CN117487797A - 一种磁珠法人全血基因组提取试剂及其应用 - Google Patents
一种磁珠法人全血基因组提取试剂及其应用 Download PDFInfo
- Publication number
- CN117487797A CN117487797A CN202311570183.6A CN202311570183A CN117487797A CN 117487797 A CN117487797 A CN 117487797A CN 202311570183 A CN202311570183 A CN 202311570183A CN 117487797 A CN117487797 A CN 117487797A
- Authority
- CN
- China
- Prior art keywords
- magnetic bead
- whole blood
- solution
- reagent
- human whole
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000011324 bead Substances 0.000 title claims abstract description 52
- 238000000605 extraction Methods 0.000 title claims abstract description 50
- 210000004369 blood Anatomy 0.000 title claims abstract description 31
- 239000008280 blood Substances 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 28
- 238000005406 washing Methods 0.000 claims abstract description 37
- 239000003480 eluent Substances 0.000 claims abstract description 17
- 230000009089 cytolysis Effects 0.000 claims abstract description 11
- 108010067770 Endopeptidase K Proteins 0.000 claims abstract description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 6
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 37
- 239000007788 liquid Substances 0.000 claims description 26
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 21
- 235000019441 ethanol Nutrition 0.000 claims description 14
- 239000007853 buffer solution Substances 0.000 claims description 11
- 150000003839 salts Chemical class 0.000 claims description 10
- 230000003196 chaotropic effect Effects 0.000 claims description 7
- 239000002736 nonionic surfactant Substances 0.000 claims description 7
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 6
- 239000002738 chelating agent Substances 0.000 claims description 6
- 150000002500 ions Chemical class 0.000 claims description 6
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- -1 polytetrafluoroethylene Polymers 0.000 claims description 5
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 4
- 229940040526 anhydrous sodium acetate Drugs 0.000 claims description 4
- 239000003945 anionic surfactant Substances 0.000 claims description 4
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 4
- 229940044631 ferric chloride hexahydrate Drugs 0.000 claims description 4
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims description 4
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 150000001298 alcohols Chemical class 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- IDOQDZANRZQBTP-UHFFFAOYSA-N 2-[2-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=CC=C1OCCO IDOQDZANRZQBTP-UHFFFAOYSA-N 0.000 claims description 2
- IEQAICDLOKRSRL-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-dodecoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO IEQAICDLOKRSRL-UHFFFAOYSA-N 0.000 claims description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- 229920004929 Triton X-114 Polymers 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 claims description 2
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 claims description 2
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 2
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 2
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 2
- 239000000376 reactant Substances 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 239000007974 sodium acetate buffer Substances 0.000 claims description 2
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 2
- 235000009518 sodium iodide Nutrition 0.000 claims description 2
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 claims description 2
- 229910001488 sodium perchlorate Inorganic materials 0.000 claims description 2
- HFQQZARZPUDIFP-UHFFFAOYSA-M sodium;2-dodecylbenzenesulfonate Chemical compound [Na+].CCCCCCCCCCCCC1=CC=CC=C1S([O-])(=O)=O HFQQZARZPUDIFP-UHFFFAOYSA-M 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims 2
- 239000006174 pH buffer Substances 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 230000000052 comparative effect Effects 0.000 description 15
- 108020004707 nucleic acids Proteins 0.000 description 12
- 102000039446 nucleic acids Human genes 0.000 description 12
- 150000007523 nucleic acids Chemical class 0.000 description 12
- 239000006166 lysate Substances 0.000 description 6
- 238000007400 DNA extraction Methods 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 238000002205 phenol-chloroform extraction Methods 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- 238000007886 magnetic bead extraction Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000005185 salting out Methods 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- 101150072531 10 gene Proteins 0.000 description 1
- 101150029062 15 gene Proteins 0.000 description 1
- 101150028074 2 gene Proteins 0.000 description 1
- 101150096316 5 gene Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
- B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28009—Magnetic properties
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28016—Particle form
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Plant Pathology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明属于基因提取试剂的技术领域,具体涉及一种磁珠法人全血基因组提取试剂及其应用。所述提取试剂的组分包括蛋白酶K液、磁珠液、裂解结合液、洗涤液1、洗涤液2和洗脱液;所述磁珠液为中的磁珠为是二氧化硅包覆的四氧化三铁磁珠,本发明试剂试剂可实现自动化提取,所得基因组DNA在浓度≥20ng/μL、A260/A280≥1.7且≤2.1、A260/A230≥1.6,且提取时间由目前常规50~90分钟缩短至40分钟以内。
Description
技术领域
本发明属于基因提取试剂的技术领域,具体涉及一种磁珠法人全血基因组提取试剂及其应用。
背景技术
基因组DNA是遗传信息的重要载体,同时也是当前科学研究前沿中分子生物学领域重要的研究对象。基因组DNA提取作为上游实验的重点,其提取所得基因组DNA的浓度、纯度与完整性,会对下游的各类检测会产生重要的影响,如文库构建、基因测序、PCR扩增、酶切实验和基因分子杂交等。因此,基因组DNA的提取与纯化步骤显得尤其重要。
现有技术中,对基因组提取方法使用的较多的,如苯酚-氯仿法、盐析法、吸附柱提取法与磁珠提取法。其中,苯酚-氯仿法中,虽然所得基因组DNA纯度较高,但试剂毒性较强,对实验操作人员存在一定危害;盐析法通常依附吸附柱提取法,通过高浓度的无机盐沉淀基因组DNA,排除醇类残留所造成的危害,但所得核酸易高浓度无机盐残留,对下游实验造成影响;吸附柱提取法所得基因组DNA蛋白与盐残留会较低,但同时核酸浓度也会偏低,且自动化程度较难实现;磁珠提取法自动化程度较高,易于普及与推广,但因为方法学原因,此方案要得到高纯度核酸比较困难。且在现有方法中,全血基因组DNA提取时间在50~90分钟,提取时间过长。
有专利CN109207474A公开了一种外周血基因组DNA快速提取方法,包括裂解液、蛋白酶K、磁珠、75%乙醇与无酶水,其仅针对所得基因组DNA与A260/280比值进行验证,无法准确判断所得基因组DNA的完整性以及A260/230比值是否达到要求;还有CN111996190A公开了一种全血基因组DNA提取试剂盒,裂解液、调节珠、纳米磁珠、洗涤液A、洗涤液B和洗脱液,其仅公开了提取试剂盒的配制方法,无任何数据表明其提取所得基因组DNA的浓度、纯度以及完整性方面均达到要求;CN102888397公开了一种利用磁珠提取全血基因组DNA的试剂盒及应用,此发明提取时间过长,需用时1小时以上,且仅针对所得基因组DNA与A260/280比值与基因组DNA的完整性进行验证,无法准确判断所得A260/230比值是否达到要求。
发明内容
本针对上述问题,本发明的目的在于提供一种磁珠法人全血基因组提取试剂,可实现自动化提取,所得基因组DNA在浓度、纯度以及完整度方面均达到要求(浓度≥20ng/μL、A260/A280≥1.7且≤2.1、A260/A230≥1.6),且提取时间由目前常规50~90分钟缩短至40分钟以内。
本发明解决技术问题采用如下技术方案:
一种磁珠法人全血基因组提取试剂,所述提取试剂的组分包括蛋白酶K液、磁珠液、裂解结合液、洗涤液1、洗涤液2和洗脱液;
所述裂解结合液的组分包括:离液盐2~5mmol/L、非离子表面活性剂0.5~10%、阴离子表面活性剂0.1~5%、pH缓冲液5~10%、醇类10~50%、离子螯合剂0.1~10mmol/L;
所述洗涤液1的组分按重量百分比计包括:离液盐2~5mmol/L,非离子表面活性剂0.5~10%、pH缓冲液10~100mmol/L、醇类10~50%;
所述洗涤液2的组分按重量百分比计包括:pH缓冲液10~100mmol/L、醇类10~50%;
所述洗脱液的的组分包括:离子螯合剂0.1~10mmol/L、pH缓冲液10~100mmol/L。
本发明采用的洗涤液1的配比作为抑制物祛除剂,非离子表面活性剂适当的使用有助于抑制物的洗脱祛除,当用量过多会使所得核酸模板纯度下降,用量过少则无法有效清除抑制物。
优选地,所述蛋白酶K液的浓度为20 mg/mL;
优选地,所述磁珠液中的磁珠为经二氧化硅包覆的四氧化三铁磁珠,制备工艺为:
将六水合氯化铁、无水乙酸钠与PEG2000溶于乙二醇中,将溶解物倒入聚四氟乙烯高压反应釜中,200℃反应8~10小时,将反应物倒出分离出Fe3O4颗粒后分散于去离子水中,倒入三口烧瓶中,往里加入氨水、无水乙醇与正硅酸四乙酯后,搅拌反应3小时分离处理,得到所述磁珠液。
优选地,所述六水合氯化铁、无水乙酸钠、PEG2000的质量比为10~15:8~12:0.3~0.6;
优选地,所述氨水、无水乙醇、正硅酸四乙酯的体积比为5:10:2
所述离液盐包括异硫氰酸胍、盐酸胍、高氯酸钠、高碘酸钠、碘化钠、氯化钠、氯化锂中的一种或多种。
所述非离子表面活性剂按重量百分比计包括:PEG200 5~15%、Triton X-114 20~30%、Tween-20 15~25%、Brij-35 10~20%、AEO-7 20~40%。
所述阴离子表面活性剂包括SLS、TLS、SDS、LDS、SDBS的一种或多种组合。
所述pH缓冲液为Tris-HCl缓冲液、柠檬酸-柠檬酸钠缓冲液与醋酸-醋酸钠缓冲液中的一种。
所述醇类按重量百分比计包括:乙醇40%、异丙醇60%。
所述离子螯合剂包括EDTA、EDTA·Na2、EGTA、8-HQ中一种或多种。
本发明还提供了一种磁珠法人全血基因组提取试剂的应用,将其用于人全血样品的基因组提取,提取流程包括如下步骤:
在96孔板中,在第1/7列加入裂解液、蛋白酶K液以及人全血样本,在第2/8列加入洗涤液1以及磁珠液,在第3/9列加入洗涤液1、在第4/10列加入洗涤液2、在第5/11列加入洗涤液2、在第6/12列加入1洗脱液;
首先取第2/8列的磁珠,浸入第1/7列中混合10分钟,温度70℃,之后浸入第2/8列中混合3分钟,温度为室温,之后浸入第3/9列中2分钟,室温,之后浸入第4/10列中1分钟,室温,之后浸入第5/11列中1分钟,室温,之后浸入第6/12列中10分钟,温度为70℃,最后弃去磁珠,将所得洗脱液进行琼脂糖凝胶电泳、Qubit与紫外分光光度计检测。
本发明的有益效果如下:
本发明的磁珠法人全血基因组提取试剂,通过试剂的组成配比使得提取试剂可依附全自动核酸提取工作站,所采用的裂解液组分中离液盐与表面活性剂的复合应用,增强对细胞膜溶解能力以及蛋白质的分解能力,使组分对样本的裂解能力有大幅度的提升;本发明组分配比均需要匹配当前使用的磁珠才可起最大的作用,因不同磁珠表面化学基团成分与含量均不相同,磁珠对核酸的结合需依靠盐离子与醇类物质的作用,当醇类物质过多导致溶剂中盐类物质可溶解度下降,盐离子浓度下降可导致磁珠表面电荷量减少,核酸负载量液可能随之减少,反之当盐离子浓度过高,醇类物质比例减少,溶液中核酸沉淀能力下降,磁珠亦无法充分有效的与核酸结合。提取所得基因组DNA的纯度在A260/280比值在与A260/230比值均显示核酸纯度较高,而基因组DNA在琼脂糖凝胶电泳中核酸条带清晰明亮。
相比市面上的全血基因组DNA提取试剂盒提取时间约为50~90分钟,本发明的提取试剂能够在40分钟内获得更高质量的基因组DNA,对于传统苯酚-氯仿法,本发明所述提取试剂的毒性更低,保护实验操作人员的健康,相比吸附柱提取法,本发明所述提取试剂可实现自动化操作,提高实验效率。
附图说明
图1为对比例1、对比例2和实施例1中各个样本的凝胶电泳图;
图2为对比例1基因提取的紫外分光光度计图表;
图3为对比例2基因提取的紫外分光光度计图表;
图4为实施例1基因提取的紫外分光光度计图表。
具体实施方式
下面结合具体实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1.
首先制备裂解结合液、洗涤液1、洗涤液2、洗脱液,分别如表1~表4所示。
表1
裂解结合液
表2 洗涤液1
表3 洗涤液2
表4 洗脱液
取96孔板,在第1/7列加入500μL裂解液、20μL蛋白酶K液以及200μL人全血样本;在第2/8列加入500 μL洗涤液1以及10 μL磁珠液;在第3/9列加入500μL洗涤液1;在第4/10列加入500μL洗涤液2;在第5/11列加入500μL洗涤液2;在第6/12列加入100μL洗脱液。
在达安Smart-32提取仪中进行如下表所示的提取操作。
表5 基因提取流程
实施例2.
首先制备裂解结合液、洗涤液1、洗涤液2、洗脱液,分别如表6~表9所示。
表6 裂解结合液
表7 洗涤液1
表8 洗涤液2
表9 洗脱液
取96孔板,在第1/7列加入500 μL裂解液、20 μL蛋白酶K液以及200 μL人全血样本;在第2/8列加入400 μL洗涤液1以及10 μL磁珠液;在第3/9列加入500 μL洗涤液1;在第4/10列加入500 μL洗涤液2;在第5/11列加入400 μL洗涤液2;在第6/12列加入100 μL洗脱液。
在达安Smart-32提取仪中进行如下表所示的提取操作。
表10 基因提取流程
实施例3.
首先制备裂解结合液、洗涤液1、洗涤液2、洗脱液,分别如表11~表14所示。
表11 裂解结合液
表12 洗涤液1
表13 洗涤液2
表14 洗脱液
取96孔板,在第1/7列加入500 μL裂解液、20 μL蛋白酶K液以及200 μL人全血样本;在第2/8列加入600 μL洗涤液1以及10 μL磁珠液;在第3/9列加入500 μL洗涤液1;在第4/10列加入600 μL洗涤液2;在第5/11列加入500 μL洗涤液2;在第6/12列加入100 μL洗脱液。
在达安Smart-32提取仪中进行如下表所示的提取操作。
表15 基因提取流程
对比例1
天根磁珠法血液基因组提取试剂盒(DP329)。
对比例2
美基磁珠法血液基因组提取试剂盒(D6310)。
将实施例1、实施例2、实施例3、对比例1和对比例2对人全血的基因组提取过程进行对比,每组选取3个人全血样本,结果如下所示:
表16 提取结果
如图2~图4所示,分别为对比例1、对比例2和实施例1基因组提取使用超微量紫外分光光度计进行检测的结果图,在检测前先使用纯净的洗脱液去背景值,然后取2μL所得核酸进行检测。结合表16以及附图结果显示,实施例1在OD值A260/A280和A260/A230比值中,优于对比例1与对比例2,且基因组DNA浓度较高(Conc≥50 ng/uL),而且在80V,1%琼脂糖凝胶电泳(如图1所示,泳道2~4为对比例1,泳道6~8为对比例2,泳道10~12为实施例1,泳道5、9为DL10000 DNA marker)中,实施例1基因组条带光亮清晰;对比例1虽基因组DNA浓度较高且条带明亮清晰,但在OD值A260/A230比值低;对比例2在基因组DNA浓度与OD值A260/A280和A260/A230比值中,均低于实施例1。
表16中的数值来源于紫外分光光度计的测量,而图2~4为表16中数值的曲线图,当曲线图成明显的S型时,表示核酸纯度高,反之则表示核酸纯度低。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (10)
1.一种磁珠法人全血基因组提取试剂,其特征在于,所述提取试剂的组分包括蛋白酶K液、磁珠液、裂解结合液、洗涤液1、洗涤液2和洗脱液。
2.根据权利要求1所述的一种磁珠法全血基因组提取试剂,其特征在于,所述裂解结合液包括:离液盐2~5mmol/L、非离子表面活性剂0.5~10%、阴离子表面活性剂0.1~5%、pH缓冲液5~10%、醇类10~50%、离子螯合剂0.1~10mmol/L;
所述洗涤液1的组分按重量百分比计包括:离液盐2~5mmol/L,非离子表面活性剂0.5~10%、pH缓冲液10~100mmol/L、醇类10~50%;
所述洗涤液2的组分按重量百分比计包括:pH缓冲液10~100mmol/L、醇类50~80%;
所述洗脱液的的组分包括:离子螯合剂0.1~10mmol/L、pH缓冲液10~100mmol/L。
3.根据权利要求1所述的磁珠法人全血基因组提取试剂,其特征在于,所述离液盐包括异硫氰酸胍、盐酸胍、高氯酸钠、高碘酸钠、碘化钠、氯化锂中的一种或多种。
4.根据权利要求1所述的磁珠法人全血基因组提取试剂,其特征在于,所述非离子表面活性剂按重量百分比计包括:PEG200 5~15%、Triton X-114 20~30%、Tween-20 15~25%、Brij-35 10~20%、AEO-7 20~40%。
5.根据权利要求1所述的磁珠法人全血基因组提取试剂,其特征在于,所述阴离子表面活性剂包括SLS、TLS、SDS、LDS、SDBS的一种或多种组合。
6.根据权利要求1所述的磁珠法人全血基因组提取试剂,其特征在于,所述pH缓冲液为Tris-HCl缓冲液、柠檬酸-柠檬酸钠缓冲液与醋酸-醋酸钠缓冲液中的一种。
7.根据权利要求1所述的磁珠法人全血基因组提取试剂,其特征在于,所述醇类按重量百分比计包括:乙醇40%、异丙醇60%。
8.根据权利要求1所述的磁珠法人全血基因组提取试剂,其特征在于,所述离子螯合剂包括EDTA、EDTA•Na2、EGTA、8-HQ中的一种或多种。
9.根据权利要求1所述的一种磁珠法人全血基因组提取试剂,其特征在于,所述磁珠液为中的磁珠为是二氧化硅包覆的四氧化三铁磁珠,制备工艺为:
将六水合氯化铁、无水乙酸钠与PEG2000溶于乙二醇中,将溶解物倒入聚四氟乙烯高压反应釜中,200℃反应8~10小时,将反应物倒出分离出Fe3O4颗粒后分散于去离子水中,倒入三口烧瓶中,往里加入氨水、无水乙醇与正硅酸四乙酯后,搅拌反应3小时分离处理,得到所述磁珠液。
10.根据权利要求9所述的一种磁珠法人全血基因组提取试剂,其特征在于,所述六水合氯化铁、无水乙酸钠、PEG2000的质量比为10~15:8~12:0.3~0.6;
所述氨水、无水乙醇、正硅酸四乙酯的体积比为5:10:2。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311570183.6A CN117487797A (zh) | 2023-11-23 | 2023-11-23 | 一种磁珠法人全血基因组提取试剂及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311570183.6A CN117487797A (zh) | 2023-11-23 | 2023-11-23 | 一种磁珠法人全血基因组提取试剂及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117487797A true CN117487797A (zh) | 2024-02-02 |
Family
ID=89681192
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311570183.6A Pending CN117487797A (zh) | 2023-11-23 | 2023-11-23 | 一种磁珠法人全血基因组提取试剂及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117487797A (zh) |
-
2023
- 2023-11-23 CN CN202311570183.6A patent/CN117487797A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107663521B (zh) | 血浆游离核酸提取试剂盒及其应用 | |
CN107937391B (zh) | 人粪便中脱落细胞核酸提取裂解液及其制备方法和应用 | |
EP2574670B1 (en) | Method for purifying nucleic acid at ultrahigh speed | |
CN103820431A (zh) | 基于纳米磁珠的核酸提取纯化方法及试剂盒 | |
CN109694863B (zh) | 用于核酸提取的裂解液、清洗液、核酸提取试剂盒及核酸的提取方法 | |
CN112011594A (zh) | 一种游离核酸提取试剂盒及其提取方法 | |
US20150184149A1 (en) | Agent and kit for isolating nucleic acids | |
CN110951725A (zh) | 一种基于磁珠法的一步法核酸提取工艺 | |
US20170029808A1 (en) | Method for recovering short-chain nucleic acids | |
CN112941067A (zh) | 一种全血核酸提取用裂解结合液及其试剂盒和应用 | |
CN117487797A (zh) | 一种磁珠法人全血基因组提取试剂及其应用 | |
CN116162618A (zh) | 一种从核酸溶液中分离dna和rna的方法及试剂组合 | |
CN113308459A (zh) | 一种磁珠法全血核酸提取裂解液、试剂盒及提取方法 | |
CN114196668B (zh) | 一种用于捕获Poly A(+)RNA的磁珠、其制备方法及应用 | |
CN114958830A (zh) | 一种同时提取病原体dna和rna的试剂盒及其应用 | |
CN114350649A (zh) | 一种核酸提取试剂盒及核酸提取方法 | |
CN114807120A (zh) | 一种高灵敏度病毒核酸提取试剂盒 | |
CN111808850A (zh) | 细菌核酸提取裂解液、制备方法及应用 | |
CN118109459B (zh) | 一种无需蛋白酶k处理的磁珠法提取RNA的试剂盒及提取方法 | |
CN116355893B (zh) | 一种磁珠法核酸提取试剂盒及其应用 | |
JP3922420B2 (ja) | 改良されたリボ核酸の単離方法 | |
CN115532242B (zh) | 腐殖酸吸附剂及其制备方法土壤dna提取试剂盒 | |
CN116926064B (zh) | 一种核酸提取试剂盒及其制备方法和应用 | |
CN116555248B (zh) | 一种细菌中高分子量dna提取试剂盒及提取方法 | |
CN118240814A (zh) | 一种完全区分dna和rna的纯化方法及rna纯化试剂盒 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |