WO2016023430A1 - 基因组dna测序文库液相杂交诱捕富集溶液及杂交方法 - Google Patents

基因组dna测序文库液相杂交诱捕富集溶液及杂交方法 Download PDF

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WO2016023430A1
WO2016023430A1 PCT/CN2015/086005 CN2015086005W WO2016023430A1 WO 2016023430 A1 WO2016023430 A1 WO 2016023430A1 CN 2015086005 W CN2015086005 W CN 2015086005W WO 2016023430 A1 WO2016023430 A1 WO 2016023430A1
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sequencing library
hybridization
genomic dna
dna sequencing
enrichment
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邵华武
邵阳
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邵华武
邵阳
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  • the invention belongs to the field of molecular biology, in particular to a formula and a hybridization method for liquid phase hybridization trapping and enrichment solution of a genomic DNA sequencing library.
  • NGS Next Generation Sequencing
  • the enrichment based on the PCR principle is to use a PCR primer for the target region to amplify the targeting sequence, and then sequence the specific PCR product. This method typically requires many cycles of denaturation, annealing and extension, often resulting in non-specific products and amplification bias. In addition, for the operation of hundreds or thousands of single-plex PCR reactions, dedicated automation equipment or partitioned equipment (such as the technology of Fruidigm and RainDance) is required. Another option is to perform limited multiplex PCR in one reaction. Therefore, PCR-based enrichment does not easily expand the target region and is therefore often used for smaller target regions (in the range of a few kilobases to several megabytes).
  • the selective cyclization method is also called a molecular inversion probe (MIP).
  • MIP molecular inversion probe
  • Each of the cyclized probes used for enrichment possesses a single-stranded DNA oligonucleic acid strand, each of which contains a non-contiguous sequence complementary to the target sequence, and in an opposite linear sequence.
  • the specific hybridization of the probe to the target sequence forms a circular DNA structure.
  • This structure is further formed by a gap filling and ligation reaction to form a closed circular single-stranded DNA.
  • a PCR reaction directed against a consensus sequence on the loop will ultimately amplify these targeted enriched regions to produce a targeted enriched sequencing library.
  • the greatest advantage of the selective cyclization method is its high specificity, but its most prominent disadvantage is that the capture uniformity for each target region is poor, much lower than the hybrid capture method. Moreover, its cost is high, and the target area that can be detected is limited in size (in the range of several kilobases to several megabytes).
  • the hybrid trapping method is that when the target nucleotide sequence in the input DNA sample specifically hybridizes with the customized DNA probe complementary thereto, the target DNA sequence is physically captured and separated. Because hybridization trapping method can easily synthesize a large number of specific DNA probes and carry them simultaneously in one reaction system, and multiple DNA sequencing library samples can be mixed and processed simultaneously by connecting different coding sequencing joints, thus hybrid trapping Most of the methods are used for medium to large target areas (1 to 60 Mb) or more samples. With the wide application of whole exon sequencing, hybrid trapping methods are being applied more and more, and various experimental procedures and commercial reagents are also being applied.
  • Hybrid trapping enrichment can be divided into solid phase chip hybridization and liquid phase hybridization.
  • solid phase chip hybridization a genomic DNA sequencing library is hybridized to a DNA probe immobilized on a chip. Non-specific DNA library fragments that cannot be bound are washed off the chip, while the targeted enriched DNA library is later eluted for sequencing.
  • liquid phase hybridization the target sequence of the DNA sequencing library specifically binds to a customized biotinylated oligonucleotide capture probe. Subsequent addition of the streptavidin magnetic beads will bind to the biotinylated probe such that the specific target sequence to which it binds is separated by magnetic adsorption. The enriched DNA library was eluted for sequencing.
  • the solid phase chip hybridization method has many advantages over PCR amplification, the cost of the chip is relatively high, and the test process requires expensive hardware equipment such as a chip incubator. In addition, since chips that simultaneously start to hybridize must simultaneously elute the enriched DNA library, the number of chips that can be simultaneously processed at a time is also very limited. Finally, in order to obtain a sufficient amount of DNA library required for a hybridization enrichment reaction, we need about 10-15 ug of input DNA to prepare a genomic library, which requires a large amount of starting material for the experimental material. In contrast, liquid phase hybridization requires only a small amount of input DNA library because of its ease of handling, especially when the sample is limited, and is used by more and more people.
  • Liquid phase hybridization promotes the full progress of the hybridization reaction due to the excess of its capture probe relative to the DNA library.
  • the homogeneity and specificity of liquid phase hybridization are higher than that of solid phase chip hybridization.
  • liquid phase hybrid capture does not require special equipment, only a laboratory thermal cycler is required, and a large number of samples can be processed simultaneously in a 96-well plate mode.
  • the DNA capture probe used is designed to hybridize to the target target region sequence
  • the enrichment is dependent on the physical and chemical properties of the nucleotide, such as the molecular mass of the nucleotide, its GC content, secondary structure, Melting and annealing temperatures, as well as nucleotide concentration and salt concentration. All of these factors have an important impact on the binding kinetics and specificity of the hybridization reaction. Techniques based on hybridization principles often require higher melting temperatures close to the probe used and require longer incubation times to increase specificity. Standard incubation times range from 10 hours to 72 hours. An important disadvantage of long incubation times is the increased time to obtain targeted enriched sequencing libraries, which affects subsequent sequencing.
  • One aspect of the invention relates to a genomic DNA sequencing library hybrid trapping enrichment solution, characterized in that it comprises sodium phosphate buffer, sodium citrate buffer (SSC), sodium dodecyl sulfate (SDS), ethylenediamine four Acetic acid (EDTA) and Denhardt solutions, the components are packaged separately.
  • SSC sodium citrate buffer
  • SDS sodium dodecyl sulfate
  • EDTA ethylenediamine four Acetic acid
  • Denhardt solutions the components are packaged separately.
  • the genomic DNA sequencing library hybrid capture enrichment solution contains or does not contain other components.
  • the sodium phosphate buffer is a 0.4-0.6 M sodium phosphate buffer having a pH between 6.8 and 7.2.
  • the concentration of SDS is 0.8-1.2%; the concentration of EDTA is 1-3 mM.
  • the genomic DNA sequencing library hybrid trapping and enriching solution comprises 1 part of sodium phosphate buffer, 2 parts of sodium citrate buffer (SSC), 1 part of SDS, 1 part of EDTA and 4 parts of Denhardt solution
  • the 20X SSC formulation 3 M NaCl, 300 mM sodium citrate; the pH is adjusted to 7.0.
  • a 50X Denhardt solution the solution contains 1% polysucrose (Ficoll, Model 400), 1% polyvinylpyrrolidone, and 1% bovine serum albumin (BSA).
  • Another aspect of the present invention also relates to a hybridization method characterized by hybridization of a hybridization trap enrichment solution using the above genomic DNA sequencing library.
  • the hybridization method comprises the following steps (Fig. 1):
  • Fragmentation of genomic DNA into 350-550 bp mechanical disruption, such as sonication, hydroshocking, or enzymatic fragmentation; the length of the fragment required depends on the length of sequencing to be subsequently employed);
  • a genome sequencing library was prepared according to a standard experimental procedure according to the subsequent NGS sequencing platform.
  • Biotin-modified single-stranded nucleotide capture probe 3pMole 2-5 ⁇ g of human Cot-1 DNA The respective ends of the closed polymer mixture are 1 mM each. No nuclease deionized water, the total volume is increased to 10 ⁇ L
  • the hybridization solution has a simple formula and is easy to obtain, which reduces production cost and is easy to preserve.
  • the method of the present invention is simple and easy, and only requires a thermal cycler conventionally used in laboratories. Overnight hybridization facilitates the design of the protocol, and the selection of the hybridization temperature of 65 °C is suitable for the commonly used single-stranded nucleotide capture probes of 80-120 bases in length.
  • the hybridization solution formulation and hybridization method of the present invention are applicable to capture enrichment of genomic DNA sequencing libraries prepared by a variety of different methods (e.g., Illumina kit, NEB kit, KAPA kit, etc.). It is also possible to simultaneously enrich multiple library samples in one hybridization reaction. Total input library samples only Need no less than 250 ng of DNA.
  • the background value generated at the time of hybridization can be well controlled, the enrichment signal can be improved, and the hybridization effect can be optimized.
  • the whole genome genomic DNA sequencing library of normal human B lymphocyte lineage is subjected to targeted enrichment (about 1 Mb) of a specific human B lymphocyte lineage, and the hybridization temperature is shortened, the hybridization time is shortened, and the target gene is hybridized.
  • Significantly increased signal Figure 2A
  • non-specific background values were significantly reduced (Fig. 2B).
  • the hybridization solution formulation and method can significantly increase the enrichment factor of the target gene and effectively reduce the background value on the basis of shortening the operation time (Fig. 2C).
  • the improved hybridization solution formula and the corresponding hybridization method are low in cost, simple and convenient, effectively shorten the hybridization time, and can accurately extract the required DNA fragments, reduce the influence of the background value on the signal value, and optimize the hybridization target. Enrichment effect.
  • Figure 1 Flowchart of hybridization trap enrichment of genomic DNA sequencing library using the hybrid solution formulation of the present invention.
  • Fragmentation of genomic DNA (2) Construction of a genomic DNA sequencing library.
  • FIG. 1 Comparative experiment using the hybridization solution and commercialized hybridization solution for targeted enrichment.
  • a and B are the hybridization methods for the hybridization solution formulation without hybridization temperature and hybridization reaction time, thereby optimizing the hybridization method using the hybridization solution.
  • C is a comparative test using the hybridization solution and the corresponding hybridization method with a commercialized hybridization solution and a hybridization method thereof.
  • the enrichment factor of the target gene and the non-target gene was detected by real-time quantitative PCR (realtime-qPCR).
  • the test results are the mean ⁇ standard error (mean ⁇ SEM) obtained from three independent replicates.
  • a genomic DNA sequencing library hybrid entrapment enrichment solution was prepared, which consisted of sodium phosphate buffer, sodium citrate buffer (SSC), SDS, EDTA and Denhardt solutions. It can be stored for a long time at -20 °C.
  • the specific formulation of the 2X concentration solution is as follows:
  • the Denhardt solution is a mixture of blocking reagents for hybridization.
  • the solution contained 1% polysucrose (Ficoll, type 400), 1% polyvinylpyrrolidone, and 1% bovine serum albumin (BSA).
  • a genome sequencing library was prepared according to Illumina TruSeq PCR-free.
  • a total of 500 ng of the genome sequencing library was vacuum dried in a 0.2 mL PCR tube at a high speed. Multiple genomic sequencing library samples with different index sequence linkers can be mixed in this library pool, each sequencing library being no less than 25 ng.

Abstract

本发明公开了基因组DNA测序文库液相杂交诱捕富集溶液及杂交方法。所述杂交富集溶液包含磷酸钠缓冲液，柠檬酸钠缓冲液，SDS，EDTA和Denhardt溶液。

Description

基因组DNA测序文库液相杂交诱捕富集溶液及杂交方法 技术领域
本发明属于分子生物学领域，具体而言，涉及一种基因组DNA测序文库液相杂交诱捕富集溶液的配方及杂交方法。
背景技术
在过去的几年中，DNA测序技术出现了一个根本性的转变，从传统的Sanger测序发展到了所谓的“下一代测序”技术(Next Generation Sequencing,NGS)。其突出特点为大规模并行测序。NGS技术允许数以百万甚至亿万的测序反应同时进行，从而达到测序通量的巨幅增长。NGS技术平台需要构建适用于大规模并行测序的片段文库。构建测序文库的过程包括将DNA片段化，随后的DNA修复和末端处理(平端或A突出端)。最后，根据具体的技术平台而选择的特异性接头连接。
尽管与传统的Sanger测序法相比，NGS技术相关的成本大幅降低，全基因组测序仍然是成本较高的。此外，有许多应用并不需要全基因组测序，而是需要对于一个或多个样品的特定区域进行测序。因此，如果感兴趣的区域只是基因组的一部分并或者大量数目的样品需要被分析，我们常常倾向于只对测序文库的一部分特异子集进行测序，而不是对整个基因组文库进行测序，从而减少不必要的成本和劳动力。例如有效的全外显子(所有基因的编码部分)测序是目前比较主要的应用，但是针对较小的基因组或基因组区域的富集测序也被广泛应用。在靶向富集的过程中，不感兴趣的DNA片段被最大限度的去除,目标区域被从最初的全基因组测序文库中富集出来，使得后续NGS测序所产生的测序结果主要集中在感兴趣的目标区域。
为了更好地应用NGS进行靶向测序，多个在测序前进行靶向富集的流程已经被研制出来。通常要进行多个步骤的富集以提供最终的目标测序文库。目前， 常用的靶向富集方法包括PCR扩增法(PCR amplification)，选择性环化法(selective circularization)，以及杂交诱捕法(hybrid capture)。不同的靶向富集技术具有不同的性能和易用性。每种方法的最重要的特性,反过来也反映了靶向富集的最大挑战，包括得到靶向富集测序文库所消耗的时间，获得有用的测序结果的每个靶碱基的总成本，富集的参数，包括特异性(测序结果中目标区与非目标区的比例)，覆盖率(测序深度)，在所有靶向目标区域的覆盖均匀度，方法流程的可重复性以及所需要的输入DNA的总量。
基于PCR原理的富集顾名思义，是利用针对目标区域的PCR引物来扩增靶向序列，进而对特异性的PCR产物进行测序。这种方法通常需要许多循环的变性，退火和延伸，常造成非特异性产物和扩增的偏向性。另外，对于运行数以百计或数以千计的单重PCR反应，需要运用专用自动化设备或分区化设备(例如Fruidigm和RainDance公司的技术)。其他的选择为在一个反应中进行有限的多重PCR。因此，基于PCR的富集并不能很容易的扩大目标区域，因而多用于较小的目标区域(几千碱基至几兆范围内)。
选择性环化法也叫分子倒位探针(MIP)。每一个用于富集的环化探针都拥有一个单链DNA寡核酸链，其两端分别含有与靶序列互补的不相连序列，并呈相反的线性顺序。探针与靶序列的特异性杂交形成了环状DNA结构。这种结构被进一步通过间隙填充和连接反应而形成封闭的环状单链DNA。针对环上的一段共有序列而进行的PCR反应会最终扩增这些靶向富集的区域而产生靶向富集的测序文库。选择性环化法的最大优点在于其高特异性，但是其最突出的缺点是针对每个目标区域的捕获均一性较差，远低于杂交捕获法。而且其成本较高，所能检测的目标区域的大小有限(几千碱基至几兆范围内)。
杂交诱捕法是当输入DNA样品中的目标核苷酸序列与定制的与其互补的DNA探针特异性杂交时，目标DNA序列就会被物理性的捕获和分离出来。由于杂交诱捕法可以很容易的合成大量特异性DNA探针，并在一个反应体系中同时进行，而且多种DNA测序文库样品可通过连接不同编码的测序接头而混合在一起同时处理，因而杂交诱捕的方法多用于中型到大型的目标区域(1至60Mb)或较多样品。随着全外显子测序的广泛应用,杂交诱捕法被越来越多的应用起来，针对其的各种实验流程及商品化试剂也越来越多。
杂交诱捕富集又可分为固相芯片杂交和液相杂交。在固相芯片杂交中，基因组DNA测序文库与固定在芯片上的DNA探针相杂交。不能结合的非特异性DNA文库片段被从芯片上洗去，而靶向富集的DNA文库稍后被洗脱下来用于测序。在液相杂交中，DNA测序文库的靶序列与定制的生物素标记的寡核苷酸捕获探针特异性的结合。随后加入链霉亲和素磁珠将会与生物素标记探针结合，从而使与之结合的特异性靶序列利用磁力吸附而分离出来。富集后的DNA文库被洗脱下来用于测序。
固相芯片杂交法虽具有许多优于PCR扩增富集的地方，但是芯片的成本比较高，且试验流程需要昂贵的硬件设备，例如芯片孵育箱。另外，由于同时开始杂交的芯片必须同时一起洗脱富集的DNA文库，每次可以同时处理的芯片数量也是非常有限的。最后，为了获得一个杂交富集反应所需的足够的DNA文库量，我们需要大约10-15ug的输入DNA来制备基因组文库，其对实验材料起始量的要求是巨大的。相比之下，液相杂交因其易于操作，又只需要少量的输入DNA文库，特别是当样品有限时，而被越来越多的人所应用。液相杂交由于其捕获探针相对于DNA文库是过量的，从而推动了杂交反应的充分进行。另外液相杂交的均一性和特异性均高于固相芯片杂交。最后，液相杂交捕获不需要特殊的仪器，只需要实验室常规的热循环仪，而且可以以96孔板的模式同时处理大量样品。
由于所用的DNA捕获探针被设计成能够与目标靶区域序列互补杂交，使得富集从而依赖于核苷酸的物理和化学特性，例如核苷酸的分子质量，其GC含量，二级结构，熔化和退火温度，以及核苷酸的浓度和盐浓度。所有的这些因素都对杂交反应的结合动力学和特异性产生重要影响。基于杂交原理的技术常常需要较高的接近于所用探针的熔化温度，并需要较长的孵育时间来提高特异性。标准的孵育时间范围从10小时到72小时不等。长孵育时间的一个重要缺点是增长了得到靶向富集测序文库的时间，从而影响了后续的测序。另外，较高的孵育温度和长孵育时间所造成的如水分蒸发的问题，可能会改变杂交混合物中的重要参数，例如盐浓度，尤其是当杂交反应的体积比较小的时候。目前许多公司已经开发了商品化的液相杂交诱捕富集基因组DNA测序文库的试剂盒。但其杂交溶液及杂交方法多针对于本公司所生产的捕获探针，其捕获探针 长度从60到150个碱基不等，且配方不公开，价格昂贵。每一种试剂盒的杂交温度，孵育时间，及输入文库量均不相同。因而很有必要发明一种低成本，操作简单，可重复性高，周期短，高特异性，又适用于多种方法构建的基因组DNA测序文库及捕获探针的杂交溶液及其相应地杂交方法，以满足日益增长的对靶向下一代测序的要求。
发明内容
本发明的目的是提供一种能够以较低成本，简单，快速，高效的实现杂交诱捕富集基因组DNA测序文库的杂交溶液及采用此杂交溶液进行杂交的方法。为了达到上述目的，拟采用如下技术方案：
本发明一方面涉及一种基因组DNA测序文库杂交诱捕富集溶液，其特征在于其包含磷酸钠缓冲液,柠檬酸钠缓冲液(SSC)，十二烷基硫酸钠(SDS),乙二胺四乙酸(EDTA)及登哈特(Denhardt)溶液，所述的组分独立包装。
在本发明的一个优选实施方式中，基因组DNA测序文库杂交诱捕富集溶液含有或者不含有其它组分。
在本发明的另一个优选实施方式中，所述的磷酸钠缓冲液的是0.4-0.6M磷酸钠缓冲液,其pH值介于6.8-7.2之间。
在本发明的另一个优选实施方式中，SDS的浓度是0.8-1.2％；EDTA的浓度是1-3mM。
在本发明的另一个优选实施方式中，所述的基因组DNA测序文库杂交诱捕富集溶液包括1份磷酸钠缓冲液,2份柠檬酸钠缓冲液(SSC)，1份SDS,1份EDTA及4份Denhardt溶液
在本发明的一个优选实施方式中，1M磷酸钠缓冲液(pH7.0)的配方：57.7ml 1M Na2HPO4与42.3ml 1M NaH2PO4混合
在本发明的一个优选实施方式中，20X SSC配方：3M NaCl，300mM柠檬酸钠；调节PH值至7.0。
在本发明的一个优选实施方式中，50X Denhardt溶液：该溶液中含有1％聚蔗糖(Ficoll，400型)，1％聚乙烯吡咯烷酮(polyvinylpyrrolidone)，和1％牛血清白蛋白(BSA)。
本发明另一方面还涉及一种杂交方法，其特征在于采用上述基因组DNA测序文库杂交诱捕富集溶液富集。
在本发明的一个优选实施方式中，所述的杂交方法包括如下步骤(图1)：
(1)将基因组DNA片段化为350-550bp(机械破碎，如超声处理，水力剪切，或酶促片段化均可；其所需片段长度取决于后续拟采用的测序长度)；
(2)根据后续所使用的NGS测序平台按照标准实验流程制备基因组测序文库。
(3)将总量250-2000ng基因组测序文库池于0.2mL PCR管中干燥，此文库池中可混合带有不同索引序列接头的多个基因组测序文库样品。
(4)将以下成分加入已烘干的测序文库并静置10分钟使其充分溶解：
生物素修饰的单链核苷酸捕获探针3pMole
2-5μg人类Cot-1DNA
相应两端封闭聚合物混合物各1mM
无核酸酶去离子水，将总体积补至10μL
(5)加入10μL上述基因组DNA测序文库杂交诱捕富集溶液，并用移液枪充分混合。
(6)将上述反应体系放入热循环仪运行下列程序:
Figure PCTCN2015086005-appb-000001
本杂交溶液配方简单，容易获得，降低了生产成本且易保存。
本发明的方法简便易行，只需要实验室常规使用的热循环仪。过夜杂交方便于流程的设计，且65℃杂交温度的选择适用于常用的80-120个碱基长度的单链核苷酸捕获探针。
本发明的杂交溶液配方和杂交方法可适用于多种不同方法制备(例如Illumina试剂盒，NEB试剂盒，KAPA试剂盒等)的基因组DNA测序文库的捕获富集。并可以同时在一个杂交反应中富集多个文库样品。总输入文库样品只 需要不少于250ng的DNA。
通过使用本杂交溶液和杂交方法，可以很好地控制杂交时产生的背景值，提高富集信号，优化杂交效果。实验证明，利用本杂交溶液对正常人B淋巴细胞系的全基因组DNA测序文库进行一组特定基因的靶向富集(约1Mb)，在提高杂交温度，缩短杂交时间的同时，目标基因的杂交信号显著增加(如图2A)，同时非特异性背景值显著降低(如图2B)。与某商品化杂交缓冲溶液相比，本杂交溶液配方及方法在缩短操作时间的基础上能够显著提高目标靶基因的富集倍数并有效降低背景值(如图2C)。
改进的杂交溶液配方及相应杂交方法成本较低，简便易行，有效缩短了杂交时间，并能够精准的提取所需要的DNA片段，减小背景值对信号值所产生的影响，优化杂交靶向富集效果。
附图说明：
图1、利用本发明杂交溶液配方进行基因组DNA测序文库杂交诱捕富集的流程图。(1)基因组DNA片段化。(2)构建基因组DNA测序文库。(3)混合带有不同索引序列接头的多个基因组测序文库样品。(4)加入阻断剂和封闭聚合物。(5)配置杂交反应体系。(6)在热循环仪中进行目标靶序列与探针的杂交。
图2、利用本杂交溶液与商品化的杂交溶液进行靶向富集的对比试验。A和B为本杂交溶液配方在不用杂交温度和杂交反应时间下的表现，从而优化使用本杂交溶液的杂交方法。C为使用本杂交溶液及相应杂交方法与某商品化杂交溶液及其杂交方法的对比试验。目标基因与非目标基因的富集倍数通过实时定量PCR(realtime-qPCR)进行检测。检测结果为三次独立重复试验所得平均值±标准误差(mean±SEM)。
具体实施方式
结合实施实例，详细说明本发明的实施方式，但本发明的技术范围不受限于下述实施方式，在不改变其要点的前提下，可做各种改变进行实施。
实施例1
制备基因组DNA测序文库杂交诱捕富集溶液，它是由磷酸钠缓冲液,柠檬酸钠缓冲液(SSC)，SDS,EDTA及Denhardt溶液组成。在-20℃下可长期保存。其2X浓度溶液的具体配方如下：
0.5M磷酸钠缓冲液,pH7.0  (1)
1％SDS
2mM EDTA
2X SSC,pH7.0  (2)
4X Denhardt溶液  (3)
(1)1M磷酸钠缓冲液(pH7.0)的配方：57.7ml 1M Na2HPO4与42.3ml 1M NaH2PO4混合
(2)20X SSC配方：3M NaCl，300mM柠檬酸钠；调节PH值至7.0。
(3)50X Denhardt溶液：Denhardt溶液是一种用于杂交的封闭试剂的混合物。该溶液中含有1％聚蔗糖(Ficoll，400型)，1％聚乙烯吡咯烷酮(polyvinylpyrrolidone)，和1％牛血清白蛋白(BSA)。
上述杂交溶液进行杂交的方法，按如下的步骤(图一)：
(1)将基因组DNA片段化为550bp(Covaris超声破碎)。
(2)根据Illumina TruSeq PCR-free制备基因组测序文库。
(3)将总量500ng基因组测序文库池于0.2mL PCR管中真空高速烘干。此文库池中可混合带有不同索引序列接头的多个基因组测序文库样品，每个测序文库不少于25ng。
(4)将以下成分加入已烘干的测序文库并使其充分溶解(约10分钟)：
Figure PCTCN2015086005-appb-000002
(5)加入10μL 2X杂交溶液(如2X杂交溶液在室温下解冻有沉淀析出，请于55℃水域加热10分钟溶解)，并用移液枪充分混合。杂交反应总体积 为20ul。
(6)将上述反应体系放入热循环仪运行下列程序:
Figure PCTCN2015086005-appb-000003
以上所述是本发明的优选实施例，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种基因组DNA测序文库杂交诱捕富集溶液，其特征在于其包含磷酸钠缓冲液,柠檬酸钠缓冲液(SSC)，SDS,EDTA及Denhardt溶液，所述的组分独立包装。
  2. 根据权利要求1所述的基因组DNA测序文库杂交诱捕富集溶液，基因组DNA测序文库杂交诱捕富集溶液含有或者不含有其它组分。
  3. 根据权利要求1所述的基因组DNA测序文库杂交诱捕富集溶液，所述的磷酸钠缓冲液的是0.4-0.6M磷酸钠缓冲液,其pH值介于6.8-7.2之间。
  4. 根据权利要求1所述的基因组DNA测序文库杂交诱捕富集溶液，SDS的浓度是0.8-1.2％；EDTA的浓度是1-3mM。
  5. 根据权利要求1所述的基因组DNA测序文库杂交诱捕富集溶液，所述的基因组DNA测序文库杂交诱捕富集溶液包括1份磷酸钠缓冲液,2份柠檬酸钠缓冲液(SSC)，1份SDS,1份EDTA及4份Denhardt溶液
  6. 根据权利要求1所述的基因组DNA测序文库杂交诱捕富集溶液，1M磷酸钠缓冲液(pH7.0)的配方：57.7ml 1M Na2HPO4与42.3ml 1M NaH2PO4混合
  7. 根据权利要求1所述的基因组DNA测序文库杂交诱捕富集溶液，20X SSC配方：3M NaCl，300mM柠檬酸钠；调节PH值至7.0。
  8. 根据权利要求1所述的基因组DNA测序文库杂交诱捕富集溶液，50X Denhardt溶液：该溶液中含有1％聚蔗糖(Ficoll，400型)，1％聚乙烯吡咯烷酮(polyvinylpyrrolidone)，和1％牛血清白蛋白(BSA)。
  9. 一种杂交方法，其特征在于采用上述基因组DNA测序文库杂交诱捕富集 溶液富集。
  10. 根据权利要求9所述的杂交方法，所述的杂交方法包括如下步骤：
    (1)将基因组DNA片段化为350-550bp(机械破碎，如超声处理，水力剪切，或酶促片段化均可；其所需片段长度取决于后续拟采用的测序长度)；
    (2)根据后续所使用的NGS测序平台按照标准实验流程制备基因组测序文库。
    (3)将总量250-2000ng基因组测序文库池于0.2mL PCR管中真空高速干燥，此文库池中可混合带有不同索引序列接头的多个基因组测序文库样品；
    (4)将以下成分加入已烘干的测序文库并静置10分钟使其充分溶解：
    生物素修饰的单链核苷酸捕获探针3pMole 2-5μg人类Cot-1DNA 相应两端封闭聚合物混合物各1mM 无核酸酶去离子水，将总体积补至10μL
    (5)加入10μL上述基因组DNA测序文库杂交诱捕富集溶液，并用移液枪充分混合；
    (6)将上述反应体系放入热循环仪运行下列程序:
    Figure PCTCN2015086005-appb-100001
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