WO2019200580A1 - 一种同时捕获基因组目标区域正反义双链的平行液相杂交捕获方法 - Google Patents

一种同时捕获基因组目标区域正反义双链的平行液相杂交捕获方法 Download PDF

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WO2019200580A1
WO2019200580A1 PCT/CN2018/083711 CN2018083711W WO2019200580A1 WO 2019200580 A1 WO2019200580 A1 WO 2019200580A1 CN 2018083711 W CN2018083711 W CN 2018083711W WO 2019200580 A1 WO2019200580 A1 WO 2019200580A1
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dna
probe set
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sense strand
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陈轶群
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上海迪赢生物科技有限公司
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Priority to US16/619,309 priority Critical patent/US20210040540A1/en
Priority to PCT/CN2018/083711 priority patent/WO2019200580A1/zh
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12Q1/6869—Methods for sequencing
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  • the invention belongs to the field of biotechnology, and particularly relates to high-throughput sequencing library preparation, target region probe design, preparation of sense strand and antisense strand probe, double-strand liquid phase hybridization enrichment capture and sequencing method.
  • next-generation sequencing is the most important tool in genetic testing.
  • the high sensitivity and high accuracy of next-generation sequencing technologies enable large-scale identification of genetic mutations and sequencing of new species.
  • next-generation sequencing technology brings greater efficiency to the genome sequencing process, whole-genome sequencing still faces this costly problem. Therefore, targeted enrichment sequencing of target regions can be rapidly developed.
  • Targeted enrichment sequencing is the use of various means to capture specific target regions of interest from whole genome libraries for further deep sequencing and analysis. This can greatly reduce the cost of sequencing, and at the same time greatly increase the sequencing depth of the target region, enabling more detailed analysis. Therefore, targeted enrichment sequencing can be more cost-effective for various fields of genetic testing.
  • large-scale targeted enrichment methods mainly include multiplex PCR method, liquid phase hybridization method, solid phase chip capture method, molecular inversion probe method and the like.
  • liquid phase hybridization is the most efficient and widely used.
  • commercially available liquid phase hybridization methods whether genetic RNA probes, DNA probes, long probes or short probes, capture only a single strand of each library molecule during hybridization. That is, only the sense strand or the antisense strand is captured, and then double-stranded library analysis is performed by post-PCR to obtain a double-stranded library analysis and then sequenced.
  • the invention provides a method for capturing DNA of interest, which can simultaneously capture the sense strand and the antisense strand of the DNA of interest, thereby improving the capture efficiency of the target DNA, and can be used for more efficient detection of low initial or low frequency mutations.
  • the present invention first provides a method of capturing a DNA of interest, the method comprising: capturing the DNA of interest using a sense strand probe set and an antisense strand probe set targeting a DNA of interest to accomplish the object. DNA capture;
  • the sense strand probe set consists of n sense strand probes, n is greater than or equal to 1; the antisense strand probe set consists of m antisense strand probes, m being greater than or equal to 1.
  • Each probe of the sense strand probe set binds to one strand of the DNA of interest; each probe of the antisense strand probe set binds to another strand of the DNA of interest.
  • n can be equal to m.
  • both the sense strand probe set and the antisense strand probe set can cover the entire sequence of the DNA of interest.
  • Both the sense strand probe and the antisense strand probe can be RNA.
  • each probe in the sense strand probe set or the antisense strand probe binds to the DNA of interest, and the probes are stacked in a tile shape, that is, in the Any two adjacent probes on the DNA of interest satisfy one or more nucleotides downstream of the upstream probe that overlap (same) upstream of the downstream probe, and when combined with the DNA of interest, two phases The overlapping portion of the adjacent probe will be selected to bind to the DNA of interest.
  • each probe in the sense strand probe set and each probe in the antisense strand probe set may contain a recognition sequence of a transcriptase and/or a recognition sequence of a sequencing primer.
  • the transcriptase may be a T7 RNA polymerase.
  • the sequencing primer can be P3 and/or P5.
  • the recognition sequence of the T7 RNA polymerase is position 1-22 of sequence 1 in the sequence listing.
  • the single probe in the sense strand probe set and the antisense strand probe set may have a length of 120-220 nt.
  • each probe in the sense strand probe set may have a length of 181 nt.
  • Each probe in the antisense strand probe set can be 184 nt in length.
  • each of the sense strand probe set and the antisense strand probe set may be labeled with biotin.
  • the capture of the DNA of interest can be carried out in a liquid phase.
  • the DNA of interest may be an exon of EGFR, ALK, KRAS and/or BRAF.
  • the method for preparing the sense strand probe and the antisense strand probe comprises:
  • the recognition sequence of the transcriptase is added to the 5' end of the other strand of the initial probe by PCR, followed by reverse transcription to obtain the antisense strand probe set.
  • the two sequencing primers can be the P5 and the P3, respectively.
  • PCR1 represents the PCR amplification of the first step
  • PCR2 represents the PCR amplification of the second step.
  • Figure 2 is a schematic diagram of bidirectional capture.
  • Figure 3 is a graph comparing the performance of liquid phase hybridization between a positive and negative sense strand probe parallel liquid phase hybridization and current standard probes. Wherein, the unit of the abscissa is %; A is the result of the method of the present invention, and B is the result of the Agilent standard hybrid system.
  • each set of probes can cover the entire sequence of the exon of the target gene, and when it binds to the exon of the target gene, any two adjacent original probes can satisfy the 60bp nucleus downstream of the upstream original probe.
  • the glycosidic acid overlaps upstream of the downstream original probe (ie, the 60 bp nucleotide downstream of the upstream original probe is identical to the upstream sequence of the downstream original probe).
  • sequence of the probe of ALK is the sequence 1-99 in the sequence listing
  • sequence of the probe of EGFR is the sequence 100-182 in the sequence listing
  • sequence of the probe of KRAS is the sequence of sequence 183-234 in the sequence listing
  • probe of BRAF The sequence is sequence 235-248 in the sequence listing.
  • each probe is added 5'-ATGCGACGTCGCAGT-3' sequence to the 5' end of each original probe obtained in step 1, and add 5'-CTGCCTGGTCCGACA-3' to the 3' end.
  • the total length of each probe is 150 bp, which will be obtained.
  • the single probe is referred to as the initial probe, and the probe set composed of each initial probe is referred to as the initial probe set, that is, the liquid phase capture initial probe set.
  • step 3 Combine all the initial probes obtained in step 2 (probe pool synthesis based on CustomArray's OligoArray instrument). The number of moles of each probe in the initial probe set was equal.
  • Example 2 Bidirectional amplification and labeling of the probe set of Example 1
  • the probe set of Example 1 was bidirectionally enlarged and labeled, and the flow is shown in FIG.
  • the Herculase kit used was supplied by Agilent under the product number 600677. The specific method is as follows:
  • the initial probe set obtained in Example 1 was diluted to 152.8 ⁇ L to obtain an initial probe solution, and PCR amplification was carried out in the first step, and the reaction system was enlarged (total volume was 200 ⁇ L) as follows:
  • Herculase II Fusion DNA Polymerase 2 ⁇ L of Herculase II Fusion DNA Polymerase (reagent in Herculase kit);
  • the above system was mixed and divided into four PCR tubes, 50 ⁇ L per tube.
  • the reaction was carried out according to the following reaction conditions:
  • reaction products obtained in the four PCR tubes were combined, and then 360 ⁇ L of AMpure magnetic beads were added thereto for purification, and rinsed with 500 ⁇ L of ethanol. Finally, it was eluted with deionized water to obtain 152.8 ⁇ L of the first step amplification probe solution.
  • the reaction system for preparing the sense strand probe (total volume 200 ⁇ L) is as follows:
  • Herculase II Fusion DNA Polymerase 2 ⁇ L of Herculase II Fusion DNA Polymerase (reagent in Herculase kit);
  • sequence of the P5-T7 primer is GGATTCTAATACGACTCACTATAGGG ATGCGACGTCGCAGT (sequence 249 in the sequence listing ).
  • the reaction system for preparing the antisense strand probe (total volume 200 ⁇ L) is as follows:
  • Herculase II Fusion DNA Polymerase 2 ⁇ L of Herculase II Fusion DNA Polymerase (reagent in Herculase kit);
  • the first step is to amplify the probe solution.
  • sequence of the P3-T7 primer is GGATTCTAATACGACTCACTATAGGGTGGA CTGCCTGGTCCGACA (sequence 250 in the sequence listing ).
  • each reaction system was mixed and divided into four PCR tubes, each having 50 ⁇ L.
  • the reaction was carried out according to the following reaction conditions:
  • PCR tubes containing the PCR reaction product of the sense strand probe and four PCR tubes containing the PCR reaction product of the antisense strand probe were obtained.
  • Four PCR tubes containing the PCR reaction product of the sense strand probe and four PCR tubes containing the PCR reaction product of the antisense strand probe were separately combined, and then the two probes were purified according to the following steps: 360uL was added to the probe after the combination.
  • the AMpure magnetic beads (Beckman, article number A63880) were mixed and allowed to stand at room temperature for 5 min, placed on a magnetic stand for 2 min to be clarified, and the supernatant was aspirated. Add 500 uL of ethanol to the magnetic beads and mix for 2 min.
  • the ethanol was thoroughly taken out and dried at room temperature for 5 min. Finally, 25 uL of nuclease-free water was added to resuspend the magnetic beads. After standing for 2 min, the supernatant was aspirated and transferred to a new tube to obtain the target probe.
  • the sense strand probe PCR reaction product and the antisense strand probe PCR reaction product obtained in step 2 were subjected to in vitro transcription and biotin labeling as follows:
  • 10x Transcription Buffer 10x Transcription Buffer consisting of solvent and solute, solvent 0.4M Tris-HCl (pH 8.0), solute and its concentration are 60mM MgCl 2 , 100mM DTT (dithiothreitol), 20mM Spermidine);
  • T7 RNA polymerase 1.5 ⁇ L T7 RNA polymerase (T7 RNA Polymerase, 20 U/ml, Roche);
  • the labeling system was gently pipetted and incubated at 37 ° C for 16 hours to obtain a transcription-labeled reaction product
  • the final RNA yield was measured using NanoDrop RNA-40 and Qubit RNA Kit, and the probe distribution was detected using a 2100 RNA Kit, and the size of the obtained RNA was confirmed to be about 150 nt.
  • the concentration of the probe in the labeled probe solution was adjusted to 200 ng/ ⁇ L with nuclease-free water, and SUPERase-In RNase inhibitor (Ambion) was added thereto at a final concentration of 1 U/ul of the probe, and stored at -80 °C. Finally, a biotin-labeled sense strand capture probe solution and a biotin-labeled antisense strand capture probe solution were obtained.
  • Example 3 Parallel liquid phase hybridization capture and sequencing of positive and negative sense strand probes
  • PCR amplification was performed according to the following PCR conditions.
  • step 2 Place 11-12 ⁇ L of the eluted product obtained in step 1 on a PCR machine, incubate at 95 ° C for 5 min, and maintain at 65 ° C for at least 5 min to obtain a reaction product;
  • step 4 the hybridization system was sealed and sealed at 65 ° C for 16 hours to obtain a hybridization product.
  • the 5 ⁇ Herculase II Reaction Buffer, Herculase II Fusion DNA Polymerase and dNTP mixture are reagents in the Herculase kit.
  • the amplification primer sequences are as follows:
  • YYYYYYYY represents an index sequence of 8 bp in size for distinguishing samples, such as GCCACATA, CTGGCATA, etc., and the sequence only needs to satisfy different DYPostPCR-I in different samples.
  • amplification products 98 ° C for 2 min; 98 ° C for 30 s, 57 ° C for 30 s, 72 ° C for 60 s, 16 cycles; 72 ° C for 10 min.
  • step 7 After the end of step 7, 40 ⁇ L of water is added to the obtained amplification product, and then 90 ⁇ L of AMPure magnetic beads are added, purified and eluted with water to 20 ⁇ L to obtain a purified product, that is, a library after capture, and the preparation process of the library after capture is as follows.
  • Figure 2 shows.
  • step 8 The purified product obtained in step 8 was sequenced and sequenced using Illumina HiSeq NGS platform, using a 2x150 bp double-end sequencing mode. The amount of data required for sequencing is calculated based on the size of the Panel (the entire area in which the probe is designed).
  • the experiment proves that the method for capturing the target DNA of the present invention can significantly improve the capture efficiency and detection sensitivity of the DNA for liquid phase hybridization.
  • a significant increase in library complexity after capture ensures that the original variability information is obtained as much as possible at the initial amount, especially for low starting samples and low frequency mutation detection.
  • the method for capturing DNA of interest of the invention has wide application value in the fields of cancer mutation detection, targeted medication guidance, fetal genetic defects and early screening of birth defects of infants.

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Abstract

提供一种同时捕获基因组目标区域正反义双链的平行液相杂交捕获方法，该方法包括：利用靶向目的DNA的正义链探针组和反义链探针组捕获目的DNA，完成目的DNA的捕获；正义链探针组由n条正义链探针组成，n大于等于1；反义链探针组由m条反义链探针组成，m大于等于1；正义链探针组和反义链探针组均能覆盖目的DNA的全部序列；正义链探针组中各探针和反义链探针组中各探针均含有转录酶的识别序列和/或测序引物的识别序列。实验证明：可以显著提高液相杂交目的DNA的捕获效率和检测灵敏度，在癌症突变检测、靶向用药指导、胎儿遗传缺陷及婴儿出生缺陷的早期筛查等领域有着广泛的应用价值。

Description

一种同时捕获基因组目标区域正反义双链的平行液相杂交捕获方法 技术领域
本发明属于生物技术领域，具体涉及高通量测序文库制备，目标区域探针的设计、正义链和反义链探针的制备、双链液相杂交富集捕获和测序方法。
背景技术
目前，基因检测已经成为临床和科研工作的重要工具。新一代测序(NGS，Next Generation Sequencing)技术是基因检测中最重要的工具。新一代测序技术的高灵敏度和高准确性使得人们能够大规模的鉴定遗传突变和进行新物种的测序。尽管新一代测序技术为基因组测序过程带来了更高的效率，但是全基因测序仍然面临这成本过高的问题。因此对目标区域的靶向富集测序得以快速兴起发展，靶向富集测序就是利用各种手段从全基因组文库中捕获特定感兴趣的目标区域，进而进行深度测序和分析。这样可以使得测序成本大幅降低，同时使得目标区域的测序深度大幅提高，可以实现更加精细的分析。因此靶向富集测序可以更经济高效的用于基因检测的各个领域。
目前大规模靶向富集方法主要包括多重PCR法，液相杂交法，固相芯片捕获法，分子倒置探针法等等。对于大多数癌症和儿科遗传病检测等领域的较大捕获区间而言，液相杂交法的效率最高，应用也最广泛。但是目前市面上已有商品化的液相杂交法无论是基因RNA探针还是DNA探针、长探针还是短探针，在杂交过程中都是只捕获每个文库分子的单条链进行捕获，也就是只捕获正义链或反义链，然后再Post-PCR过程中补平得到双链文库分析再进行测序。因此无论捕获效率多高，永远都有一半的分子未被捕获到，这样使得目前很多应用受到严重限制，比如液态活检(Liquid Biopsy)、临床穿刺样本、极少量FFPE样本等微量样本的情况，或者癌症早期检测的低频突变等等。因此，如何在极低起始量的情况实现高效的捕获，或者普通起始量的情况下实现超低频突变检测成为最紧迫的问题。
发明公开
本发明提供一种捕获目的DNA的方法，该方法可以同时捕获目的DNA的正义链和反义链，进而提高了目的DNA的捕获效率，可以用于低起始量或低频突变的更有效检测。
为解决上述技术问题，本发明首先提供了捕获目的DNA的方法，所述方法包括：利用靶向目的DNA的正义链探针组和反义链探针组捕获所述目的DNA，完成所述目的DNA的捕获；
所述正义链探针组由n条正义链探针组成，n大于等于1；所述反义链探针组由m条反义链探针组成，m大于等于1。
所述正义链探针组的各探针均与所述目的DNA中的一条链结合；所述反义链探针组的各探针均与所述目的DNA中的另一条链结合。
n可等于m。
上述方法中，所述正义链探针组和所述反义链探针组均能覆盖所述目的DNA的全部序列。
所述正义链探针和所述反义链探针均可为RNA。
在n大于1或m大于1时，所述正义链探针组或所述反义链探针中各探针在与所述目的DNA结合时，探针间呈叠瓦片式，即在所述目的DNA上任何两个相邻的探针均满足上游探针的下游有一个或多个核苷酸与下游探针的上游重叠(相同)，在与所述目的DNA结合时，两个相邻探针的重叠部分会择其一与所述目的DNA结合。
上述方法中，所述正义链探针组中各探针和所述反义链探针组中各探针均可含有转录酶的识别序列和/或测序引物的识别序列。
上述方法中，所述转录酶可为T7RNA聚合酶。所述测序引物可为P3和/或P5。
所述T7RNA聚合酶的识别序列为序列表中序列1的第1-22位。所述P3的序列可为序列表中序列2的26-46位；所述P5的序列可为序列表中序列1的第23-41位。
上述方法中，所述正义链探针组和所述反义链探针组中的单条探针的长度可为120-220nt。
上述方法中，所述正义链探针组中各探针的长度可为181nt。所述反义链探针组中各探针的长度可为184nt。
上述方法中，所述正义链探针组和所述反义链探针组中的各探针均可标记有生物素。
上述方法中，所述目的DNA的捕获可在液相中进行。
在本发明的一个实施例中，所述目的DNA可为EGFR、ALK、KRAS和/或BRAF的外显子。
所述正义链探针和所述反义链探针的制备方法包括：
1)根据所述目的DNA制备n条原始探针，各原始探针在与所述目的DNA结合时，探针间呈叠瓦片式，即在所述目的DNA上任何两个相邻的探针均满足上游探针的下游有一个或多个核苷酸与下游探针的上游重叠(相同)，在与所述目的DNA结合时，两个相邻探针的重叠部分会择其一与所述目的DNA结合；
2)利用PCR在各原始探针的两端分别添加两条测序引物的序列，得到初始探针，所述初始探针为DNA片段；
3)通过PCR在所述初始探针的一条链的5′端添加所述转录酶的识别序列，然后进行反转录，得到所述正义链探针组；
通过PCR在所述初始探针的另一条链的5′端添加所述转录酶的识别序列，然后进行反转录，得到所述反义链探针组。
所述两条测序引物可分别为所述P5和所述P3。
所述正义链探针组和所述反义链探针组的下述任一应用，也属于本发明的保护范围：
X1)在捕获目的DNA中的应用；
X2)在制备捕获目的DNA产品中的应用；
X3)在目的DNA测序中的应用；
X4)在制备目的DNA测序产品中的应用；
X5)在遗传病检测中的应用；
X6)在制备遗传病检测产品中的应用；
X7)在癌症检测中的应用；
X8)在制备癌症检测产品中的应用；
X9)在液态活检中的应用；
X10)在制备液态活检产品中的应用；
X11)在胎儿遗传缺陷早期筛查中的应用；
X12)在制备胎儿遗传缺陷早期筛查产品中的应用；
X13)在婴儿出生缺陷早期筛查中的应用；
X14)在制备婴儿出生缺陷早期筛查产品中的应用；
X15)在检测低频或超低频突变中的应用；
X16)在制备检测低频或超低频突变产品中的应用。
所述捕获目的DNA的方法的下述任一应用，也属于本发明的保护范围：
X1)在捕获目的DNA中的应用；
X2)在目的DNA测序中的应用；
X3)在遗传病检测中的应用；
X4)在癌症检测中的应用；
X5)在液态活检中的应用；
X6)在胎儿遗传缺陷早期筛查中的应用；
X7)在婴儿出生缺陷早期筛查中的应用；
X8)在检测低频或超低频突变中的应用。
附图说明
图1为对实施例1的探针组进行双向放大和标记的流程图。其中，PCR1表示第一步的PCR放大，PCR2表示第二步的PCR放大，
图2为双向捕获示意图。
图3为正反义链探针平行液相杂交捕获与目前标准探针液相杂交性能比较图。其中，横坐标的单位为％；A为本发明的方法的结果，B为安捷伦标准的杂交体系的结果。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。下述实施例中的实验方法，如无 特殊说明，均为常规方法。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。下述实施例中，如无特殊说明，序列表中各核苷酸序列的第1位均为相应DNA的5′末端核苷酸，末位均为相应DNA的3′末端核苷酸。
实施例1：液相捕获初始探针组的制备
1，根据目标基因(EGFR，ALK,KRAS，BRAF)外显子的序列，设计四组叠瓦片式原始探针，这四组探针中所有原始探针长度均为120mer，且均为单链DNA，每组探针能覆盖目标基因外显子的全部序列，在与目标基因外显子结合时，任何两个相邻的原始探针均可满足上游原始探针的下游有60bp的核苷酸与下游原始探针的上游重叠(即上游原始探针的下游有60bp的核苷酸与下游原始探针的上游序列相同)。ALK的探针的序列为序列表中序列1-99，EGFR的探针的序列为序列表中序列100-182，KRAS的探针的序列为序列表中序列183-234，BRAF的探针的序列为序列表中序列235-248。
2，在步骤1得到的各原始探针的5′端添加5′-ATGCGACGTCGCAGT-3′序列，3′端添加5′-CTGCCTGGTCCGACA-3′，每个探针的总长度均为150bp，将得到的单个探针记为初始探针，各初始探针所组成的探针组记为初始探针组，即液相捕获初始探针组。
3，合并步骤2得到的所有初始探针(基于CustomArray公司的OligoArray仪器进行探针池合成)。初始探针组中各探针的摩尔数均相等。
实施例2：对实施例1的探针组进行双向放大和标记
对实施例1的探针组进行双向放大和标记，流程如图1所示。所用Herculase试剂盒为安捷伦公司产品，货号为600677。具体方法如下：
1、将实施例1得到的初始探针组稀释到152.8μL得到初始探针溶液，进行第一步的PCR放大，放大反应体系(总体积为200μL)如下：
1.6μL P5引物(10μM)(序列为AGGG ATGCGACGTCGCAGT)；
1.6μL P3引物(10μM)(序列为GTGGA CTGCCTGGTCCGACA)；
2μL dNTP混合物(各dNTP的浓度均为100mM)(Herculase试剂盒试剂)；
40μL 5×Herculase II Reaction Buffer(Herculase试剂盒中试剂)；
2μL Herculase II Fusion DNA Polymerase(Herculase试剂盒中试剂)；
152.8μL初始探针溶液。
将上述体系混匀后分在四个PCR管中，每管50μL。按照如下反应条件进行反应：
Figure PCTCN2018083711-appb-000001
Figure PCTCN2018083711-appb-000002
将四个PCR管中得到的反应产物合并，然后向其中添加360μL的AMpure磁珠进行纯化，500μL乙醇漂洗。最后用去离子水洗脱，得到152.8μL第一步放大探针溶液。
2、将探针组进行第二步的PCR放大：
制备正义链探针的反应体系(总体积200μL)如下：
1.6μL P5-T7引物(10μM)；
1.6μL P3引物(10μM)；
2μL dNTP混合物(各dNTP的浓度均为100mM)(Herculase试剂盒试剂)；
40μL 5×Herculase II Reaction Buffer(Herculase试剂盒中试剂)；
2μL Herculase II Fusion DNA Polymerase(Herculase试剂盒中试剂)；
152.8μL第一步放大探针溶液。
其中，P5-T7引物的序列为GGATTCTAATACGACTCACTATAGGG ATGCGACGTCGCAGT(序列表中序列249)。
制备反义链探针的反应体系(总体积200μL)如下：
1.6μL P5引物(10μM)；
1.6μL P3-T7引物(10μM)；
2μL dNTP混合物(各dNTP的浓度均为100mM)(Herculase试剂盒试剂)；
40μL 5×Herculase II Reaction Buffer(Herculase试剂盒中试剂)；
2μL Herculase II Fusion DNA Polymerase(Herculase试剂盒中试剂)；
152.8μL 第一步放大探针溶液。
其中，P3-T7引物的序列为GGATTCTAATACGACTCACTATAGGGTGGA CTGCCTGGTCCGACA(序列表中序列250)。
针对上述的两个反应体系，将每个反应体系混匀后分别分在四个PCR管中，每管50μL。按照如下反应条件进行反应：
Figure PCTCN2018083711-appb-000003
Figure PCTCN2018083711-appb-000004
反应结束后，得到含有正义链探针PCR反应产物的四个PCR管和含有反义链探针PCR反应产物的四个PCR管。分别合并含有正义链探针PCR反应产物的四个PCR管和含有反义链探针PCR反应产物的四个PCR管，然后按照下述步骤纯化两种探针：合并后向探针中加入360uL的AMpure磁珠(贝克曼公司，货号A63880)混匀后室温放置5min，置于磁力架上2min待溶液澄清，吸取上清液，在磁珠中加入500uL乙醇混匀后等待2min，待溶液澄清后彻底取出乙醇，室温晾干5min。最后加入25uL无核酸酶水重悬磁珠，静置2min后吸取上清转移到新管中，即得到目的探针。
3、探针的标记
按照如下方法对步骤2得到的正义链探针PCR反应产物和反义链探针PCR反应产物进行体外转录和生物素标记：
取1μg PCR反应产物，用无核酸酶水(Nuclease-free water)调整定容到28.2μL，然后向其中添加如下体积的如下试剂，得到体外转录标记体系(总体积为50μL)：
5μL 10x转录缓冲液(10x Transcription Buffer，该缓冲液由溶剂和溶质组成，溶剂为0.4M Tris-HCl(pH 8.0)，溶质及其浓度分别为60mM MgCl 2,100mM DTT(dithiothreitol),20mM亚精胺(spermidine))；
12.5μL生物素RNA Labeling Mix(生物素标记混合液，罗氏公司，货号11685597910)；
1.5μL T7RNA聚合酶(T7RNA Polymerase，20U/ml，Roche)；
0.3μL焦磷酸酶(Pyrophosphatase，0.1U/ml,NEB)；
2.5μL SUPERase-In RNase inhibitor(20U/ml,Life Technologies，RNA酶抑制剂)。
将标记体系轻轻吹打混匀，37℃孵育16小时，得到转录标记反应产物；
向转录标记反应产物中添加1μL的TURBO DNase(2U/μL，Ambion)在37℃消化处理15min，再用Qiagen的RNeasy Mini Kit纯化，用50μL的无核酸酶水洗脱，洗脱两次，合并两次洗脱产物，得到100μL标记后的探针溶液。
使用NanoDrop的RNA-40和Qubit RNA Kit测定最终的RNA产量，使用2100RNA Kit检测探针的分布，确认得到的RNA的大小为150nt左右。
用无核酸酶水调整标记后的探针溶液中探针的浓度至200ng/μL，按照探针终浓度为1U/ul向其加入SUPERase-In RNase inhibitor(Ambion)，-80度保存。最终得到生物素标记正义链捕获探针溶液和生物素标记反义链捕获探针溶液。
实施例3：正反义链探针的平行液相杂交捕获及测序
1、取30ng血浆游离DNA标准品(Horizon公司，货号HD780)进行建库，得到基因组DNA文库。文库的构建所用试剂盒为(无锡迪赢生物科技有限公司，货号D8010A)，方法如下：
在30ng的血浆游离DNA标准品中补水定容至54.7uL，再加入9.8uL的末端修复缓冲液(蓝盖1号管)和5.5uL的末端修复酶(蓝盖2号管)，吹打混合均匀，放置于PCR仪上20度孵育30min，不适用热盖。加入120uL的AMPure磁珠(确保在室温平衡30分钟以上)混合均匀。室温放置5min，注意此时不要放在磁力架上。放置于磁力架上静置澄清后弃上清液。加入200uL的80％乙醇(当天配置)，静置1分钟后弃上清。再次加入200uL的80％乙醇(当天配置)，静置30秒后弃上清，快速离心后弃去残余乙醇，室温放置3分钟。从磁力架上取下管子，加入42uL的无核酸酶水重悬磁珠，再加入6uL加尾缓冲液(绿盖3号管)和2uL的加尾酶(绿盖4号管)，用旋涡混匀仪充分混合均匀，30度孵育30min，不加热盖。加入90uL的纯化结合液(黄盖5号管，室温平衡30分钟)充分混合均匀。从磁力架上取下管子，加入30uL的无核酸酶水重悬磁珠，在加入15uL的连接缓冲液液(橙盖6号管)和5uL的接头混合液(棕盖7号管)。用旋涡混匀仪充分混合均匀。放置于PCR仪上20℃孵育15min，不加热盖，加入70uL的纯化结合液(黄盖5号管)，纯化，最后用20uL的无核酸酶水洗脱。在新的PCR管中加入25uL的PCR混合液(粉盖8号管)，5uL的Pre-PCR引物混合液(白盖9号管)和20uL的洗脱DNA。混合均匀。按照如下PCR条件执行PCR扩增。
步骤温度时间
S1    98℃    45s
S2    98℃    15s
S3    65℃    30s
S4    72℃    30s
S5    重复S2-S4 6-8次
S6    72℃    5min
S7    4℃    保持
在PCR管中加入70uL的AMPure磁珠，最后用30uL的无核酸酶水洗脱。
取750ng的基因组DNA文库加水补足50μL，利用1.8X磁珠纯化后用下列试剂进行洗脱，首先加入6.4μL的无核酸酶水，然后加入2.5μL human Cot-1DNA(Thermo Fisher公司，货号15279011，1mg/ml)，2.5μL鲑精DNA(salmon sperm DNA，Thermo Fisher公司，货号15632-011，10mg/ml)和0.6μL的Blocker 3(无锡迪赢生物科技公司，货号D8014A)。
2、将步骤1得到的洗脱产物11-12μL放置于PCR仪上，95℃孵育5min，65℃ 保持至少5min，得到反应产物；
3、根据样本量在1.5mL EP管中制备如下混合液：向EP管中依次加入6.63μL的20x SSPE，0.27μL的0.5M EDTA，2.65μL的50xDenhardt’s(Thermo Fisher公司，货号750018)，3.45μL的1％SDS，旋涡剧烈震荡2s后spin(离心快甩)，如果有沉淀则放置于65℃孵育5min；然后再向该EP管中加入1μL的SUPERase-In RNase inhibitor(20U/μL)以及实施例2的正义链捕获探针溶液1μL和反义链捕获探针溶液1μL，旋涡剧烈震荡5s中后Spin，混合均匀得到混合液。
4、加16μL上述步骤3的溶液到步骤2的反应产物中，轻轻吹打10次，得到杂交体系。
5、步骤4结束后，将杂交体系密封盖子后65℃杂交16小时，得到杂交产物。
6、准备50μL的Dynabeads MyOne Streptavidin T1beads(Life Technologies)，使用Bead结合液洗脱，将得到的200μL的磁珠悬液加入步骤5的杂交产物中，在旋转混匀仪上室温孵育30min后于磁力架上吸附，弃掉上清，加入200μL的高盐洗脱液(无锡迪赢生物科技有限公司，D8013A)室温孵育15min，磁力架吸附弃掉上清，再加入65℃预热的低盐洗脱液(无锡迪赢生物科技有限公司，D8013A)，共计洗三次。最后加入31.5μL的水到磁珠中，得到纯化产物。
7、向步骤6获得的纯化后产物中依次加入10μL的5×Herculase II Reaction Buffer、1μL的Herculase II Fusion DNA Polymerase、0.5μL的dNTP混合物(各dNTP的浓度均为100mM)、1μL的12.5×SYBR Green和1μL的DYPostPCR-U。然后每个样本加入5μL的不同的DYPostPCR-I，得到不同扩增体系。
5×Herculase II Reaction Buffer、Herculase II Fusion DNA Polymerase和dNTP混合物均为Herculase试剂盒中试剂。
扩增引物序列如下：
DYPostPCR-U：
5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3′；
DYPostPCR-I：
5′-CAAGCAGAAGACGGCATACGAGATYYYYYYYYGTGACTGGAGTT*C-3′。YYYYYYYY代表用于区分样本的大小为8bp的Index序列，比如GCCACATA，CTGGCATA等，该序列只需满足使不同样本中的DYPostPCR-I不同即可。
DYPostPCR-U和DYPostPCR-I序列中的*表示硫代修饰。
将不同扩增体系均在如下条件下进行反应得到扩增产物：98℃2min；98℃30s，57℃30s，72℃60s，16个循环；72℃10min。
8、步骤7结束后，向得到的扩增产物中添加40μL水，然后加入90μL AMPure 磁珠，纯化并用水洗脱至20μL，得到纯化产物，即捕获后文文库，捕获后文库的制备流程如图2所示。
9、将步骤8得到的纯化产物上机测序，使用Illumina公司HiSeq NGS平台进行测序，采用2x150bp双端测序模式。根据Panel(设计探针的全部区域)大小计算出测序所需数据量。
结果如图3所示。从图中可以看出：与业内公认的安捷伦标准的杂交体系(Agilent G9611A)比较，本发明的利用正义链捕获探针和反义链捕获探针同时捕获目的DNA的捕获效率、文库复杂度(特定测序深度下文库中唯一Reads的比例)和对捕获的DNA测序的大于1000x覆盖度均明显大于安捷伦标准的杂交体系。
工业应用
实验证明：本发明的捕获目的DNA的方法可以显著提高液相杂交目的DNA的捕获效率和检测灵敏度。捕获后文库复杂度的显著提高可以确保起始量情况下可以尽可能得到更多的原始变异信息，尤其适合低起始量样本和低频突变检测。本发明的捕获目的DNA的方法在癌症突变检测、靶向用药指导、胎儿遗传缺陷及婴儿出生缺陷的早期筛查等领域有着广泛的应用价值。

Claims (10)

  1. 捕获目的DNA的方法，包括：利用靶向目的DNA的正义链探针组和反义链探针组捕获所述目的DNA，完成所述目的DNA的捕获；
    所述正义链探针组由n条正义链探针组成，n大于等于1；所述反义链探针组由m条反义链探针组成，m大于等于1。
  2. 根据权利要求1所述的方法，其特征在于：所述正义链探针组和所述反义链探针组均能覆盖所述目的DNA的全部序列。
  3. 根据权利要求1或2所述的方法，其特征在于：所述正义链探针组中各探针和所述反义链探针组中各探针还均含有转录酶的识别序列和/或测序引物的识别序列。
  4. 根据权利要求3所述的方法，其特征在于：所述转录酶为T7RNA聚合酶；所述测序引物为P3和/或P5。
  5. 根据权利要求1-4中任一所述的方法，其特征在于：所述正义链探针组和所述反义链探针组中的单条探针的长度为120-220nt。
  6. 根据权利要求1-5中任一所述的方法，其特征在于：所述正义链探针组中各探针的长度为181nt；和/或，所述反义链探针组中各探针的长度为184nt。
  7. 根据权利要求1-6中任一所述的方法，其特征在于：所述正义链探针组和所述反义链探针组中的各探针均标记有生物素。
  8. 根据权利要求1-7中任一所述的方法，其特征在于：所述目的DNA的捕获在液相中进行。
  9. 权利要求1-7中任一所述正义链探针组和所述反义链探针组的下述任一应用：
    X1)在捕获目的DNA中的应用；
    X2)在制备捕获目的DNA产品中的应用；
    X3)在目的DNA测序中的应用；
    X4)在制备目的DNA测序产品中的应用；
    X5)在遗传病检测中的应用；
    X6)在制备遗传病检测产品中的应用；
    X7)在癌症检测中的应用；
    X8)在制备癌症检测产品中的应用；
    X9)在液态活检中的应用；
    X10)在制备液态活检产品中的应用；
    X11)在胎儿遗传缺陷早期筛查中的应用；
    X12)在制备胎儿遗传缺陷早期筛查产品中的应用；
    X13)在婴儿出生缺陷早期筛查中的应用；
    X14)在制备婴儿出生缺陷早期筛查产品中的应用；
    X15)在检测低频或超低频突变中的应用；
    X16)在制备检测低频或超低频突变产品中的应用。
  10. 权利要求1-8中任一所述方法的下述任一应用：
    X1)在捕获目的DNA中的应用；
    X2)在目的DNA测序中的应用；
    X3)在遗传病检测中的应用；
    X4)在癌症检测中的应用；
    X5)在液态活检中的应用；
    X6)在胎儿遗传缺陷早期筛查中的应用；
    X7)在婴儿出生缺陷早期筛查中的应用；
    X8)在检测低频或超低频突变中的应用。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102808027A (zh) * 2012-08-16 2012-12-05 苏州工业园区为真生物医药科技有限公司 检测egfr基因突变位点的试剂盒
CN102827830A (zh) * 2012-08-14 2012-12-19 盛司潼 一种捕获核酸片段的方法
CN103667442A (zh) * 2013-09-13 2014-03-26 西南民族大学 一种针对微量样本的转录组高通量测序方法
CN106086013A (zh) * 2016-06-30 2016-11-09 厦门艾德生物医药科技股份有限公司 一种用于核酸富集捕获的探针及设计方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040091888A1 (en) * 2002-03-20 2004-05-13 Takeshi Nishio Method for identification of S genotype in brassicaceae
CA2618163A1 (en) * 2008-02-07 2009-08-07 K. W. Michael Siu Head and neck cancer biomarkers
US20170342495A1 (en) * 2016-05-11 2017-11-30 Sinai Health System Markers of preterm birth

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102827830A (zh) * 2012-08-14 2012-12-19 盛司潼 一种捕获核酸片段的方法
CN102808027A (zh) * 2012-08-16 2012-12-05 苏州工业园区为真生物医药科技有限公司 检测egfr基因突变位点的试剂盒
CN103667442A (zh) * 2013-09-13 2014-03-26 西南民族大学 一种针对微量样本的转录组高通量测序方法
CN106086013A (zh) * 2016-06-30 2016-11-09 厦门艾德生物医药科技股份有限公司 一种用于核酸富集捕获的探针及设计方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
WANG ET AL., PROCEEDINGS OF 2016 ANNUAL CONFERENCE OF CHINA PLANT PROTECTION SOCIETY, 10 November 2013 (2013-11-10) *
XU ET AL., NATIOANL MEDICAL JOURNAL OF CHINA, 10 October 2006 (2006-10-10), pages 2606 - 2610 *

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