WO2019019219A1 - 核酸均一化方法及其试剂盒和应用 - Google Patents

核酸均一化方法及其试剂盒和应用 Download PDF

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WO2019019219A1
WO2019019219A1 PCT/CN2017/096420 CN2017096420W WO2019019219A1 WO 2019019219 A1 WO2019019219 A1 WO 2019019219A1 CN 2017096420 W CN2017096420 W CN 2017096420W WO 2019019219 A1 WO2019019219 A1 WO 2019019219A1
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nucleic acid
homogenization method
adsorption
group
kit
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PCT/CN2017/096420
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French (fr)
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张捷
李强
王凯
王宁
倪晶奕
卢忠鹰
邵俊斌
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上海之江生物科技股份有限公司
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Priority to EP17919474.1A priority Critical patent/EP3660163A4/en
Priority to US16/632,374 priority patent/US20200407774A1/en
Publication of WO2019019219A1 publication Critical patent/WO2019019219A1/zh

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2523/00—Reactions characterised by treatment of reaction samples
    • C12Q2523/30—Characterised by physical treatment
    • C12Q2523/308—Adsorption or desorption

Definitions

  • the invention belongs to the field of high-throughput sequencing, and particularly relates to a nucleic acid homogenization method and a kit and application thereof, which can realize rapid and stable equal dilution of nucleic acid, PCR product or high-throughput sequencing library concentration, and realize the same
  • the scale application has laid the foundation.
  • Nucleic acid screening or diagnosis has been widely used in clinical, disease prevention and control, food safety testing, import and export quarantine testing and other scenarios.
  • application scenarios such as high-throughput sequencing, in order to continuously parallel analysis, increase the amount of experimental detection, and reduce the cost of sequencing, different samples are usually labeled and mixed together for sequencing. This requires ensuring the concentration of different samples before mixing. Basically consistent, eventually different samples can produce the same amount of data.
  • Nucleic acid homogenization refers to the process of making several nucleic acids equal in amount per nucleic acid. Isocratic dilution of traditional nucleic acid concentration is usually achieved by continuous dilution after quantification. The method of quantification or fluorescent dye and fluorescent probe is usually used for quantification. If the absorbance is quantified, it is usually subjected to other substances (protein, polysaccharide, non-target fragment nucleic acid). Significant influence on the target product, and the quantitative deviation of the target product is accurate; the quantitative results are accurate by the fluorescent dye method or the fluorescent probe method, but the serial dilution operation for multiple samples is complicated, time-consuming and laborious, especially special fluorescence.
  • Magnetic beads as a kind of nanospheres, are widely used in the extraction of nucleic acids.
  • the binding of nucleic acids to magnetic beads relies primarily on electrostatic, hydrophobic and hydrogen bonding.
  • the cells or tissues are released by the lysate, and the DNA/RNA is released.
  • the surface-modified superparamagnetic silica nanomagnetic beads "specifically bind" to the nucleic acid to form a "nucleic acid-magnetic bead complex".
  • the composite is then separated by the action of an external magnetic field.
  • the non-specifically adsorbed impurities are washed away by the eluent, desalted, and purified, and the nucleic acid substance to be extracted is obtained.
  • the object of the present invention is to provide a nucleic acid homogenization method, a kit and an application thereof, which are used for solving the cumbersome method of nucleic acid homogenization in the prior art, having large deviations, and failing to realize automation. defect.
  • nucleic acid homogenization method comprising at least the following steps:
  • nucleic acid adsorption materials with the same saturation amount of nucleic acid to several nucleic acid solutions, and ensuring that each The nucleic acid adsorption material added to the nucleic acid solution can reach the nucleic acid adsorption saturation state;
  • the nucleic acid is eluted separately from the nucleic acid adsorbing material obtained by the separation.
  • the nucleic acid adsorbing material may selectively coat any one or more of a carboxyl group, an amino group, a hydroxyl group or a silicon group.
  • each of the nucleic acid solutions is added with the same and equal amount of nucleic acid adsorbing material.
  • the nucleic acid adsorbent material is a nanosphere or glass particle.
  • the nucleic acid adsorbing material is monodisperse nanospheres or monodisperse glass particles.
  • the nanospheres are magnetically adsorbed.
  • the nanospheres are formed by coating ferrous acid with Fe 3 O 4 .
  • the nano microspheres have an average particle diameter of 0.5 to 2 micrometers.
  • the method further comprises the step of adding a solvent to the nucleic acid in the step (4).
  • the method further comprises the step (4) of adding a solvent to the nucleic acid.
  • the solvent refers to a common solvent used for purifying and dispersing nucleic acids.
  • kits comprising a nucleic acid adsorbing material, the particulate material being coated with any one or more of a carboxyl group, an amino group, a hydroxyl group or a silicon group.
  • the nucleic acid adsorbing material is a nanosphere or glass particle.
  • the nucleic acid adsorbent material is a monodisperse nanosphere or a monodisperse glass particle.
  • the nanospheres can be magnetically adsorbed.
  • the nanospheres are formed by coating oleic acid with Fe 3 O 4 .
  • the particulate material has a diameter of from 0.5 to 2 microns.
  • the particulate material is monodisperse.
  • the kit further comprises at least one of a volume fraction of 70-85% ethanol, water or Tris-HCL buffer.
  • the Tris-HCL buffer has a pH of 7.0 to 8.5.
  • Another aspect of the invention provides the use of the above kit for a nucleic acid homogenization method.
  • nucleic acid homogenization method of the present invention has the following beneficial effects:
  • the homogenization of nucleic acids can be quickly realized, especially the equal dilution of a plurality of nucleic acids, which can be realized very quickly, and the deviation is small, and is particularly suitable for high-throughput determination of nucleic acids.
  • one or more of the method steps recited in the present invention are not exclusive of other method steps that may be present before or after the combination step, or that other method steps can be inserted between the steps specifically mentioned, unless otherwise It should be understood that the combined connection relationship between one or more devices/devices referred to in the present invention does not exclude that other devices/devices may exist before or after the combined device/device or Other devices/devices can also be inserted between the two devices/devices unless otherwise stated.
  • each method step is merely a convenient means of identifying the various method steps, and is not intended to limit the order of the various method steps or to limit the scope of the invention, the relative In the case where the technical content is not substantially changed, it is considered to be a scope in which the present invention can be implemented.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention employ molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. Conventional technology. These techniques are well described in the existing literature. For details, see Sambrook et al.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons , New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, and the like.
  • the ultrasonic/magnetic separation was alternately alternated three times to obtain a stable, uniformly dispersed 1 wt% nanosphere polymerization.
  • Body suspension pre-LibNorm. Under the catalysis of ammonia water, glycerol and pre-LibNorm were added at 1:1:1 v/v in tetraethyl orthosilicate, and reacted at room temperature for 24 hours. The product was washed alternately with 100% ethanol/ddH 2 O three times, and vacuum dried at 40 ° C to obtain the final product, thereby obtaining a hydrophilic, monodisperse, high magnetic amount nano magnetic composite microsphere LibNorm (referred to as "nano microsphere"). ). The average particle diameter of the nanospheres was determined to be 0.5 to 2 ⁇ m.
  • LibNorm magnetic microspheres Due to the good hydrophilic properties of LibNorm magnetic microspheres, it can be stably dispersed into a uniform nanosphere suspension in the pre-formed diluted matrix LibNorm buffer.
  • Example 2 Dilute the nanospheres in Example 1 and dilute the gradient: 10x, 30x, 90x, 120x, 240x, 360x, 480x, 540x, 600x, 720x, 900x (one replicate per gradient), using the established procedure iNaSP automated instrument to 20 ⁇ L of 20ng/ ⁇ L nucleic acid sample was diluted in proportion (add 45 ⁇ L of nanosphere to nucleic acid sample, capture for 5min at room temperature, discard the supernatant, add 90 ⁇ L 10mM Tris pH8.5 elution buffer, then Add the same amount of solvent), check the concentration with Qubit3.0, the results are as follows:
  • Figure 1 The second group of automated isodose dilution adsorption curves
  • Nanosphere Automation Group 1 1.39 1.46 1.58 2 1.40 1.48 1.55 3 1.45 1.45 1.54 4 1.43 1.52 1.57 5 1.39 1.49 1.55 6 1.50 1.51 1.57 7 1.47 1.47 1.54 8 1.46 1.45 1.56
  • the results in Table 3 show that the nano-microsphere group (manual operation, automation group) can achieve the effect of non-nano microspheres proportionally diluted nucleic acid, and the nano microsphere group (manual operation) is basically equivalent to the nano microsphere automation group.
  • the concentration difference between the other equal dilution treatments is smaller, that is, more stable.
  • Example 6 Human saliva sample microbial 16S rDNA community microecological high-throughput sequencing library equal dilution results
  • 16S rDNA PCR Prepare PCR system - 10 ⁇ buffer 5 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, dNTP (10mM) 1 ⁇ L, Dsc Tagase 0.5 ⁇ L, H 2 O 12.5 ⁇ L, Amplicon PCR Forward/Reverse Primer (1 ⁇ M 0.5 ⁇ L each, DNA template 1 ⁇ L (both per person); PCR conditions were -95 ° C for 3 minutes; 95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds, 25 cycles; then 72 ° C for 5 minutes .
  • Nanosphere Automation Group 1 1.48 1.60 1.61 2 1.50 1.59 1.58 3 1.51 1.55 1.57 4 1.33 1.54 1.54 5 1.29 1.44 1.58 6 1.47 1.52 1.60 7 1.51 1.55 1.55 8 1.38 1.46 1.59
  • Example 7 Mouse intestinal tissue nucleic acid glass beads are diluted in equal proportion
  • the results in Table 5 show that the glass bead set (manual operation, automatic operation) can achieve the effect of non-glass microbeads proportionally diluted nucleic acid, and the glass microbead set (manual operation) is basically equivalent to the glass microbead automation group.
  • the concentration difference between the other equal dilution treatments is smaller, that is, more stable.

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Abstract

本发明提供一种核酸均一化方法及其试剂盒和应用，所述方法至少包括以下步骤：将核酸饱和吸附量相同的核酸吸附材料分别加入到若干份核酸溶液中，并保证每份核酸溶液中加入的核酸吸附材料都能达到核酸吸附饱和状态；分离饱和吸附核酸的核酸吸附材料；从分离获得的核酸吸附材料中分别洗脱核酸。本发明所公开的核酸均一化方法可实现核酸、PCR产物或高通量测序文库浓度等比例稀释。

Description

核酸均一化方法及其试剂盒和应用 技术领域
本发明属于高通量测序领域，具体涉及一种核酸均一化方法及其试剂盒和应用，该方法可实现核酸、PCR产物或高通量测序文库浓度的快速、稳定等比例稀释，为其实现规模化应用奠定了基础。
背景技术
核酸的筛查或诊断，已被广泛应用于临床、疾病防控、食品安全检测、进出口检疫检测等多种场景中。对于某些应用场景，如高通量测序，为了连续平行分析、增加实验检测量、降低测序成本，通常会将不同样品标记好后混合在一起进行测序，这需要保证不同的样品混样前浓度基本一致，最终不同样品可产生同样的数据量。
核酸均一化是指将若干份核酸制成每份核酸含量相等的过程。传统的核酸浓度的等比例稀释通常采用定量后连续稀释来实现，定量通常采用吸光度或荧光染料、荧光探针的方法，若采用吸光度定量则通常会受到其他物质(蛋白、多糖、非目的片段核酸和其他杂质)的显著影响，造成目的产物定量的较大偏差；采用荧光染料法或荧光探针法定量结果准确，但对多样品的连续稀释操作，则操作复杂，费时费力，尤其特殊的荧光定量仪器(如Qubit或各种荧光定量PCR仪)和对应试剂的引入，会极大的增加核酸样品等比例稀释的成本。另外，大量样品的自动等比例稀释操作也几乎不可能实现，这对临床、疾控等时效性要求高的应用场景局限性极大。
磁珠，作为纳米微球的一种，被广泛的应用于核酸的提取。核酸结合到磁珠上主要依靠静电作用、疏水作用和氢键作用。细胞或组织在裂解液作用下，其中的DNA/RNA被释放出来。此时经过表面修饰的超顺磁性氧化硅纳米磁珠即与核酸进行“特异性结合”，形成“核酸-磁珠复合物”。然后在外加磁场的作用下，复合物即分离出来。最后经过洗脱液洗去非特异性吸附的杂质、去盐、纯化后，即得到欲提取的核酸物质。
如何利用磁珠或者其他核酸吸附材料实现对核酸的均一化处理，尚未有文献报道。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种核酸均一化方法及其试剂盒和应用，用于解决现有技术中核酸均一化方法繁琐，偏差大，无法实现自动化等的缺陷。
为实现上述目的及其他相关目的本发明提供一种核酸均一化方法，至少包括以下步骤：
(1)将核酸饱和吸附量相同的核酸吸附材料分别加入到若干份核酸溶液中，并保证每 份核酸溶液中加入的核酸吸附材料都能达到核酸吸附饱和状态；
(2)分离饱和吸附核酸的核酸吸附材料；
(3)从分离获得的核酸吸附材料中分别洗脱核酸。
优选地，所述核酸吸附材料可选择性地包被羧基、氨基、羟基或者硅基任意一种或几种。
优选地，各所述核酸溶液加入的核酸吸附材料相同且等量。
优选地，所述核酸吸附材料是纳米微球或玻璃颗粒。
优选地，所述核酸吸附材料为单分散纳米微球或单分散玻璃颗粒。
优选地，所述纳米微球可被磁性吸附。
优选地，所述纳米微球是采用油酸包被Fe3O4形成的。
优选地，所述纳米微球的平均粒径是0.5～2微米。
优选地，还包括步骤(4)向核酸中加入溶剂。
进一步地，还包括步骤(4)向核酸中加入溶剂。
所述溶剂是指用于纯化、分散核酸的常用溶剂。
本发明的另一个方面还提供了试剂盒，所述试剂盒中包含核酸吸附材料，所述颗粒材料包被羧基、氨基、羟基或者硅基任意一种或几种。
优选地，所核酸吸附材料是纳米微球或玻璃颗粒。
更优选地，所述核酸吸附材料是单分散纳米微球或单分散玻璃颗粒。
更优选地，所述纳米微球可被磁性吸附。
更优选地，所述纳米微球是采用油酸包被Fe3O4形成的。
优选地，所述颗粒材料的直径是0.5～2微米。
优选地，所述颗粒材料是单分散性。
优选地，所述试剂盒中还包括体积分数70～85％乙醇、水或Tris-HCL缓冲液中的至少一种。
优选地，所述Tris-HCL缓冲液pH是7.0～8.5。
本发明的另一方面提供了上述试剂盒用于核酸均一化方法的用途。
如上所述，本发明的核酸均一化方法，具有以下有益效果：
采用本发明所述的方法，可以快速实现对核酸的均一化，尤其是对多个核酸的等比例稀释，可以十分快捷的实现，而且偏差小，特别适用于核酸的高通量测定。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。须知，下列实施例中未具体注明的工艺设备或装置均采用本领域内的常规设备或装置。此外应理解，本发明中提到的一个或多个方法步骤并不排斥在所述组合步骤前后还可以存在其他方法步骤或在这些明确提到的步骤之间还可以插入其他方法步骤，除非另有说明；还应理解，本发明中提到的一个或多个设备/装置之间的组合连接关系并不排斥在所述组合设备/装置前后还可以存在其他设备/装置或在这些明确提到的两个设备/装置之间还可以插入其他设备/装置，除非另有说明。而且，除非另有说明，各方法步骤的编号仅为鉴别各方法步骤的便利工具，而非为限制各方法步骤的排列次序或限定本发明可实施的范围，其相对关系的改变或调整，在无实质变更技术内容的情况下，当亦视为本发明可实施的范畴。
在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring Harbor Laboratory Press，1989and Third edition，2001；Ausubel等，CURRENT PROTOCOLS IN MOLECULAR BIOLOGY，John Wiley&Sons，New York，1987 and periodic updates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
以下实施例中的试剂均来自市购。
实施例一 纳米微球的制备
1.1在氮气环境下，向Fe2+/Fe3+盐溶液中加入过量氨水，80℃反应0.5小时得Fe3O4。向其中再加入20％油酸，室温孵育1.5小时，用ddH2O洗涤，直至pH7.0。磁分离去上清后100％乙醇连续洗涤3次，40℃真空干燥后得到纳米微球粉末。按1∶1w/v加入辛烷溶液，即得油酸包埋Fe3O4纳米微球混合液。再按计量加入水和表面活性剂，超声30min后形成细乳化液b1。配制0.1wt％的SDS溶液，并1∶1v/v加入苯乙烯，搅拌均匀后即乳化形成溶液b2。1∶3v/v混合b1和b2，并加入水溶性引发剂搅拌半小时，转移到75℃水浴反应18小时，即可得到“磁性复合微球”。接着，在上述磁性复合微球中加入0.5wt％的Tween-20溶液，超声30min，磁分离弃上清，超声/磁分离连续交替作用3次，得到稳定、均一分散的1wt％纳米微球聚合体悬浮液pre-LibNorm。氨水催化下，在正硅酸乙酯中1∶1∶1v/v加入丙三醇、pre-LibNorm，室温反应24小时。产物用100％乙醇/ddH2O连续交替洗涤3次，40℃真空干燥后得到终产物，即可得到亲水、单分散、高磁量的纳米磁性复合微球LibNorm(简称“纳米微球”)。经测定，所述纳米微球的平均粒径是0.5～2微米。
1.2由于LibNorm磁性微球良好的亲水性能，可在预配好的稀释基质LibNorm buffer中稳定地分散为均一的纳米微球悬浮液。
实施例二 自动化纳米微球稀释梯度验证
对实施例1中的纳米微球进行稀释，稀释梯度：10x、30x、90x、120x、240x、360x、480x、540x、600x、720x、900x(每个梯度一个复孔)，用已设好程序的iNaSP自动化仪器对20ng/μL核酸样品20μL进行等比例稀释(向核酸样品中加入纳米微球45μL，室温振荡捕获5min后，弃掉上清，加入90μL 10mM Tris pH8.5洗脱缓冲液，再加入等量溶剂)，用Qubit3.0检测浓度，结果如下：
表1iNaSP自动化操作LibNorm beads纳米微球等比例稀释结果(单位：ng/μL)
稀释度 平行1 平行2 Ave.
10x 6.68 6.72 6.70
30x 5.16 5.08 5.12
90x 3.88 4.12 4.00
120x 4.48 3.98 4.23
240x 2.81 3.05 2.93
360x 2.92 2.46 2.69
480x 2.46 2.77 2.615
540x 2.28 2.44 2.36
600x 2.12 2.44 2.28
720x 1.66 2.02 1.84
900x 2.02 1.85 1.935
Figure PCTCN2017096420-appb-000001
图1第二组自动化等比例稀释饱和吸附曲线图
根据表1实验结果，在iNaSP自动化纳米微球等比例稀释核酸测试时，LibNorm beads纳米微球的饱和吸附稀释度为120x。
实施例四 不同浓度原始样品iNaSP自动化纳米微球等比例稀释实验
用120x稀释度的纳米微球对8个不同浓度(Ct值从小到大)的核酸进行等比例稀释验证(向每份核酸样品中加入纳米微球45μL，室温振荡捕获5min后，弃掉上清，加入90μL 10mM Tris pH8.5洗脱缓冲液，再加入等量溶剂)，每个样品三个复孔。Qubit3.0检测结果如下：
表2自动化等比例稀释宫颈分泌物核酸结果(单位：ng/μL)
Figure PCTCN2017096420-appb-000002
表2结果验证了对于不同浓度的起始模板，用120x稀释的纳米微球等比例稀释后，可以实现等比例稀释要求(CV＜0.05)，即iNaSP自动化操作系统可以成功实现LibNorm beads纳米微球的自动化，且稳定性良好。同时，结果也可以看出iNaSP自动化核酸等比例稀释(自 动化组，下同)可与手动操作等效。
实施例五 小鼠小肠组织核酸LibNorm beads等比例稀释
1)称取8份小鼠小肠组织各20mg，利用
Figure PCTCN2017096420-appb-000003
96Tissue提取总核酸；
2)分别加入90μL蒸馏水于上一步提取的核酸10μL中，完成10倍稀释，获得200ng/μL核酸样品。取10倍稀释的核酸20μL分别加入120倍稀释的LibNorm beads纳米微球45μL，室温振荡捕获5min后，弃掉上清，加入90μL 10mM Tris pH8.5溶液洗脱即可，加入等量溶剂，最后利用Qubit3.0测量每个已经过等比例稀释操作的核酸浓度；
3)非纳米微球组、纳米微球自动化组为对照。Qubit3.0定量的结果如表3所示。
表3小鼠小肠组织核酸等比例稀释浓度检测结果(单位：ng/μL)
序号 非纳米微球组 纳米微球组(手动操作) 纳米微球自动化组
1 1.39 1.46 1.58
2 1.40 1.48 1.55
3 1.45 1.45 1.54
4 1.43 1.52 1.57
5 1.39 1.49 1.55
6 1.50 1.51 1.57
7 1.47 1.47 1.54
8 1.46 1.45 1.56
表3结果显示，纳米微球组(手动操作、自动化组)均能实现非纳米微球组等比例稀释核酸的效果，且纳米微球组(手动操作)与纳米微球自动化组也基本等效，尤其后者各等比例稀释处理之间浓度差异更小，即更稳定。
实施例六 人唾液样品微生物16S rDNA群落微生态高通量测序文库等比例稀释结果
1)取新鲜采集的唾液200μL，利用QIAamp DNA Mini Kit提取总核酸；
2)16S rDNA PCR：配制PCR体系——10*buffer 5μL，Mg2+(25mM)4μL，dNTP(10mM)1μL，Dsc Tag酶0.5μL，H2O 12.5μL，Amplicon PCR Forward/Reverse Primer(1μM)各0.5μL，DNA模板1μL(均为每人份)；PCR条件为——95℃3分钟；95℃30秒，55℃30秒，72℃30秒，25个循环；再72℃5分钟。
3)PCR产物经AMPure XP beads纯化后加Illumina index接头连接，接头连接PCR体系为
10*buffer 5μL，Mg2+(25mM)4μL，dNTP(10mM)1μL，Dsc Tag酶0.5μL，H2O 24.5μL，Index1和Index 2各5μL，DNA模板5μL，PCR条件为95℃3分钟；95℃30秒，55℃30秒，72℃30秒，8个循环；再72℃5分钟；
4)分别取不同浓度的测序文库20μL，加入120倍稀释的LibNorm beads纳米微球45μL，室温振 荡捕获5min后，弃掉上清，加入90μL 10mM Tris pH8.5溶液洗脱即可，向每个样品中加入等量溶剂，最后利用Qubit3.0测量每个已经过等比例稀释操作的文库浓度；
5)非纳米微球组、纳米微球自动化组为对照。Qubit3.0定量的结果如表4所示：
表4唾液样品高通量测序文库浓度检测结果(单位：ng/μL)
序号 非纳米微球组 纳米微球组(手动操作) 纳米微球自动化组
1 1.48 1.60 1.61
2 1.50 1.59 1.58
3 1.51 1.55 1.57
4 1.33 1.54 1.54
5 1.29 1.44 1.58
6 1.47 1.52 1.60
7 1.51 1.55 1.55
8 1.38 1.46 1.59
表4结果显示，纳米微球组(手动操作、自动操作)均能实现非纳米微球组等比例稀释高通量测序文库的效果，且纳米微球组(手动操作)与纳米微球自动化组也基本等效，尤其后者各等比例稀释处理之间浓度差异更小，即更稳定。
实施例七 小鼠小肠组织核酸玻璃微珠等比例稀释
1)取8份不同浓度的小鼠小肠总核酸；
2)分别加入90μL蒸馏水于上一步提取的核酸10μL中，完成10倍稀释。取10倍稀释的核酸20μL分别加入150倍稀释的玻璃微珠(直径0.5～2微米)45μL(使得每个样品中的玻璃微球吸附的核酸样品达到饱和状态)，室温振荡捕获5min后，12000rpm离心5min后弃掉上清，加入90μL10mM Tris pH8.5溶液洗脱即可，最后利用Qubit3.0测量每个已经过等比例稀释操作的核酸浓度；
3)非玻璃微珠组、玻璃微珠自动化组为对照。Qubit3.0定量的结果如表5所示：
表5小鼠小肠组织核酸玻璃微珠等比例稀释浓度检测结果(单位：ng/μL)
初始Ct值 非玻璃微珠组 玻璃微珠组(手动操作) 玻璃微珠自动化组
18.0 1.43 1.47 1.54
20.2 1.42 1.51 1.57
22.5 1.46 1.49 1.56
26.2 1.44 1.51 1.54
28.1 1.52 1.54 1.58
30.4 1.49 1.53 1.56
31.8 1.44 1.49 1.55
33.2 1.46 1.55 1.54
表5结果显示，玻璃微珠组(手动操作、自动操作)均能实现非玻璃微珠组等比例稀释核酸的效果，且玻璃微珠组(手动操作)与玻璃微珠自动化组也基本等效，尤其后者各等比例稀释处理之间浓度差异更小，即更稳定。
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法、组合物的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。

Claims (13)

  1. 一种核酸均一化方法，其特征在于，至少包括以下步骤：
    (1)将核酸饱和吸附量相同的核酸吸附材料分别加入到若干份核酸溶液中，并保证每份核酸溶液中加入的核酸吸附材料都能达到核酸吸附饱和状态；
    (2)分离饱和吸附核酸的核酸吸附材料；
    (3)从分离获得的核酸吸附材料中分别洗脱核酸。
  2. 根据权利要求1所述的核酸均一化方法，其特征在于：所述核酸吸附材料可选择性地包被羧基、氨基、羟基或者硅基任意一种或几种。
  3. 根据权利要求1所述的核酸均一化方法，其特征在于，各所述核酸溶液加入的核酸吸附材料相同且等量。
  4. 根据权利要求3所述的核酸均一化方法，其特征在于，所述核酸吸附材料是纳米微球或玻璃颗粒。
  5. 根据权利要求4所述的核酸均一化方法，其特征在于，所述核酸吸附材料为单分散纳米微球或单分散玻璃颗粒。
  6. 根据权利要求3所述的核酸均一化方法，其特征在于：所述纳米微球可被磁性吸附。
  7. 根据权利要求3所述的核酸均一化方法，其特征在于：所述纳米微球是采用油酸包被Fe3O4形成的。
  8. 根据权利要求3所述的核酸均一化方法，其特征在于：所述纳米微球的平均粒径是0.5～2微米。
  9. 根据权利要求1所述的核酸均一化方法，其特征在于：还包括步骤(4)向核酸中加入溶剂。
  10. 一种用于核酸均一化的试剂盒，其特征在于，所述试剂盒中包含核酸吸附材料，所述核酸吸附材料可选择性的包被羧基、氨基、羟基或者硅基任意一种或几种。
  11. 根据权利要求10所述的试剂盒，其特征在于，所述核酸吸附材料是纳米微球或玻璃颗粒。
  12. 根据权利要求10所述的试剂盒，其特征在于，所述试剂盒中还包括体积分数为70～85％乙醇、水或Tris-HCL缓冲液中的至少一种。
  13. 如权利要求10～12所述的试剂盒用于核酸均一化的用途。
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WOLFE: "CHROMATIN STRUCTURE AND FUNCTION", 1998, ACADEMIC PRESS

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