WO2016023431A1 - 杂交富集捕获dna测序文库洗涤溶液及洗涤方法 - Google Patents

杂交富集捕获dna测序文库洗涤溶液及洗涤方法 Download PDF

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WO2016023431A1
WO2016023431A1 PCT/CN2015/086012 CN2015086012W WO2016023431A1 WO 2016023431 A1 WO2016023431 A1 WO 2016023431A1 CN 2015086012 W CN2015086012 W CN 2015086012W WO 2016023431 A1 WO2016023431 A1 WO 2016023431A1
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washing solution
centrifuge tube
pipette
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discard
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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 hybridization enrichment capture DNA sequencing library washing solution and a washing method.
  • Double-stranded nucleic acid molecules (such as DNA, DNA/RNA, RNA/RNA) exist in a double helix conformation. This double helix structure is stabilized by hydrogen bonds between corresponding bases in the double strand (such as A+T/U or G+C) and hydrophobic forces in the base stack.
  • the principle of base complementary pairing (hybridization) is the central principle of all processes involved in nucleic acids. In a basic hybridization reaction, a nucleic acid probe or primer is designed to bind complementarily to a target sequence. This principle is exploited by hybridization trapping enrichment of genomic DNA sequencing libraries for targeted sequencing of target genomic regions or a set of genes.
  • the efficiency and accuracy of nucleic acid hybridization depends mainly on three aspects: (1) denaturing conditions (ie, separation); (2) renaturation conditions (ie, reannealing); and (3) washing conditions after hybridization.
  • denaturing conditions ie, separation
  • renaturation conditions ie, reannealing
  • washing conditions after hybridization.
  • the specific binding between the gene target sequence and the capture probe is highly dependent on the severity of these washing steps. Highly complementary DNA duplexes are much more stable in this demanding wash environment than in low complementarity. Therefore, increasing the severity of the wash can be used to remove non-specific binding between the probe and the genomic DNA.
  • the three main adjustable factors determine the harshness of the wash: 1. Temperature. As the temperature increases, non-specific binding between the probe and the genomic DNA will be denatured and isolated. 2. Salt concentration. As the salt concentration decreases, non-specific binding between the probe and the genomic DNA will be denatured and isolated. 3. Time. As the wash time is extended, non-specific binding between the probe and the genomic DNA will be denatured and isolated. Other influencing factors, including pH and the number of washes, also affect the severity of the wash.
  • a wash solution formulation and a method of washing and subsequently eluting a sequencing library using the wash solution are proposed:
  • One aspect of the invention relates to a washing solution for hybridization trapping enriched DNA sequencing libraries, characterized in that it consists solely of sodium citrate buffer (SSC) and sodium dodecyl sulfate (SDS), said components It is individually packaged.
  • SSC sodium citrate buffer
  • SDS sodium dodecyl sulfate
  • the sodium citrate buffer comprises NaCl, and sodium citrate, the pH of which is 6.8-7.2.
  • Another aspect of the invention also relates to a method of washing a library using the above washing solution
  • washing solutions includes:
  • Washing solution I 1X SSC, 0.1% SDS;
  • Washing solution II 0.1X SSC, 0.1% SDS;
  • the centrifuge tube was removed and 1 mL of the washing solution I incubated at 65 ° C was added thereto, and the mixture was mixed up and down 10 times with a pipette, and then incubated at 65 ° C for 5 minutes;
  • the centrifuge tube was removed and 1 mL of the washing solution II incubated at 65 ° C was added thereto, and the mixture was mixed up and down 10 times with a pipette, and then incubated at 65 ° C for 5 minutes;
  • the centrifuge tube was removed and 1 mL of the washing solution II incubated at 65 ° C was added thereto, and the mixture was mixed up and down 10 times with a pipette, and then incubated at 65 ° C for 5 minutes;
  • the tube was placed on a magnetic stand, and 1 mL of Wash Solution III was added from the other side of the bead. After 30 seconds, the supernatant was carefully removed and discarded, and the remaining liquid was discarded as much as possible with a pipette.
  • the sample was taken out, vortexed and mixed rapidly at 600 g for 3 seconds to ensure that there were no magnetic beads remaining on the tube wall cover;
  • the formula is simple, easy to obtain, low cost and stable and easy to store.
  • the washing method is simple, easy to operate, and does not require special instruments.
  • the washing effect is good and the background value is low (Fig. 1), thereby improving the efficiency of targeted enrichment (the target rate can be increased from 60% to 85% compared with the commercial washing solution and method).
  • the eluted library elution method is simple and easy, and does not require additional reagent elution and purification, which reduces the loss caused by further purification and reduces the time of the process.
  • Figure 1 Comparative test using the detergent solution formulation of the present invention and corresponding washing methods and commercial washing solutions.
  • the enrichment factor (background value) of 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 washing solution enriched in a DNA sequencing library is prepared by hybridization, which is composed only of sodium citrate buffer (SSC) and SDS. It can be stored for a long time at 4 °C.
  • SSC sodium citrate buffer
  • the centrifuge tube was taken out and 1 mL of the washing solution I incubated at 65 ° C was added thereto, and the mixture was mixed up and down 10 times with a pipette, and then incubated at 65 ° C for 5 minutes.
  • the centrifuge tube was taken out and 1 mL of the washing solution II incubated at 65 ° C was added thereto, and the mixture was mixed up and down 10 times with a pipette, and then incubated at 65 ° C for 5 minutes.
  • the centrifuge tube was taken out and 1 mL of the washing solution II incubated at 65 ° C was added thereto, and the mixture was mixed up and down 10 times with a pipette, and then incubated at 65 ° C for 5 minutes.
  • the centrifuge tube was removed and 1 mL of the washing solution II at room temperature was added thereto, and the mixture was mixed up and down 10 times with a pipette, and then placed at room temperature for 5 minutes by rotating the mixer.
  • centrifuge After removing the centrifuge tube, centrifuge at 600g for 3 seconds to ensure that there are no magnetic beads remaining on the tube wall cover.
  • the magnetic beads were dried at room temperature for about 3 minutes. You can see the actual drying of the magnetic beads, and prolong or shorten the drying time.
  • the sample was taken out, vortexed and then rapidly centrifuged at 600 g for 3 seconds to ensure that no magnetic beads remained on the tube wall cover.

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Abstract

提供了一种杂交富集捕获DNA测序文库的洗涤溶液，所述洗涤溶液仅由柠檬酸钠缓冲液（SSC）及SDS组成。还提供了一种使用所述洗涤溶液的洗涤方法。

Description

杂交富集捕获DNA测序文库洗涤溶液及洗涤方法 技术领域
本发明属于分子生物学领域，具体而言，涉及一种杂交富集捕获DNA测序文库洗涤溶液及洗涤方法。
背景技术
双链核酸分子(如DNA，DNA/RNA，RNA/RNA)以双螺旋的构象存在。这种双螺旋结构是由双链中相对应碱基之间的氢键(如A+T/U或G+C)以及碱基堆积中的疏水力来稳定的。碱基互补配对原则(杂交)是涉及核酸的所有进程的中心原则。在基本的杂交反应中，核酸探针或引物被设计成与靶序列互补结合。通过杂交诱捕富集基因组DNA测序文库而进行针对目标基因组区域或一组基因的靶向测序就是利用了这一原理。
核酸杂交的效率和准确性主要取决于以下三个方面：(1)变性条件(即分离)；(2)复性条件(即重新退火)；(3)杂交后的洗涤条件。一旦核酸的互补链被分离时，“复性”或“再退火”的步骤使引物或探针结合到靶核酸的样品中。该步骤也有时被称为“杂交”步骤。在杂交富集DNA测序文库时，与基因组靶序列相结合的生物素标记的探针将与链酶亲和素磁珠紧密结合而被捕获下来。在这之后，任何没有结合的，或者非特异性错误结合的DNA序列将被通过一系列的洗涤反应而被除去。基因靶序列与捕获探针之间的特异性结合很大程度上取决于这些洗涤步骤的苛刻性。互补性高的DNA双链在这种苛刻的洗涤环境中的稳定性远高于互补性低的。因此，提高洗涤的苛刻性可以用于去除探针与基因组DNA之间的非特异性结合。
3个主要的可调节因素决定了洗涤的苛刻性：1.温度。随着温度的升高，探针与基因组DNA之间的非特异性结合将被变性，分离。2.盐浓度。随着盐浓度的降低，探针与基因组DNA之间的非特异性结合将被变性，分离。3.时间。 随着洗涤时间的延长，探针与基因组DNA之间的非特异性结合将被变性，分离。其他的影响因素包括pH值和洗涤的次数也会影响洗涤的苛刻性。
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，高效的实现对利用链霉亲和素磁珠和生物素标记探针结合而进行的杂交诱捕富集DNA测序文库进行洗涤的洗涤溶液配方及采用此洗涤溶液进行洗涤和后续洗脱测序文库的方法。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杂交诱捕富集DNA测序文库的洗涤溶液，其特征在于它是仅由柠檬酸钠缓冲液(SSC)及十二烷基硫酸钠(SDS)组成，所述的组分是独立包装的。
在本发明的一个优选实施方式中，柠檬酸钠缓冲液包括NaCl，和柠檬酸钠，其pH值是6.8-7.2.
本发明另一方面还涉及采用上述洗涤溶液进行洗涤文库的方法，
采用洗涤溶液包括：
洗涤溶液I：1X SSC，0.1％SDS；
洗涤溶液II：0.1X SSC，0.1％SDS；
洗涤溶液III：0.2X SSC，10％SDS
其特征在于包括如下步骤：
准备：对于每个富集反应，将1ml洗涤溶液I和2ml洗涤溶液II提前30分钟置于65℃温育，其他洗涤溶液置于室温；
将利用Invitrogen
Figure PCTCN2015086012-appb-000001
M270链霉亲和素磁珠对生物素标记杂交探针进行的捕获反应体系于600g快速离心3秒，以确保管壁管盖上没有磁珠残留；
将离心管放在磁力分离架上，放置1分钟，待液体澄清后，小心取出并弃去上清液；
取下离心管并向其中加入65℃温育的1mL洗涤溶液I，用移液枪上下混匀10次后，于65℃孵育5分钟；
将离心管放在磁力分离架上，放置1分钟，待液体澄清后，小心取出并弃 去上清液；
取下离心管并向其中加入65℃温育的1mL洗涤溶液II，用移液枪上下混匀10次后，于65℃孵育5分钟；
将离心管放在磁力分离架上，放置1分钟，待液体澄清后，小心取出并弃去上清液；
取下离心管并向其中加入65℃温育的1mL洗涤溶液II，用移液枪上下混匀10次后，于65℃孵育5分钟；
将离心管放在磁力分离架上，放置1分钟，待液体澄清后，小心取出并弃去上清液；
取下离心管并向其中加入室温的1mL洗涤溶液II，用移液枪上下混匀10次后，于室温放置旋转混匀器旋转5min；
取下离心管后于600g快速离心3秒，确保管壁管盖上没有磁珠残留；
将离心管放在磁力分离架上，待液体澄清后，小心取出并弃去上清液，并用P10移液枪尽量弃去所有剩余的液体；
保持离心管置于磁力架上，从磁珠的另一面管壁加入1mL洗涤溶液III，计时30秒后，小心取出并弃去上清液，并用移液枪尽量弃去剩余的液体。
待磁珠干燥之后，加入22.5μL无酶水重悬磁珠，用移液枪上下混匀10次后置于98℃加热10min；
加热后取出样品，涡旋混合后于600g快速离心3秒，确保管壁管盖上没有磁珠残留；
将离心管置于磁力架上待澄清后，立即取出20μL含有杂交诱捕富集的DNA文库样品上清液用于后续的富集后扩增。
本洗涤溶液配方及其洗涤方法的优势在于：
1.配方简单，易于获得，成本较低且稳定易于保存。
2.洗涤方法简单，易于操作，不需要特殊的仪器。
3.洗涤效果好，背景值低(如图1)，从而提高了靶向富集的效率(与商品化洗涤溶液及方法相比中靶率可从60％提高到85％)。
4.洗涤后的文库洗脱方法简单易行，不需要额外的试剂洗脱和纯化，既减少了进一步纯化所带来的损失也缩减了工艺流程的时间。
附图说明：
图1、利用本发明洗涤溶液配方及相应洗涤方法与商品化洗涤溶液的对比试验。非目标基因的富集倍数(背景值)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
配制杂交诱捕富集DNA测序文库的洗涤溶液，它是仅由柠檬酸钠缓冲液(SSC)及SDS组成。在4℃下可长期保存。
1)所需试剂：20X SSC(3M NaCl，300mM柠檬酸钠，调节PH值至7.0)
             10％SDS
2)洗涤溶液I：1X SSC
              0.1％SDS
3)洗涤溶液II：0.1X SSC
              0.1％SDS
4)洗涤溶液III：0.2XSSC
               10％SDS
采用上述洗涤溶液进行洗涤文库的方法以及后续文库的洗脱，按如下的步骤：
准备：对于每个富集反应，将1ml洗涤溶液I和2ml洗涤溶液II提前30分钟置于65℃温育。其他洗涤溶液置于室温。
将利用Invitrogen
Figure PCTCN2015086012-appb-000002
M270链霉亲和素磁珠对生物素标记杂交探针进行的捕获反应体系于600g快速离心3秒，以确保管壁管盖上没有磁珠残留。
将离心管放在磁力分离架上，放置1分钟，待液体澄清后，小心取出并弃去上清液。
取下离心管并向其中加入65℃温育的1mL洗涤溶液I，用移液枪上下混匀10次后，于65℃孵育5分钟。
将离心管放在磁力分离架上，放置1分钟，待液体澄清后，小心取出并弃去上清液。
取下离心管并向其中加入65℃温育的1mL洗涤溶液II，用移液枪上下混匀10次后，于65℃孵育5分钟。
将离心管放在磁力分离架上，放置1分钟，待液体澄清后，小心取出并弃去上清液。
取下离心管并向其中加入65℃温育的1mL洗涤溶液II，用移液枪上下混匀10次后，于65℃孵育5分钟。
将离心管放在磁力分离架上，放置1分钟，待液体澄清后，小心取出并弃去上清液。
取下离心管并向其中加入室温的1mL洗涤溶液II，用移液枪上下混匀10次后，于室温放置旋转混匀器旋转5min。
取下离心管后于600g快速离心3秒，确保管壁管盖上没有磁珠残留。
将离心管放在磁力分离架上，待液体澄清后，小心取出并弃去上清液，并用P10移液枪尽量弃去所有剩余的液体。
保持离心管置于磁力架上，从磁珠的另一面管壁加入1mL洗涤溶液III(不要弄乱磁珠)，计时30秒后，小心取出并弃去上清液，并用P10移液枪尽量弃去剩余的液体。
室温干燥磁珠约3分钟。可看实际磁珠干燥情况，适当延长或者缩短干燥时间。
待磁珠干燥之后，加入22.5μL无酶水重悬磁珠，用移液枪上下混匀10次后置于98℃加热10min。
加热后取出样品，涡旋混合后于600g快速离心3秒，确保管壁管盖上没有磁珠残留。
将离心管置于磁力架上待澄清后，立即取出20μL含有杂交诱捕富集的DNA文库样品上清液加入到0.2mL PCR管中，用于后续富集DNA文库的扩增。
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 (4)

  1. 一种杂交诱捕富集DNA测序文库的洗涤溶液，其特征在于它是仅由柠檬酸钠缓冲液(SSC)及SDS组成，所述的组分是独立包装的。
  2. 根据权利要求1所述的洗涤溶液，柠檬酸钠缓冲液包括NaCl，和柠檬酸钠，其pH值是6.8-7.2.
  3. 根据权利要求2所述的洗涤溶液，其特征在于包括如下洗涤溶液：
    洗涤溶液I：1X SSC,0.1％SDS；
    洗涤溶液II：0.1X SSC,0.1％SDS；
    洗涤溶液III：0.2X SSC,10％SDS
  4. 采用权利要求3所述的洗涤溶液进行洗涤文库的方法，其特征在于包括如下步骤：
    准备：对于每个富集反应，将1ml洗涤溶液I和2ml洗涤溶液II提前30分钟置于65℃温育，其他洗涤溶液置于室温；
    将利用Invitrogen 
    Figure PCTCN2015086012-appb-100001
     M270链霉亲和素磁珠对生物素标记杂交探针进行的捕获反应体系于600g快速离心3秒，以确保管壁管盖上没有磁珠残留；
    将离心管放在磁力分离架上，放置1分钟，待液体澄清后，小心取出并弃去上清液；
    取下离心管并向其中加入65℃温育的1mL洗涤溶液I，用移液枪上下混匀10次后，于65℃孵育5分钟；
    将离心管放在磁力分离架上，放置1分钟，待液体澄清后，小心取出并弃去上清液；
    取下离心管并向其中加入65℃温育的1mL洗涤溶液II，用移液枪上下混匀10次后，于65℃孵育5分钟；
    将离心管放在磁力分离架上，放置1分钟，待液体澄清后，小心取出并弃去上清液；
    取下离心管并向其中加入65℃温育的1mL洗涤溶液II，用移液枪上下混匀10次后，于65℃孵育5分钟；
    将离心管放在磁力分离架上，放置1分钟，待液体澄清后，小心取出并弃去上清液；
    取下离心管并向其中加入室温的1mL洗涤溶液II，用移液枪上下混匀10次后，于室温放置旋转混匀器旋转5min；
    取下离心管后于600g快速离心3秒，确保管壁管盖上没有磁珠残留；
    将离心管放在磁力分离架上，待液体澄清后，小心取出并弃去上清液，并用P10移液枪尽量弃去所有剩余的液体；
    保持离心管置于磁力架上，从磁珠的另一面管壁加入1mL洗涤溶液III，计时30秒后，小心取出并弃去上清液，并用移液枪尽量弃去剩余的液体。
    待磁珠干燥之后，加入22.5μL无酶水重悬磁珠，用移液枪上下混匀10次后置于98℃加热10min；
    加热后取出样品，涡旋混合后于600g快速离心3秒，确保管壁管盖上没有磁珠残留；
    将离心管置于磁力架上待澄清后，立即取出20μL含有杂交诱捕富集的DNA文库样品上清液用于后续的富集后扩增。
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