Index80,或DNA Index81~DNA Index90,或DNA Index91~DNA Index100,或DNA Index101~DNA Index110,或DNA Index111~DNA Index120,或DNA Index121~DNA Index130,或DNA Index131~DNA Index140,或DNA Index141~DNA Index150,或DNA Index151~DNA Index161,或者他们任何两个或多个的组合。 |
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说明书全文 | 一种基于接头连接的DNA PCR-Free标签文库构建方法技术领域[0001] 本发明涉及核酸测序技术领域,特别是基于接头连接的DNA PCR-Free标签文库技术领域。另外,本发明还涉及标签技术,以及实现多个样品在同一反应体系中进行文库构建的方法。本发明的方法特别适用于第二代测序技术,尤其是solexa测序技术。 背景技术[0002] Illumina公司提供的Solexa DNA测序平台,可在一个反应中同时加入四种带荧光标记的核苷酸,采用边合成边测序(Sequencing By Synthesis,SBS),具有所需样品量少,高通量,高精确性,拥有简单易操作的自动化平台和功能强大等特点[1-4]。文库构建首先需要将目的片段进行末端修复,在目的片段的3’末端连接“A”碱基,将3’末端带有“A”碱基的目的片段与DNA接头(也称为adapter)连接,通过PCR反应将目的片段进行扩增,最后回收含有DNA接头的目的片段文库,见图1。含有DNA接头的目的片段文库与测序芯片上面的DNA接头进行杂交,通过桥式PCR进行扩增后,最后边合成边测序。在每个循环过程里,荧光标记的核苷和聚合酶被加入到单分子阵列中。互补的核苷和核苷酸片断的第一个碱基配对,通过酶加入到引物上。多余的核苷被移走。这样每个单链DNA分子通过互补碱基的配对被延伸,针对每种碱基的特定波长的激光激发结合上的核苷的标记,这个标记会释放出荧光,最后收集到的荧光信号来翻译成碱基序列。目前这种DNA建库方法可以根据需求运用于各种研究领域,如基因组的De Novo测序,基因组重测序、转录组测序和表观基因组测序等。 [0003] 基于上述建库方法illumina公司也推出了DNA标签(也称为index)建库方法,如图2所示。在DNA标签建库流程中,PCR过程 使用了3条PCR标签引物,通过PCR导入标签来构建DNA标签文库[5]。专利申请WO2005068656A1和WO2008093098A2中公开了一种使用标签序列标记核酸样品的来源从而可以将样品进行混合测序的方法,可以通过PCR的过程将特定的核苷酸序列(即,标签序列)通过PCR导入到文库中,PCR标签引物序列见表1。这些带有标签的文库可以根据需求进行任意混合,然后通过solexa测序仪器进行测序,最后将数据按标签标签序列进行分类。 [0004] 但是illumina公司提供的标签文库制备的方法存在着一些缺陷:第一、目前illumina公司只提供12个长度为6bp的标签序列,标签的数量较少,随着solexa测序通量的增加,不能对大量样本进行混合测序将是一个巨大的缺陷;第二、目前illumina公司提供的标签建库方法是通过PCR反应将标签序列导入到目的片段文库中,需要3条PCR引物对目的片段进行扩增(两条公用引物和一条PCR标签引物,如表1),而且PCR扩增效率不高。第三、illumina公司提供的标签建库方法中接头不包含标签序列,每一个标签文库需要通过一个PCR反应来导入标签序列,然后针对每一个标签文库都需要切胶回收,将切胶回收后的标签目的片段文库随后进行混合,这样不仅费时费力,而且费用也较高。 [0005] 因此,对标签的序列及标签引入方法进行优化和改进,使标签引入的效率提高,扩大标签序列的数量,才能满足高通量的建库的要求,以适应测序通量不断提高的现状,使测序仪器的产能充分利用,降低测序的成本。 [0006] 表1 illumina公司提供的标签序列及相对应的PCR标签引物序列 [0007] [0008] 发明内容[0009] 鉴于目前illumina公司的solexa测序平台提供的DNA标签文库制备方法的不足,本发明改良了DNA标签文库的制备方法,通过连接DNA PCR-Free标签接头来导入标签序列,不需要经过PCR反应便可成功的构建DNA标签文库,并应用于solexa DNA测序。 [0010] 通过测试含标签序列的DNA PCR-Free标签接头的连接效率和标签核酸序列的识别率,优化并筛选出161条长度为8bp的DNA标签序列(如表2,8bp的DNA标签序列)及DNA PCR-Free标签接头序列。 [0011] 这些长度为8bp的标签之间的差异在4个碱基,即至少4个碱基序列不同。当标签的8个碱基中的任意一个碱基出现测序错误或合成错误,都不影响到标签的最终识别。 [0012] 基于目前illumina公司的solexa测序平台提供的DNA标签文库制备方法,本发明改良了标签序列,分别设计了长度为8bp独特的标签序列,将标签嵌入DNA接头中,可以分别本发明设计的161个DNAPCR-Free标签接头用于构建标签文库的方法,不仅能提高了DNA标 签文库的制备的效率,增大了DNA样品的测序通量,也能提高标签的识别率,极大的降低了单个样品的solexa测序费用。 [0013] 标签设计首先需要考虑标签序列之间的可识别性和识别率的问题,然后需要考虑标签序列混合之后的每个位点的GT与AC碱基含量的平衡问题,最后考虑数据产出的可重复性和准确性。在设计标签的过程中,本发明充分考虑到以上几个因素,同时避免了标签的核酸序列出现3或3个以上连续的碱基,这样可以降低序列在合成过程中或测序过程中的错误率。同时尽量避免标签引物接头自身形成发夹结构,提高DNA标签接头的连接效率。 [0014] 本发明对illumina提供的DNA接头序列进行优化,将标签设计为长度为8bp的特定序列,通过含有所述标签序列的DNA PCR-Free标签接头的连接将标签序列导入目的文库中。这样将建库方法优化以后,不需要通过PCR反应来构建DNA标签文库。目前为止,通过这些DNA PCR-Free标签接头导入标签的建库方法及标签序列,并没有相关的报道。如图3所示为illumina公司的DNA标签建库流程图,图4为优化后基于接头连接的DNA PCR-Free标签文库构建方法实验流程图。 [0015] 表2长度为8bp的DNA标签序列(DNA Index-1)及PCR-Free标签序列(PCR-Free Index-N),PCR-Free Index-N与DNA PCR-Free接头1.0等摩尔退火后形成PCR-Free标签接头。退火条件为:DNA PCR-Free接头1.0与PCR-Free Index-N等摩尔量混合后,95℃10min;70℃10min;65℃10min;60℃10min;55℃10min;50℃10min;4℃∞。升温至95℃后,所有降温速度控制为每秒0.1℃缓慢降温,让两条序列结合在一起形成Y型结构的PCR-Free标签接头。 [0016] [0017] [0018] [0019] [0020] [0021] [0022] [0023] [0024] [0025] [0026] [0027] [0028] 使用Lasergene的PrimerSelect软件预测DNA PCR-Free标签接头形成的最稳定“Y型”DNA PCR-Free标签接头(the most stable dimer overrall)及能量值。 [0029] DNA PCR-Free index1接头 [0030] The most stable dimer overall:12bp,-22.8kcal/mol [0031] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCATTGCTTATCTCGTATGCCGTCTTCTGCTTG 3′ [0032] |||||||||||| : ::: :: : ::: : [0033] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0034] DNA PCR-Free index2接头 [0035] The most stable dimer overall:12bp,-22.8kcal/mol [0036] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTCGGATTATCTCGTATGCCGTCTTCTGCTTG 3′ [0037] |||||||||||| : ::: ::: : [0038] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0039] DNA PCR-Free index3接头 [0040] The most stable dimer overall:12bp,-22.8kcal/mol [0041] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCATCATTATCTCGTATGCCGTCTTCTGCTTG 3′ [0042] |||||||||||| : ::: ::: : [0043] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0044] DNA PCR-Free index4接头 [0045] The most stable dimer overall:12bp,-22.8kcal/mol [0046] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCTCCTGTATCTCGTATGCCGTCTTCTGCTTG 3′ [0047] |||||||||||| : ::: : :: : ::: : [0048] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0049] DNA PCR-Free index5接头 [0050] The most stable dimer overall:12bp,-22.8kcal/mol [0051] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGCTCGGTATCTCGTATGCCGTCTTCTGCTTG 3′ [0052] |||||||||||| : ::: : ::: : [0053] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0054] DNA PCR-Free index6接头 [0055] The most stable dimer overall:12bp,-22.8kcal/mol [0056] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAACAGGTATCTCGTATGCCGTCTTCTGCTTG 3′ [0057] |||||||||||| : ::: : : : ::: : [0058] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0059] DNA PCR-Free index7接头 [0060] The most stable dimer overall:12bp,-22.8kcal/mol [0061] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTCAAGGTATCTCGTATGCCGTCTTCTGCTTG 3′ [0062] |||||||||||| : ::: : ::: : [0063] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0064] DNA PCR-Free index8接头 [0065] The most stable dimer overall:12bp,-22.8kcal/mol [0066] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCTAACGTATCTCGTATGCCGTCTTCTGCTTG 3′ [0067] |||||||||||| : ::: : :: ::: : [0068] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0069] DNA PCR-Free index9接头 [0070] The most stable dimer overall:12bp,-22.8kcal/mol [0071] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACGTAGTATCTCGTATGCCGTCTTCTGCTTG 3′ [0072] |||||||||||| : ::: : : : ::: : [0073] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0074] DNA PCR-Free index10接头 [0075] The most stable dimer overall:12bp,-22.8kcal/mol [0076] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTAAGAGTATCTCGTATGCCGTCTTCTGCTTG 3′ [0077] |||||||||||| : ::: : : ::: : [0078] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0079] DNA PCR-Free index11接头 [0080] The most stable dimer overall:12bp,-22.8kcal/mol [0081] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTACCTTCTATCTCGTATGCCGTCTTCTGCTTG 3′ [0082] |||||||||||| : ::: : :: ::: : [0083] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0084] DNA PCR-Free index12接头 [0085] The most stable dimer overall:12bp,-22.8kcal/mol [0086] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGTCTCTATCTCGTATGCCGTCTTCTGCTTG 3′ [0087] |||||||||||| : ::: : ::: : [0088] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0089] DNA PCR-Free index13接头 [0090] The most stable dimer overall:12bp,-22.8kcal/mol [0091] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGAGATCTATCTCGTATGCCGTCTTCTGCTTG 3′ [0092] |||||||||||| : ::: ::: : [0093] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0094] DNA PCR-Free index14接头 [0095] The most stable dimer overall:12bp,-22.8kcal/mol [0096] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCAGCGCTATCTCGTATGCCGTCTTCTGCTTG 3′ [0097] |||||||||||| : ::: ::: : [0098] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0099] DNA PCR-Free index15接头 [0100] The most stable dimer overall:12bp,-22.8kcal/mol [0101] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACATGAACCTATCTCGTATGCCGTCTTCTGCTTG 3′ [0102] |||||||||||| : ::: : ::: : [0103] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0104] DNA PCR-Free index16接头 [0105] The most stable dimer overall:12bp,-22.8kcal/mol [0106] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACACCAGACTATCTCGTATGCCGTCTTCTGCTTG 3′ [0107] |||||||||||| : ::: ::: : [0108] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0109] DNA PCR-Free index17接头 [0110] The most stable dimer overall:12bp,-22.8kcal/mol [0111] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTATAACTATCTCGTATGCCGTCTTCTGCTTG 3′ [0112] |||||||||||| : ::: ::: : [0113] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0114] DNA PCR-Free index18接头 [0115] The most stable dimer overall:12bp,-22.8kcal/mol [0116] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCGGAAGTATCTCGTATGCCGTCTTCTGCTTG 3′ [0117] |||||||||||| : ::: : ::: : [0118] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0119] DNA PCR-Free index19接头 [0120] The most stable dimer overall:12bp,-22.8kcal/mol [0121] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTAGTTATATCTCGTATGCCGTCTTCTGCTTG 3′ [0122] |||||||||||| : ::: : ::: : [0123] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0124] DNA PCR-Free index20接头 [0125] The most stable dimar overall:12bp,-22.8kcal/mol [0126] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCTTATATATCTCGTATGCCGTCTTCTGCTTG 3′ [0127] |||||||||||| : ::: : ::: : [0128] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0129] DNA PCR-Free index21接头 [0130] The most stable dimer overall:12bp,-22.8kcal/mol [0131] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAATCGATATCTCGTATGCCGTCTTCTGCTTG 3′ [0132] |||||||||||| : ::: :: ::: : [0133] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0134] DNA PCR-Free index22接头 [0135] The most stable dimer overall:12bp,-22.8kcal/mol [0136] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAATAAGATATCTCGTATGCCGTCTTCTGCTTG 3′ [0137] |||||||||||| : ::: :: ::: : [0138] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0139] DNA PCR-Free index23接头 [0140] The most stable dimer overall:12bp,-22.8kcal/mol [0141] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTATGCCATATCTCGTATGCCGTCTTCTGCTTG 3′ [0142] |||||||||||| : ::: :: : ::: : [0143] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0144] DNA PCR-Free index24接头 [0145] The most stable dimer overall:12bp,-22.8kcal/mol [0146] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACATTCTAATATCTCGTATGCCGTCTTCTGCTTG 3′ [0147] |||||||||||| : ::: ::: ::: : [0148] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0149] DNA PCR-Free index25接头 [0150] The most stable dimer overall:12bp,-22.8kcal/mol [0151] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAATGTTGATCTCGTATGCCGTCTTCTGCTTG 3′ [0152] |||||||||||| : ::: : : ::: : [0153] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0154] DNA PCR-Free index26接头 [0155] The most stable dimer overall:12bp,-22.8kcal/mol [0156] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTACTTGATCTCGTATGCCGTCTTCTGCTTG 3′ [0157] |||||||||||| : ::: : : : ::: : [0158] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0159] DNA PCR-Free index27接头 [0160] The most stable dimer overall:12bp,-22.8kcal/mol [0161] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACATTCACTGATCTCGTATGCCGTCTTCTGCTTG 3′ [0162] |||||||||||| : ::: :: : : ::: : [0163] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0164] DNA PCR-Free index28接头 [0165] The most stable dimer overall:12bp,-22.8kcal/mol [0166] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCATATGATCTCGTATGCCGTCTTCTGCTTG 3′ [0167] |||||||||||| : ::: : : ::: : [0168] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0169] DNA PCR-Free index29接头 [0170] The most stable dimer overall:12bp,-22.8kcal/mol [0171] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCTTAATGATCTCGTATGCCGTCTTCTGCTTG 3′ [0172] |||||||||||| : ::: : : : ::: : [0173] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0174] DNA PCR-Free index30接头 [0175] The most stable dimer overall:12bp,-22.8kcal/mol [0176] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGATATGGATCTCGTATGCCGTCTTCTGCTTG 3′ [0177] |||||||||||| : ::: : :: ::: : [0178] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0179] DNA PCR-Free index31接头 [0180] The most stable dimer overall:12bp,-22.8kcal/mol [0181] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGATGGATCTCGTATGCCGTCTTCTGCTTG 3′ [0182] |||||||||||| : ::: : :: ::: : [0183] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0184] DNA PCR-Free index32接头 [0185] The most stable dimer overall:12bp,-22.8kcal/mol [0186] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGATCGGATCTCGTATGCCGTCTTCTGCTTG 3′ [0187] |||||||||||| : ::: : :::: ::: : [0188] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0189] DNA PCR-Free index33接头 [0190] The most stable dimer overall:12bp,-22.8kcal/mol [0191] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAACCGGATCTCGTATGCCGTCTTCTGCTTG 3′ [0192] |||||||||||| : ::: ::: ::: : [0193] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0194] DNA PCR-Free index34接头 [0195] The most stable dimer overall:12bp,-22.8kcal/mol [0196] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTAAGTCGATCTCGTATGCCGTCTTCTGCTTG 3′ [0197] |||||||||||| : ::: : ::: : [0198] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0199] DNA PCR-Free index35接头 [0200] The most stable dimer overall:12bp,-22.8kcal/mol [0201] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTATTCGCGATCTCGTATGCCGTCTTCTGCTTG 3′ [0202] |||||||||||| : ::: :: : ::: : [0203] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0204] DNA PCR-Free index36接头 [0205] The most stable dimer overall:12bp,-22.8kcal/mol [0206] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAAGCACGATCTCGTATGCCGTCTTCTGCTTG 3′ [0207] |||||||||||| : ::: :: : ::: : [0208] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0209] DNA PCR-Free index37接头 [0210] The most stable dimer overall:12bp,-22.8kcal/mol [0211] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCCAGTAGATCTCGTATGCCGTCTTCTGCTTG 3′ [0212] |||||||||||| : ::: : ::: : [0213] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0214] DNA PCR-Free index38接头 [0215] The most stable dimer overall:12bp,-22.8kcal/mol [0216] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGTCTAGATCTCGTATGCCGTCTTCTGCTTG 3′ [0217] |||||||||||| : ::: : ::: : [0218] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0219] DNA PCR-Free index39接头 [0220] The most stable dimer overall:12bp,-22.8kcal/mol [0221] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGCGCTAGATCTCGTATGCCGTCTTCTGCTTG 3′ [0222] |||||||||||| : ::: : ::: : [0223] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0224] DNA PCR-Free index40接头 [0225] The most stable dimer overall:12bp,-22.8kcal/mol [0226] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCTGTGAGATCTCGTATGCCGTCTTCTGCTTG 3′ [0227] |||||||||||| : ::: : : : ::: : [0228] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0229] DNA PCR-Free index41接头 [0230] The most stable dimer overall:12bp,-22.8kcal/mol [0231] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAACTAAGATCTCGTATGCCGTCTTCTGCTTG 3′ [0232] |||||||||||| : ::: : :: : ::: : [0233] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0234] DNA PCR-Free index42接头 [0235] The most stable dimer overall:12bp,-22.8kcal/mol [0236] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACATAGGAAGATCTCGTATGCCGTCTTCTGCTTG 3′ [0237] |||||||||||| : ::: : ::: : [0238] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0239] DNA PCR-Free index43接头 [0240] The most stable dimer overall:12bp,-22.8kcal/mol [0241] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTACAAGATCTCGTATGCCGTCTTCTGCTTG 3′ [0242] |||||||||||| : ::: : : ::: : [0243] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0244] DNA PCR-Free in dex44接头 [0245] The most stable dimer overall:12bp,-22.8kcal/mol [0246] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATGGTTCATCTCGTATGCCGTCTTCTGCTTG 3′ [0247] |||||||||||| : ::: ::: ::: : [0248] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0249] DNA PCR-Free index45接头 [0250] The most stable dimer overall:12bp,-22.8kcal/mol [0251] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCACATTCATCTCGTATGCCGTCTTCTGCTTG 3′ [0252] |||||||||||| : ::: : ::: : [0253] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0254] DNA PCR-Free index46接头 [0255] The most stable dimer overall:12bp,-22.8kcal/mol [0256] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCTTGGTCATCTCGTATGCCGTCTTCTGCTTG 3′ [0257] |||||||||||| : ::: : ::: : [0258] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0259] DNA PCR-Free index47接头 [0260] The most stable dimer overall:12bp,-22.8kcal/mol [0261] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGAGGATCATCTCGTATGCCGTCTTCTGCTTG 3′ [0262] |||||||||||| : ::: ::: : [0263] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0264] DNA PCR-Free index48接头 [0265] The most stable dimer overall:12bp,-22.8kcal/mol [0266] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTCCATCATCTCGTATGCCGTCTTCTGCTTG 3′ [0267] |||||||||||| : ::: :: ::: : [0268] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0269] DNA PCR-Free index49接头 [0270] The most stable dimer overall:12bp,-22.8kcal/mol [0271] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACTAATCATCTCGTATGCCGTCTTCTGCTTG 3′ [0272] |||||||||||| : ::: : ::: : [0273] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0274] DNA PCR-Free index50接头 [0275] The most stable dimer overall:12bp,-22.8kcal/mol [0276] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAAGGCGCATCTCGTATGCCGTCTTCTGCTTG 3′ [0277] |||||||||||| : ::: : :: ::: : [0278] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0279] DNA PCR-Free index51接头 [0280] The most stable dimer overall:12bp,-22.8kcal/mol [0281] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAATAGAGCATCTCGTATGCCGTCTTCTGCTTG 3′ [0282] |||||||||||| : ::: :: : ::: : [0283] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0284] DNA PCR-Free index52接头 [0285] The most stable dimer overall:12bp,-22.8kcal/mol [0286] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTGTTCCATCTCGTATGCCGTCTTCTGCTTG 3′ [0287] |||||||||||| : ::: : : ::: : [0288] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0289] DNA PCR-Free index53接头 [0290] The most stable dimer overall:12bp,-22.8kcal/mol [0291] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTCCTCCATCTCGTATGCCGTCTTCTGCTTG 3′ [0292] |||||||||||| : ::: :: ::: : [0293] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0294] DNA PCR-Free index54接头 [0295] The most stable dimer overall:12bp,-22.8kcal/mol [0296] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCGACTCCATCTCGTATGCCGTCTTCTGCTTG 3′ [0297] |||||||||||| : ::: : ::: : [0298] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0299] DNA PCR-Free index55接头 [0300] The most stable dimer overall:12bp,-22.8kcal/mol [0301] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTACAGGCCATCTCGTATGCCGTCTTCTGCTTG 3′ [0302] |||||||||||| : ::: : ::: : [0303] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0304] DNA PCR-Free index56接头 [0305] The most stable dimer overall:12bp,-22.8kcal/mol [0306] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTAAGCCATCTCGTATGCCGTCTTCTGCTTG 3′ [0307] |||||||||||| : ::: : : ::: : [0308] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0309] DNA PCR-Free index57接头 [0310] The most stable dimer overall:12bp,-22.8kcal/mol [0311] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAATTACCATCTCGTATGCCGTCTTCTGCTTG 3′ [0312] |||||||||||| : ::: : : ::: : [0313] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0314] DNA PCR-Free index58接头 [0315] The most stable dimer overall:12bp,-22.8kcal/mol [0316] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACATAACACCATCTCGTATGCCGTCTTCTGCTTG 3′ [0317] |||||||||||| : ::: ::: : [0318] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0319] DNA PCR-Free index59接头 [0320] The most stable dimer overall:12bp,-22.8kcal/mol [0321] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGTAGGACATCTCGTATGCCGTCTTCTGCTTG 3′ [0322] |||||||||||| : ::: : ::: : [0323] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0324] DNA PCR-Free index60接头 [0325] The most stable dimer overall:12bp,-22.8kcal/mol [0326] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGTAGGACATCTCGTATGCCGTCTTCTGCTTG 3′ [0327] |||||||||||| : ::: : ::: : [0328] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0329] DNA PCR-Free index61接头 [0330] The most stable dimer overall:12bp,-22.8kcal/mol [0331] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTCTCGACATCTCGTATGCCGTCTTCTGCTTG 3′ [0332] |||||||||||| : ::: ::: : [0333] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0334] DNA PCR-Free index62接头 [0335] The most stable dimer overall:12bp,-22.8kcal/mol [0336] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGGTTAACATCTCGTATGCCGTCTTCTGCTTG 3′ [0337] |||||||||||| : ::: : ::: : [0338] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0339] DNA PCR-Free index63接头 [0340] The most stable dimer overall:12bp,-22.8kcal/mol [0341] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTGCAACATCTCGTATGCCGTCTTCTGCTTG 3′ [0342] |||||||||||| : ::: : : ::: : [0343] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0344] DNA PCR-Free index64接头 [0345] The most stable dimer overall:12bp,-22.8kcal/mol [0346] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTCAATTAATCTCGTATGCCGTCTTCTGCTTG 3′ [0347] |||||||||||| : ::: ::: : [0348] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0349] DNA PCR-Free index65接头 [0350] The most stable dimer overall:12bp,-22.8kcal/mol [0351] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAAGTCTAATCTCGTATGCCGTCTTCTGCTTG 3′ [0352] |||||||||||| : ::: : :: ::: : [0353] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0354] DNA PCR-Free index66接头 [0355] The most stable dimer overall:12bp,-22.8kcal/mol [0356] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAACCTAATCTCGTATGCCGTCTTCTGCTTG 3′ [0357] |||||||||||| : ::: : ::: : [0358] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0359] DNA PCR-Free index67接头 [0360] The most stable dimer overall:12bp,-22.8kcal/mol [0361] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTACCATAATCTCGTATGCCGTCTTCTGCTTG 3′ [0362] |||||||||||| : ::: : ::: : [0363] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0364] DNA PCR-Free index68接头 [0365] The most stable dimer overall:12bp,-22.8kcal/mol [0366] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGACACATAATCTCGTATGCCGTCTTCTGCTTG 3′ [0367] |||||||||||| : ::: :: ::: : [0368] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0369] DNA PCR-Free index69接头 [0370] The most stable dimer overall:12bp,-22.8kcal/mol [0371] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGATAATAATCTCGTATGCCGTCTTCTGCTTG 3′ [0372] |||||||||||| : ::: ::: : [0373] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0374] DNA PCR-Free index70接头 [0375] The most stable dimer overall:12bp,-22.8kcal/mol [0376] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGCGGTGAATCTCGTATGCCGTCTTCTGCTTG 3′ [0377] |||||||||||| : ::: : ::: : [0378] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0379] DNA PCR-Free index71接头 [0380] The most stable dimer overall:12bp,-22.8kcal/mol [0381] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTACTATGAATCTCGTATGCCGTCTTCTGCTTG 3′ [0382] |||||||||||| : ::: : : ::: : [0383] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0384] DNA PCR-Free index72接头 [0385] The most stable dimer overall:12bp,-22.8kcal/mol [0386] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGTTGGAATCTCGTATGCCGTCTTCTGCTTG 3′ [0387] |||||||||||| : ::: : : ::: : [0388] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0389] DNA PCR-Free index73接头 [0390] The most stable dimer overall:12bp,-22.8kcal/mol [0391] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTGAGGAATCTCGTATGCCGTCTTCTGCTTG 3′ [0392] |||||||||||| : ::: : : ::: : [0393] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0394] DNA PCR-Free index74接头 [0395] The most stable dimer overall:12bp,-22.8kcal/mol [0396] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCGCCGAATCTCGTATGCCGTCTTCTGCTTG 3′ [0397] |||||||||||| : ::: :: ::: : [0398] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0399] DNA PCR-Free index75接头 [0400] The most stable dimer overall:12bp,-22.8kcal/mol [0401] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTATAGAATCTCGTATGCCGTCTTCTGCTTG 3′ [0402] |||||||||||| : ::: : : : ::: : [0403] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0404] DNA PCR-Free ind ex76接头 [0405] The most stable dimer overall:12bp,-22.8kcal/mol [0406] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCATGAGAATCTCGTATGCCGTCTTCTGCTTG 3′ [0407] |||||||||||| : ::: : ::: : [0408] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0409] DNA PCR-Free index77接头 [0410] The most stable dimer overall:12bp,-22.8kcal/mol [0411] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCACCTCAATCTCGTATGCCGTCTTCTGCTTG 3′ [0412] |||||||||||| : ::: : ::: : [0413] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0414] DNA PCR-Free index78接头 [0415] The most stable dimer overall:12bp,-22.8kcal/mol [0416] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACACCTTGCAATCTCGTATGCCGTCTTCTGCTTG 3′ [0417] |||||||||||| : ::: : ::: : [0418] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0419] DNA PCR-Free index79接头 [0420] The most stable dimer overall:12bp,-22.8kcal/mol [0421] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACATACTCCAATCTCGTATGCCGTCTTCTGCTTG 3′ [0422] |||||||||||| : ::: ::: ::: : [0423] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0424] DNA PCR-Free index80接头 [0425] The most stable dimer overall:12bp,-22.8kcal/mol [0426] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTCGACAATCTCGTATGCCGTCTTCTGCTTG 3′ [0427] |||||||||||| : ::: : :: ::: : [0428] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0429] DNA PCR-Free index81接头 [0430] The most stable dimer overall:12bp,-22.8kcal/mol [0431] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCATCATAAATCTCGTATGCCGTCTTCTGCTTG 3′ [0432] |||||||||||| : ::: ::: ::: : [0433] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0434] DNA PCR-Free index82接头 [0435] The most stable dimer overall:12bp,-22.8kcal/mol [0436] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACATGAAATCTCGTATGCCGTCTTCTGCTTG 3′ [0437] |||||||||||| : ::: : : ::: : [0438] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0439] DNA PCR-Free index83接头 [0440] The most stable dimer overall:12bp,-22.8kcal/mol [0441] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACATGAGGAAATCTCGTATGCCGTCTTCTGCTTG 3′ [0442] |||||||||||| : ::: ::: : [0443] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0444] DNA PCR-Free index84接头 [0445] The most stable dimer overall:12bp,-22.8kcal/mol [0446] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCCTCCAAATCTCGTATGCCGTCTTCTGCTTG 3′ [0447] |||||||||||| : ::: : ::: : [0448] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0449] DNA PCR-Free index85接头 [0450] The most stable dimer overall:12bp,-22.8kcal/mol [0451] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGACAAATCTCGTATGCCGTCTTCTGCTTG 3′ [0452] |||||||||||| : ::: : ::: : [0453] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0454] DNA PCR-Free index86接头 [0455] The most stable dimer overall:12bp,-22.8kcal/mol [0456] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTCCAGAAATCTCGTATGCCGTCTTCTGCTTG 3′ [0457] |||||||||||| : ::: : : ::: : [0458] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0459] DNA PCR-Free index87接头 [0460] The most stable dimer overall:12bp,-22.8kcal/mol [0461] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCTATCGATCTCGTATGCCGTCTTCTGCTTG 3′ [0462] |||||||||||| : ::: : ::: : [0463] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0464] DNA PCR-Free index88接头 [0465] The most stable dimer overall:12bp,-22.8kcal/mol [0466] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTACTGTTATCTCGTATGCCGTCTTCTGCTTG 3′ [0467] |||||||||||| : ::: :: ::: : [0468] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0469] DNA PCR-Free index89接头 [0470] The most stable dimer overall:12bp,-22.8kcal/mol [0471] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACACACGCGGATCTCGTATGCCGTCTTCTGCTTG 3′ [0472] |||||||||||| : ::: : ::: ::: : [0473] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0474] DNA PCR-Free index90接头 [0475] The most stable dimer overall:12bp,-22.8kcal/mol [0476] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTATCCAGAATCTCGTATGCCGTCTTCTGCTTG 3′ [0477] |||||||||||| : ::: ::: : ::: : [0478] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0479] DNA PCR-Free index91接头 [0480] The most stable dimer overall:12bp,-22.8kcal/mol [0481] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAGGAATAATCTCGTATGCCGTCTTCTGCTTG 3′ [0482] |||||||||||| : ::: : ::: : [0483] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0484] DNA PCR-Free index92接头 [0485] The most stable dimer overall:12bp,-22.8kcal/mol [0486] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAACGTGAATCTCGTATGCCGTCTTCTGCTTG 3′ [0487] |||||||||||| : ::: :: : : ::: : [0488] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0489] DNA PCR-Free index93接头 [0490] The most stable dimer overall:12bp,-22.8kcal/mol [0491] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGCACAGATCTCGTATGCCGTCTTCTGCTTG 3′ [0492] |||||||||||| : ::: : : : ::: : [0493] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0494] DNA PCR-Free index94接头 [0495] The most stable dimer overall:12bp,-22.8kcal/mol [0496] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACATTGCGTTATCTCGTATGCCGTCTTCTGCTTG 3′ [0497] |||||||||||| : ::: : ::: : [0498] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0499] DNA PCR-Free index95接头 [0500] The most stable dimer overall:12bp,-22.8kcal/mol [0501] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCGTAAGCATCTCGTATGCCGTCTTCTGCTTG 3′ [0502] |||||||||||| : ::: : ::: : [0503] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0504] DNA PCR-Free index96接头 [0505] The most stable dimer overall:12bp,-22.8kcal/mol [0506] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGTCACGATCTCGTATGCCGTCTTCTGCTTG 3′ [0507] |||||||||||| : ::: : ::: : [0508] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0509] DNA PCR-Free index97接头 [0510] The most stable dimer overall:12bp,-22.8kcal/mol [0511] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCGAAGTAATCTCGTATGCCGTCTTCTGCTTG 3′ [0512] |||||||||||| : ::: : ::: : [0513] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0514] DNA PCR-Free index98接头 [0515] The most stable dimer overall:12bp,-22.8kcal/mol [0516] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGACTGCGATCTCGTATGCCGTCTTCTGCTTG 3′ [0517] |||||||||||| : ::: : :: : ::: : [0518] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0519] DNA PCR-Free index99接头 [0520] The most stable dimer overall:12bp,-22.8kcal/mol [0521] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAGCATTGATCTCGTATGCCGTCTTCTGCTTG 3′ [0522] |||||||||||| : ::: :: : : ::: : [0523] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0524] DNA PCR-Free index100接头 [0525] The most stable dimer overall:12bp,-22.8kcal/mol [0526] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCGCCGTGATCTCGTATGCCGTCTTCTGCTTG 3′ [0527] |||||||||||| : ::: : : ::: : [0528] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0529] DNA PCR-Free index101接头 [0530] The most stable dimer overall:12bp,-22.8kcal/mol [0531] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGCGGCGATCTCGTATGCCGTCTTCTGCTTG 3′ [0532] |||||||||||| : ::: : : : ::: : [0533] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0534] DNA PCR-Free index102接头 [0535] The most stable dimer overall:12bp,-22.8kcal/mol [0536] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGGATGCATCTCGTATGCCGTCTTCTGCTTG 3′ [0537] |||||||||||| : ::: : : ::: : [0538] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0539] DNA PCR-Free index103接头 [0540] The most stable dimer overall:12bp,-22.8kcal/mol [0541] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCAATGGCATCTCGTATGCCGTCTTCTGCTTG 3′ [0542] |||||||||||| : ::: : : : ::: : [0543] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0544] DNA PCR-Free index104接头 [0545] The most stable dimer overall:12bp,-22.8kcal/mol [0546] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTATTCTCATCTCGTATGCCGTCTTCTGCTTG 3′ [0547] |||||||||||| : ::: : :: ::: : [0548] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0549] DNA PCR-Free index105接头 [0550] The most stable dimer overall:12bp,-22.8kcal/mol [0551] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTCATTACATCTCGTATGCCGTCTTCTGCTTG 3′ [0552] |||||||||||| : ::: : : ::: : [0553] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0554] DNA PCR-Free index106接头 [0555] The most stable dimer overall:12bp,-22.8kcal/mol [0556] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCCAAGCATCTCGTATGCCGTCTTCTGCTTG 3′ [0557] |||||||||||| : ::: : : ::: : [0558] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0559] DNA PCR-Free index107接头 [0560] The most stable dimer overall:12bp,-22.8kcal/mol [0561] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGTATACTATCTCGTATGCCGTCTTCTGCTTG 3′ [0562] |||||||||||| : ::: : : : ::: : [0563] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0564] DNA PCR-Free index108接头 [0565] The most stable dimer overall:12bp,-22.8kcal/mol [0566] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGCGTGCATCTCGTATGCCGTCTTCTGCTTG 3′ [0567] |||||||||||| : ::: : : ::: : [0568] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0569] DNA PCR-Free index109接头 [0570] The most stable dimer overall:12bp,-22.8kcal/mol [0571] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCCGACGGATCTCGTATGCCGTCTTCTGCTTG 3′ [0572] |||||||||||| : ::: ::: ::: : [0573] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0574] DNA PCR-Free index110接头 [0575] The most stable dimer overall:12bp,-22.8kcal/mol [0576] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCAGGCATATCTCGTATGCCGTCTTCTGCTTG 3′ [0577] |||||||||||| : ::: : : ::: : [0578] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0579] DNA PCR-Free index111接头 [0580] The most stable dimer overall:12bp,-22.8kcal/mol [0581] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAGCGAATCTCGTATGCCGTCTTCTGCTTG 3′ [0582] |||||||||||| : ::: : :: ::: : [0583] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0584] DNA PCR-Free index112接头 [0585] The most stable dimer overall:12bp,-22.8kcal/mol [0586] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACACTGGATCTCGTATGCCGTCTTCTGCTTG 3′ [0587] |||||||||||| : ::: : :: ::: : [0588] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0589] DNA PCR-Free index113接头 [0590] The most stable dimer overall:12bp,-22.8kcal/mol [0591] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCCTCGCATCTCGTATGCCGTCTTCTGCTTG 3′ [0592] |||||||||||| : ::: : :::: ::: : [0593] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0594] DNA PCR-Free index114接头 [0595] The most stable dimer overall:12bp,-22.8kcal/mol [0596] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCGCGCAATCTCGTATGCCGTCTTCTGCTTG 3′ [0597] |||||||||||| : ::: : ::: : [0598] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0599] DNA PCR-Free index115接头 [0600] The most stable dimer overall:12bp,-22.8kcal/mol [0601] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGCCACCTATCTCGTATGCCGTCTTCTGCTTG 3′ [0602] |||||||||||| : ::: : : ::: : [0603] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0604] DNA PCR-Free index116接头 [0605] The most stable dimer overall:12bp,-22.8kcal/mol [0606] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCATGCGGCATCTCGTATGCCGTCTTCTGCTTG 3′ [0607] |||||||||||| : ::: :: : ::: : [0608] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0609] DNA PCR-Free index117接头 [0610] The most stable dimer overall:12bp,-22.8kcal/mol [0611] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCAACAGATCTCGTATGCCGTCTTCTGCTTG 3′ [0612] |||||||||||| : ::: : : : ::: : [0613] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0614] DNA PCR-Free index118接头 [0615] The most stable dimer overall:12bp,-22.8kcal/mol [0616] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGGTATCAATCTCGTATGCCGTCTTCTGCTTG 3′ [0617] |||||||||||| : ::: ::: : [0618] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0619] DNA PCR-Free index119接头 [0620] The most stable dimer overall:12bp,-22.8kcal/mol [0621] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGGCCAATATCTCGTATGCCGTCTTCTGCTTG 3′ [0622] |||||||||||| : ::: : ::: : [0623] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0624] DNA PCR-Free index120接头 [0625] The most stable dimer overall:12bp,-22.8kcal/mol [0626] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGCCGTCCATCTCGTATGCCGTCTTCTGCTTG 3′ [0627] |||||||||||| : ::: : ::: : [0628] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0629] DNA PCR-Free index121接头 [0630] The most stable dimer overall:12bp,-22.8kcal/mol [0631] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGAGTGATCTCGTATGCCGTCTTCTGCTTG 3′ [0632] |||||||||||| : ::: : ::: : [0633] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0634] DNA PCR-Free index122接头 [0635] The most stable dimer overall:12bp,-22.8kcal/mol [0636] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACACGCAGCCATCTCGTATGCCGTCTTCTGCTTG 3′ [0637] |||||||||||| : ::: : ::: : [0638] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0639] DNA PCR-Free index123接头 [0640] The most stable dimer overall:12bp,-22.8kcal/mol [0641] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAGCTGACATCTCGTATGCCGTCTTCTGCTTG 3′ [0642] |||||||||||| : ::: :: :: ::: : [0643] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0644] DNA PCR-Free index124接头 [0645] The most stable dimer overall:12bp,-22.8kcal/mol [0646] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGATGGCTATCTCGTATGCCGTCTTCTGCTTG 3′ [0647] |||||||||||| : ::: ::: : [0648] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0649] DNA PCR-Free index125接头 [0650] The most stable dimer overall:12bp,-22.8kcal/mol [0651] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGAATCATATCTCGTATGCCGTCTTCTGCTTG 3′ [0652] |||||||||||| : ::: :: ::: : [0653] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0654] DNA PCR-Free index126接头 [0655] The most stable dimer overall:12bp,-22.8kcal/mol [0656] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACAGACATCTCGTATGCCGTCTTCTGCTTG 3′ [0657] |||||||||||| : ::: : ::: : [0658] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0659] DNA PCR-Free index127接头 [0660] The most stable dimer overall:12bp,-22.8kcal/mol [0661] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGGTCGTATCTCGTATGCCGTCTTCTGCTTG 3′ [0662] |||||||||||| : ::: : ::: ::: : [0663] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0664] DNA PCR-Free index128接头 [0665] The most stable dimer overall:12bp,-22.8kcal/mol [0666] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCGTGGAGATCTCGTATGCCGTCTTCTGCTTG 3′ [0667] |||||||||||| : ::: : : ::: : [0668] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0669] DNA PCR-Free index129接头 [0670] The most stable dimer overall:12bp,-22.8kcal/mol [0671] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTTCCGCATCTCGTATGCCGTCTTCTGCTTG 3′ [0672] |||||||||||| : ::: : :: ::: : [0673] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0674] DNA PCR-Free index130接头 [0675] The most stable dimer overall:12bp,-22.8kcal/mol [0676] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACACATGTACATCTCGTATGCCGTCTTCTGCTTG 3′ [0677] |||||||||||| : ::: ::: : [0678] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0679] DNA PCR-Free index131接头 [0680] The most stable dimer overall:12bp,-22.8kcal/mol [0681] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGGCTAAATCTCGTATGCCGTCTTCTGCTTG 3′ [0682] |||||||||||| : ::: ::: : [0683] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0684] DNA PCR-Free index132接头 [0685] The most stable dimer overall:12bp,-22.8kcal/mol [0686] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATCCTGATCTCGTATGCCGTCTTCTGCTTG 3′ [0687] |||||||||||| : ::: : : ::: : [0688] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0689] DNA PCR-Free index133接头 [0690] The most stable dimer overall:12bp,-22.8kcal/mol [0691] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGACGATATATCTCGTATGCCGTCTTCTGCTTG 3′ [0692] |||||||||||| : ::: :: ::: : [0693] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0694] DNA PCR-Free index134接头 [0695] The most stable dimer overall:12bp,-22.8kcal/mol [0696] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCTGGCCAATCTCGTATGCCGTCTTCTGCTTG 3′ [0697] |||||||||||| : ::: : : ::: : [0698] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0699] DNA PCR-Free index135接头 [0700] The most stable dimer overall:12bp,-22.8kcal/mol [0701] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGACGTCATCTCGTATGCCGTCTTCTGCTTG 3′ [0702] |||||||||||| : ::: : ::: : [0703] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0704] DNA PCR-Free index136接头 [0705] The most stable dimer overall:12bp,-22.8kcal/mol [0706] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCTCTCTAATCTCGTATGCCGTCTTCTGCTTG 3′ [0707] |||||||||||| : ::: : :::: ::: : [0708] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0709] DNA PCR-Free index137接头 [0710] The most stable dimer overall:12bp,-22.8kcal/mol [0711] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGCGTGTCATCTCGTATGCCGTCTTCTGCTTG 3′ [0712] |||||||||||| : ::: : ::: : [0713] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0714] DNA PCR-Free index138接头 [0715] The most stable dimer overall:12bp,-22.8kcal/mol [0716] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGTTGTTATCTCGTATGCCGTCTTCTGCTTG 3′ [0717] |||||||||||| : ::: : ::: : [0718] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0719] DNA PCR-Free index139接头 [0720] The most stable dimer overall:12bp,-22.8kcal/mol [0721] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGCTACGATCTCGTATGCCGTCTTCTGCTTG 3′ [0722] |||||||||||| : ::: :: : ::: : [0723] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0724] DNA PCR-Free index140接头 [0725] The most stable dimer overall:12bp,-22.8kcal/mol [0726] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGTAACCAATCTCGTATGCCGTCTTCTGCTTG 3′ [0727] |||||||||||| : ::: : : ::: : [0728] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0729] DNA PCR-Free index141接头 [0730] The most stable dimer overall:12bp,-22.8kGal/mol [0731] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGTGTTAAATCTCGTATGCCGTCTTCTGCTTG 3′ [0732] |||||||||||| : ::: : : ::: : [0733] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0734] DNA PCR-Free index142接头 [0735] The most stable dimer overall:12bp,-22.8kcal/mol [0736] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATAGCCGATCTCGTATGCCGTCTTCTGCTTG 3′ [0737] |||||||||||| : ::: ::: : : ::: : [0738] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0739] DNA PCR-Free index143接头 [0740] The most stable dimer overall:12bp,-22.8kcal/mol [0741] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAACACCGATCTCGTATGCCGTCTTCTGCTTG 3′ [0742] |||||||||||| : ::: : : : : ::: : [0743] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0744] DNA PCR-Free index144接头 [0745] The most stable dimer overall:12bp,-22.8kcal/mol [0746] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTAGTTAATCTCGTATGCCGTCTTCTGCTTG 3′ [0747] |||||||||||| : ::: : ::: : [0748] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0749] DNA PCR-Free index145接头 [0750] The most stable dimer overall:12bp,-22.8kcal/mol [0751] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTCTGCCTATCTCGTATGCCGTCTTCTGCTTG 3′ [0752] |||||||||||| : ::: : : ::: : [0753] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0754] DNA PCR-Free index146接头 [0755] The most stable dimer overall:12bp,-22.8kcal/mol [0756] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGAGTAGAATCTCGTATGCCGTCTTCTGCTTG 3′ [0757] |||||||||||| : ::: : : : ::: : [0758] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0759] DNA PCR-Free index147接头 [0760] The most stable dimer overall:12bp,-22.8kcal/mol [0761] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGCGCAGCATCTCGTATGCCGTCTTCTGCTTG 3′ [0762] |||||||||||| : ::: : ::: : [0763] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0764] DNA PCR-Free index148接头 [0765] The most stable dimar overall:12bp,-22.8kcal/mol [0766] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGCCTATAATCTCGTATGCCGTCTTCTGCTTG 3′ [0767] |||||||||||| : ::: :: ::: : [0768] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0769] DNA PCR-Free index149接头 [0770] The most stable dimer overall:12bp,-22.8kcal/mol [0771] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGCTAGTGATCTCGTATGCCGTCTTCTGCTTG 3′ [0772] |||||||||||| : ::: : ::: : [0773] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0774] DNA PCR-Free index150接头 [0775] The most stable dimer overall:12bp,-22.8kcal/mol [0776] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGAGCTCATCTCGTATGCCGTCTTCTGCTTG 3′ [0777] |||||||||||| : ::: : ::: : [0778] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0779] DNA PCR-Free index151接头 [0780] The most stable dimer overall:12bp,-22.8kcal/mol [0781] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGGATTAGATCTCGTATGCCGTCTTCTGCTTG 3′ [0782] |||||||||||| : ::: : : ::: : [0783] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0784] DNA PCR-Free index152接头 [0785] The most stable dimer overall:12bp,-22.8kcal/mol [0786] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGGACGGAATCTCGTATGCCGTCTTCTGCTTG 3′ [0787] |||||||||||| : ::: : ::: : [0788] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0789] DNA PCR-Free index153接头 [0790] The most stable dimer overall:12bp,-22.8kcal/mol [0791] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGACTGAGGATCTCGTATGCCGTCTTCTGCTTG 3′ [0792] |||||||||||| : ::: :: :: ::: : [0793] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0794] DNA PCR-Free index154接头 [0795] The most stable dimer overall:12bp,-22.8kcal/mol [0796] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGTGTTAATCTCGTATGCCGTCTTCTGCTTG 3′ [0797] |||||||||||| : ::: : ::: : [0798] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0799] DNA PCR-Free index155接头 [0800] The most stable dimer overall:12bp,-22.8kcal/mol [0801] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTCGTCCGATCTCGTATGCCGTCTTCTGCTTG 3′ [0802] |||||||||||| : ::: :: : ::: : [0803] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0804] DNA PCR-Free index156接头 [0805] The most stable dimer overall:12bp,-22.8kcal/mol [0806] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGGAGAGGATCTCGTATGCCGTCTTCTGCTTG 3′ [0807] |||||||||||| : ::: :: ::: : [0808] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0809] DNA PCR-Free index157接头 [0810] The most stable dimer overall:12bp,-22.8kcal/mol [0811] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGGAATTCATCTCGTATGCCGTCTTCTGCTTG 3′ [0812] |||||||||||| : ::: ::: : [0813] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0814] DNA PCR-Free index158接头 [0815] The most stable dimer overall:12bp,-22.8kcal/mol [0816] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTGGCGCCATCTCGTATGCCGTCTTCTGCTTG 3′ [0817] |||||||||||| : ::: ::: : [0818] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0819] DNA PCR-Free index159接头 [0820] The most stable dimer overall:12bp,-22.8kcal/mol [0821] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCTTAATATCTCGTATGCCGTCTTCTGCTTG 3′ [0822] |||||||||||| : ::: : : ::: : [0823] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0824] DNA PCR-Free index160接头 [0825] The most stable dimer overall:12bp,-22.8kcal/mol [0826] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGCGATTATCTCGTATGCCGTCTTCTGCTTG 3′ [0827] |||||||||||| : ::: : : ::: : [0828] 3′TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5′ [0829] DNA PCR-Free index161接头 [0830] The most stable dimer overall:12bp,-22.8kcal/mol [0831] 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACAACCGCAAATCTCGTATGCCGTCTTCTGCTTG 3′ [0832] |||||||||||| : ::: : : : ::: : [0834] 图1:ilumina公司提供的常规DNA建库流程示意图。 [0835] 图2:illumina公司提供的常规DNA标签建库流程示意图。 [0836] 图3:illumina公司的DNA标签建库流程图。 [0837] 图4:本发明提供的基于接头连接的DNA PCR-Free标签文库构建方法。 [0838] 图5:实施例1所构建的文库电泳检测结果。泳道1:D2000,泳道2-24:PCR-Free index接头与DNA的连接产物;泳道25:50bp ladder。所用的D2000和50bp的marke分别产自天根公司和NEB公司。 具体实施方式[0839] 下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。 [0840] 本发明一方面提供了一组DNA标签,所述DNA标签包括如下或由如下组成:表2所示的161个DNA标签或与之相差1个碱基的DNA标签中的至少10个,或至少20个,或至少30个,或至少40个,至少50个,或至少60个,或至少70个,或至少80个,或90个,或至少 100个,或至少110个,或至少120个,或至少130个,或至少140个,或至少150个,或全部 161个, [0841] 所述DNA标签优选地至少包括表2所示的161个DNA标签的DNA Index1~DNA Index10,或DNA Index11~DNA Index20,或DNA Index21~DNA Index30,或DNA Index31~DNA Index40,或DNA Index41~DNA Index50,或DNA Index51~DNA Index60,或DNA Index61~DNA Index70,或DNA Index71~DNA Index80,或DNA Index81~DNA Index90,或DNA Index91~DNA Index100,或DNA Index101~DNA Index110,或DNA Index111~DNA Index120,或DNA Index121~DNA Index130,或DNA Index131~DNA Index140,或DNA Index141~DNA Index150,或DNA Index151~DNA Index161,或者他们任何两个或多个的组合。 [0842] 在本发明的一个具体实施方式中,所述DNA标签中所述的相差1个碱基包括标签中1个碱基的取代、添加或删除。 [0843] 在本发明的一个具体实施方式中,提供了上文所述的DNA标签用于构建DNA标签文库,优选的DNA PCR-Free标签文库的用途,其中DNA标签文库的DNA标签接头包含所述DNA标签,从而构成各自相对应的DNA标签接头,所述DNA标签接头优选地用作DNA PCR-Free标签文库的DNA PCR-Free接头。 [0844] 在本发明的一个具体实施方式中,在本发明所提供的用途中,所述DNA标签插入DNA PCR-Free标签接头中,或通过或不通过连接子连接在DNA接头的3’末端,优选地插入DNA PCR-Free标签接头中。所述连接子是1-6个核苷酸序列,优选地1-3个核苷酸序列。 [0845] 在本发明的一个具体实施方式中,提供了通过上文所述的DNA标签构建的DNA标签文库,优选的DNA PCR-Free标签文库。 [0846] 本发明另一方面含有上文所述的DNA标签的一组DNA PCR-Free标签接头,其中所述DNA PCR-Free标签接头优选地用作DNA PCR-Free标签文库的接头,所述一组DNA PCR-Free标签接头由DNA PCR-Free接头1.0和PCR-Free标签序列组成,所述PCR-Free标签序列包括如下或由如下组成:表2所示161个PCR-Free标签序列或与其所包含的DNA标签序列相差1个碱基的PCR-Free标签序列中的至少10个,或至少20个,或至少30个,或至少40个,至少50个,或至少60个,或至少70个,或至少80个,或90个,或至少100个,或至少110个,或至少120个,或至少130个,或至少140个,或至少150个,或全部161个, [0847] 所述PCR-Free标签序列优选地至少包括表2所示的161个PCR-Free标签序列中的PCR-Free Index-1~PCR-Free Index-10,或PCR-Free Index-11~PCR-Free Index-20,或 PCR-Free Index-21 ~ PCR-Free Index-30,或 PCR-Free Index-31 ~PCR-Free Index-40, 或 PCR-Free Index-41 ~ PCR-Free Index-50, 或 PCR-Free Index-51 ~ PCR-Free Index-60,或 PCR-Free Index-61 ~ PCR-Free Index-70,或PCR-Free Index-71~PCR-Free Index-80,或PCR-Free Index-81~PCR-Free Index-90,或 PCR-Free Index-91 ~ PCR-Free Index-100,或 PCR-Free Index-101 ~ PCR-Free Index-110,或PCR-Free Index-111~PCR-Free Index-120,或PCR-Free Index-121~PCR-Free Index-130,或PCR-Free Index-131~PCR-Free Index-140,或PCR-Free Index-141~PCR-Free Index-150,或PCR-Free Index-151~PCR-Free Index-161,或者他们任何两个或多个的组合。 [0848] 在本发明的一个具体实施方式中,上文所述DNA PCR-Free标签接头中所述的相差1个碱基包括标签中1个碱基的取代、添加或删除。 [0849] 在本发明的一个具体实施方式中,提供了所述DNA PCR-Free标签接头用于构建DNA标签文库,优选的DNA PCR-Free标签文库的用途。 [0850] 在本发明的一个具体实施方式中,提供了通过上文所述的DNAPCR-Free标签接头构建的DNA标签文库,优选的DNA PCR-Free标签文库。 [0851] 本发明另一方面进一步提供了一种DNA标签文库的构建方法,所述方法的特征在于使用包含标签的DNA PCR-Free接头来构建DNA标签文库,优选的DNA PCR-Free标签文库,其中所述方法不包括PCR步骤。 [0852] 在本发明另一方面进一步提供的DNA标签文库,优选的DNAPCR-Free标签文库的构建方法中,包括: [0853] 1)DNA模板准备,提供n个DNA样品,n为整数且1≤n≤161的整数,优选地n为整数且2≤n≤161,所述DNA样品来自所有真核和原核DNA样品,包括但不限于人DNA样品;优选地所述DNA模板长度为250bp; [0854] 2)末端修复; [0855] 3)DNA片段3’末端加“A”碱基; [0856] 5)连接DNA标签接头,其中对DNA片段使用不同的标签接头,每一个标签接头连接到DNA片段的两端。 [0857] 在本发明的一个具体实施方式中,在上文所述方法中,所述DNA标签接头是本发明所述的DNA PCR-Free标签接头。 [0858] 在本发明的一个具体实施方式中,提供了通过本发明所述的方法构建的DNA标签文库,优选的DNA PCR-Free标签文库。 [0859] Paired End DNA寡核苷酸序列: [0860] DNA PCR-Free接头1.0: [0861] AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT [0862] DNA PCR-Free index接头:(如表2所示) [0863] 主要实验仪器及试剂 [0864] [0865] [0866] [0867] [0868] 实施例1:DNA标签实验建库方法具体 [0869] 1.1DNA模板准备 [0870] 以质粒pMD18-T(日本takara)为模板,使用Primer Premier5.0软件设计引物,PCR扩增产物长度为250bp的片段,使用NanoDrop 1000仪器(美国NanoDrop)检测扩增产物的浓度,然后根据浓度取1ug的该PCR产物作为文库构建的插入片段,补水使其体积至35μL。PCR引物序列: [0871] pMD18-T引物1:CGGGGAGAGGCGGTTTGCGTATTGG; [0872] pMD18-T引物2:TTTTGTGATGCTCGTCAGGGGGGCG。 [0873] 1.2末端修复[6] [0874] 按照下列的配比准备反应混合: [0875] pMD18-T质粒DNA模板 35μL [0876] T4 DNA连接酶缓冲液 50μL [0877] dNTPs混合液 4μL [0878] T4 DNA聚合酶 5μL [0879] Klenow DNA聚合酶 1μL [0880] T4多聚核苷酸激酶 5μL [0881] 总体积 100μL [0882] 将舒适型恒温混匀器调至20℃,反应30min,然后用QIAquick PCR纯化试剂盒进行纯化,最后将样品溶于32μL EB solution。 [0883] 1.3DNA片段3′末端加“A”碱基 [0884] 按照下列的配比准备反应混合物: [0885] 末端修复后的DNA 32μL [0886] Klenow酶缓冲液 5μL [0887] dATP(1mM) 10μL [0888] Klenow酶(3′到5′外切酶活性) 3μL [0889] 总体积 50μL [0890] 将舒适型恒温混匀器调至37℃,反应30min,然后用MiniElute PCR纯化试剂盒进行纯化,最后将样品溶于10μL EB solution。 [0891] 1.4连接PCR-Free标签接头 [0892] 按照下列的配比准备反应混合物: [0893] DNA 10μL [0894] T4 DNA连接酶缓冲液 25μL [0895] DNA PCR-Free标签接头 10μL [0896] T4 DNA连接酶 5μL [0897] 总体积 50μL [0898] 注:对于每一个DNA样品,所使用的PCR-Free标签接头可为表2中所示PCR-Free标签序列(PCR-Free Index-N)中的任意一种与DNA PCR-Free接头1.0退火后的PCR-Free标签接头。 [0899] 将舒适型恒温混匀器调至20℃,反应15min,然后用QIAquick PCR纯化试剂盒进行纯化,最后将样品溶于30μL EB solution。 [0900] 1.5PCR-Free index文库的胶回收纯化 [0901] 将连接产物于2%的琼脂糖胶中进行电泳分离;随后将目的片段条带切胶转移至Eppendorf管中。用QIAquick胶纯化试剂盒进行胶纯化回收,回收产物溶于20μL EB solution。 [0902] 1.6PCR-Free index文库检测 [0903] 1)使用Agilent 2100 Bioanalyzer检测文库产量。 [0904] 2)使用QPCR定量检测文库产量。 [0905] 构建的44个DNA PCR-Free标签文库,使用1%琼脂糖凝胶电泳结果如图5a和图5b所示,图5a中分别使用了D2000和50bp的marker;箭头所标记的为目的文库,其中泳道1和泳道25分别是D2000和50bp的marker,泳道2-24分别是DNA PCR-Free标签1文库、DNA PCR-Free标签2文库、DNA PCR-Free标签3文库、DNA PCR-Free标签4文库、DNA PCR-Free标签5文库、DNA PCR-Free标签6文库、DNA PCR-Free标签7文库、DNA PCR-Free标签8文库、DNA PCR-Free标签9文库、DNA PCR-Free标签10文库、DNA PCR-Free标签11文库、DNA PCR-Free标签12文库、DNA PCR-Free标签13文库、DNA PCR-Free标签14文库、DNA PCR-Free标签15文库、DNA PCR-Free标签16文库、DNA PCR-Free标签17文库、DNA PCR-Free标签18文库、DNA PCR-Free标签19文库、DNA PCR-Free标签20文库、DNA PCR-Free标签21文库、DNA PCR-Free标签22文库、DNA PCR-Free标签23文库。图5b中分别使用了D2000和50bp的marker;箭头所标记的为目的文库,其中泳道1和泳道25分别是D2000和50bp的marker,泳道3-23分别是DNA PCR-Free标签101文库、DNA PCR-Free标签102文库、DNAPCR-Free标签103文库、DNA PCR-Free标签104文库、DNA PCR-Free标签105文库、DNA PCR-Free标签106文库、DNA PCR-Free标签107 文库、DNA PCR-Free标签108文库、DNA PCR-Free标签109文库、DNA PCR-Free标签110文库、DNA PCR-Free标签111文库、DNA PCR-Free标签112文库、DNA PCR-Free标签113文库、DNA PCR-Free标签114文库、DNA PCR-Free标签115文库、DNA PCR-Free标签116文库、DNA PCR-Free标签117文库、DNA PCR-Free标签118文库、DNA PCR-Free标签119文库、DNA PCR-Free标签120文库、DNA PCR-Free标签121文库。由于目的片段(PCR产物)长度为250bp,接头长度为65bp且为“Y型”结构,理论上目的片段两端连接上DNA PCR-Free标签接头后的会增加130bp左右的分子量,但是由于DNA PCR-Free标签接头为特殊的“Y型”结构,其在琼脂糖中电泳的速度会比相同分子量的双链DNA电泳迁移率稍小,电泳条带显示的位置为 500bp。D2100 marker从上往下的条带分别是:2000bp、1000bp、750bp、500bp、250bp、100bp; 目的条带下方有部分条带,为目的片段的一端连接上接头的产物,其产物量不多,不影响文库构建的结果。结果表明,接头全部成功连接到pcr产物上。 [0906] Solexa测序结果统计:共测序33932756条读取结果(也称为·reads),标签完全识别即0 mismatch占96.73%,其他读取结果(other reads)占3.27%,所以测序结果标签的完全识别的序列为~96.7%,可以满足solexa DNA标签的测序要求。 [0907] 尽管本发明的具体实施方式已经得到详细的描述,本领域技术人员将会理解。根据已经公开的所有教导,可以对那些细节进行各种修改和替换,这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。 [0908] 参考文献 [0909] 1、Paired-End sequencing User Guide;illumina part#1003880 [0910] 2、Preparing samples for ChIP sequencing for DNA;illumina part#11257047 Rev.A; [0911] 3、mRNA sequencing sample preparation Guide;illumina part#1004898 Rev.D [0912] 4、Preparing 2-5kb samples for mate pair library sequencing;illumina part#1005363 Rev.B; [0913] 5、Preparing samples for multiplexed Paired-End sequencing;illumina part#1005361 Rev.B. |