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 PDFInfo
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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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- 238000005406 washing Methods 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 15
- 238000001712 DNA sequencing Methods 0.000 title claims abstract description 9
- 239000000243 solution Substances 0.000 claims abstract description 54
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 9
- 239000001509 sodium citrate Substances 0.000 claims abstract description 9
- 239000000872 buffer Substances 0.000 claims abstract description 6
- 239000011324 bead Substances 0.000 claims description 26
- 239000007788 liquid Substances 0.000 claims description 21
- 239000000203 mixture Substances 0.000 claims description 18
- 239000000523 sample Substances 0.000 claims description 18
- 239000006228 supernatant Substances 0.000 claims description 18
- 238000009396 hybridization Methods 0.000 claims description 14
- 238000007885 magnetic separation Methods 0.000 claims description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000006148 magnetic separator Substances 0.000 claims description 6
- 108010090804 Streptavidin Proteins 0.000 claims description 5
- 229960002685 biotin Drugs 0.000 claims description 4
- 235000020958 biotin Nutrition 0.000 claims description 4
- 239000011616 biotin Substances 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 230000009871 nonspecific binding Effects 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 238000010828 elution Methods 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- 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
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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
- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
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- 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
Definitions
- 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
Description
Claims (4)
- 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。
- 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.
- 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
- 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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CN104178817A (zh) * | 2014-08-13 | 2014-12-03 | 邵华武 | 杂交富集捕获dna测序文库洗涤溶液及洗涤方法 |
CN106676169B (zh) * | 2016-11-15 | 2021-01-12 | 上海派森诺医学检验所有限公司 | 一种用于乳腺癌易感基因brca1和brca2突变检测的杂交捕获试剂盒及其方法 |
CN114836526A (zh) * | 2020-08-31 | 2022-08-02 | 伯科生物科技有限公司 | 一种靶向测序方法及试剂盒 |
CN112195219B (zh) * | 2020-11-03 | 2021-05-07 | 至本医疗科技(上海)有限公司 | 小panel探针靶向捕获富集测序文库分子的降噪方法 |
CN112680795A (zh) * | 2020-12-29 | 2021-04-20 | 上海派森诺生物科技股份有限公司 | 一种mNGS法检测病原微生物的DNA文库构建方法 |
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US20140094384A1 (en) * | 2011-07-15 | 2014-04-03 | Georgetown University | Stromal antigen 2 (stag2) compositions and methods |
CN104178817A (zh) * | 2014-08-13 | 2014-12-03 | 邵华武 | 杂交富集捕获dna测序文库洗涤溶液及洗涤方法 |
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US20140094384A1 (en) * | 2011-07-15 | 2014-04-03 | Georgetown University | Stromal antigen 2 (stag2) compositions and methods |
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CN104178817A (zh) * | 2014-08-13 | 2014-12-03 | 邵华武 | 杂交富集捕获dna测序文库洗涤溶液及洗涤方法 |
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