CN110846383A - Method and kit for constructing mRNA library - Google Patents
Method and kit for constructing mRNA library Download PDFInfo
- Publication number
- CN110846383A CN110846383A CN201911222706.1A CN201911222706A CN110846383A CN 110846383 A CN110846383 A CN 110846383A CN 201911222706 A CN201911222706 A CN 201911222706A CN 110846383 A CN110846383 A CN 110846383A
- Authority
- CN
- China
- Prior art keywords
- working concentration
- reaction system
- mul
- reaction
- solution
- 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.)
- Granted
Links
- 108020004999 messenger RNA Proteins 0.000 title claims abstract description 115
- 238000000034 method Methods 0.000 title claims abstract description 74
- 238000006243 chemical reaction Methods 0.000 claims abstract description 266
- 239000000243 solution Substances 0.000 claims abstract description 87
- 102000004190 Enzymes Human genes 0.000 claims abstract description 56
- 108090000790 Enzymes Proteins 0.000 claims abstract description 56
- 238000010438 heat treatment Methods 0.000 claims abstract description 56
- 239000007853 buffer solution Substances 0.000 claims abstract description 50
- 230000003321 amplification Effects 0.000 claims abstract description 39
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 39
- 238000002156 mixing Methods 0.000 claims abstract description 24
- 239000012634 fragment Substances 0.000 claims abstract description 23
- 102000012410 DNA Ligases Human genes 0.000 claims abstract description 21
- 108010061982 DNA Ligases Proteins 0.000 claims abstract description 21
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 72
- 239000002299 complementary DNA Substances 0.000 claims description 72
- 238000006062 fragmentation reaction Methods 0.000 claims description 54
- 102100034343 Integrase Human genes 0.000 claims description 52
- 102000004594 DNA Polymerase I Human genes 0.000 claims description 43
- 108010017826 DNA Polymerase I Proteins 0.000 claims description 43
- 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 42
- 239000000872 buffer Substances 0.000 claims description 37
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 37
- 238000013467 fragmentation Methods 0.000 claims description 37
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 36
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 33
- 230000000694 effects Effects 0.000 claims description 31
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 29
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 26
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 claims description 22
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 claims description 22
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 claims description 20
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 claims description 20
- 239000003161 ribonuclease inhibitor Substances 0.000 claims description 20
- 101710203526 Integrase Proteins 0.000 claims description 19
- 241000713869 Moloney murine leukemia virus Species 0.000 claims description 18
- 101710163270 Nuclease Proteins 0.000 claims description 16
- 230000015572 biosynthetic process Effects 0.000 claims description 16
- 238000003786 synthesis reaction Methods 0.000 claims description 16
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 15
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 15
- 239000001103 potassium chloride Substances 0.000 claims description 14
- 235000011164 potassium chloride Nutrition 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 14
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 12
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 12
- 238000012937 correction Methods 0.000 claims description 11
- 238000013519 translation Methods 0.000 claims description 11
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 claims description 10
- 239000004353 Polyethylene glycol 8000 Substances 0.000 claims description 9
- 229940085678 polyethylene glycol 8000 Drugs 0.000 claims description 9
- 235000019446 polyethylene glycol 8000 Nutrition 0.000 claims description 9
- 230000026731 phosphorylation Effects 0.000 claims description 4
- 238000006366 phosphorylation reaction Methods 0.000 claims description 4
- 125000000446 sulfanediyl group Chemical group *S* 0.000 claims description 3
- 238000010276 construction Methods 0.000 abstract description 13
- 239000011324 bead Substances 0.000 description 28
- 238000010804 cDNA synthesis Methods 0.000 description 28
- 239000000203 mixture Substances 0.000 description 19
- 239000000523 sample Substances 0.000 description 14
- 239000013614 RNA sample Substances 0.000 description 11
- 238000000746 purification Methods 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 230000002194 synthesizing effect Effects 0.000 description 6
- 238000010009 beating Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 239000012148 binding buffer Substances 0.000 description 3
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 3
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 3
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 230000002349 favourable effect Effects 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 230000002195 synergetic effect Effects 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- GUAHPAJOXVYFON-ZETCQYMHSA-N (8S)-8-amino-7-oxononanoic acid zwitterion Chemical compound C[C@H](N)C(=O)CCCCCC(O)=O GUAHPAJOXVYFON-ZETCQYMHSA-N 0.000 description 2
- 238000003559 RNA-seq method Methods 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- UHZZMRAGKVHANO-UHFFFAOYSA-M chlormequat chloride Chemical compound [Cl-].C[N+](C)(C)CCCl UHZZMRAGKVHANO-UHFFFAOYSA-M 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- -1 Taq-BDNA polymerase Proteins 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- GYOZYWVXFNDGLU-XLPZGREQSA-N dTMP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)C1 GYOZYWVXFNDGLU-XLPZGREQSA-N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000010841 mRNA extraction Methods 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Images
Classifications
-
- 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)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present disclosure provides a method of constructing an mRNA library, comprising: mixing the mRNA fragments, the first mixed enzyme solution and the first buffer solution into a first reaction system, and carrying out first heat treatment on the first reaction system to obtain a first reaction solution; mixing the first reaction solution, the second mixed enzyme solution and the second buffer solution to form a second reaction system, and carrying out second heat treatment on the second reaction system to obtain a second reaction solution; mixing the second reaction solution with T4DNA ligase, a third buffer solution and a Y-shaped joint to form a third reaction system, carrying out third heat treatment on the third reaction system, and purifying after the third heat treatment to obtain a third reaction solution; and adding the premix and the amplification primers to the third reaction solution to mix a fourth reaction system, subjecting the fourth reaction system to a fourth heat treatment, and purifying after the fourth heat treatment to obtain an mRNA library. According to the present disclosure, a method and a kit for constructing an mRNA library, which are advantageous for improving library construction efficiency, can be provided.
Description
Technical Field
The present disclosure relates to a method and a kit for constructing an mRNA library.
Background
Transcriptome sequencing (RNA-seq) is a sequencing technique that comprehensively and rapidly obtains sequence information and expression information of almost all transcripts of a specific cell or tissue in a certain state by using a new generation sequencing technique. Based on the different research directions and strategies, transcriptome sequencing is divided into mRNA-seq for encoded proteins and Ribo-zero RNA-seq for various non-coding RNAs.
At present, the main process for constructing an mRNA-seq library comprises enrichment of mRNA, fragmentation of mRNA, synthesis of double-stranded cDNA (cDNA16 ℃, 1h) through reverse transcription of one strand and two strands, purification of cDNA products (about 30min), repair of the tail end of the double-stranded cDNA, reaction of adding A (20 ℃, 15 min; 65 ℃, 15min), linker connection, purification and fragmentation screening of the ligation products, PCR amplification and purification and other steps.
Disclosure of Invention
The present disclosure has been made in view of the above-described state of the art, and an object thereof is to provide a method and a kit for constructing an mRNA library, which are advantageous in improving library construction efficiency.
To this end, one aspect of the present disclosure provides a method of constructing an mRNA library, comprising: preparing and mixing an mRNA fragment, a first mixed enzyme solution having an RNase inhibitor and a reverse transcriptase, and a first buffer solution having dNTP as a first reaction system, and subjecting the first reaction system to a first heat treatment, wherein in the first heat treatment, a first cDNA strand is synthesized using the mRNA fragment as a template to obtain a first reaction solution, and the reverse transcriptase is an MMLV reverse transcriptase lacking in RNase H activity; mixing the first reaction solution with a second mixed enzyme solution containing DNA polymerase I, ribonuclease H, T4, T4 polynucleotide kinase, Taq-B DNA polymerase, and Klenow fragments, which lack nuclease activity for nick translation from the 5 'end to the 3' end and nuclease activity for correction from the 3 'end to the 5' end, and a second buffer solution containing dntps as a second reaction system, and subjecting the second reaction system to a second heat treatment in which a second cDNA strand is synthesized using the first cDNA strand as a template, followed by end repair and end addition a of cDNA composed of the first cDNA strand and the second cDNA strand to form a second reaction solution; mixing the second reaction solution with T4DNA ligase, a third buffer solution containing dimethyl sulfoxide, and a Y-shaped linker to obtain a third reaction system, subjecting the third reaction system to a third heat treatment in which cDNA is ligated to the Y-shaped linker, and purifying the third heat treatment to obtain a third reaction solution; and adding a premixed solution containing an amplification enzyme and an amplification primer to the third reaction solution and mixing them to form a fourth reaction system, subjecting the fourth reaction system to a fourth heat treatment, subjecting cDNA to an amplification reaction in the fourth heat treatment, and purifying the cDNA after the fourth heat treatment to obtain an mRNA library.
In the present disclosure, in the first reaction system, rnase inhibitor can prevent mRNA from being degraded, and MMLV reverse transcriptase lacking rnase H activity can increase the yield of first strand cDNA synthesis, thereby increasing the efficiency of first strand cDNA synthesis. In addition, the second strand synthesis, end repair and end A addition of cDNA are all performed in the second reaction system, and in the second reaction system, DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase, and nuclease activity for nick translation lacking from 5 'end to 3' end and Klenow fragment for correction lacking from 3 'end to 5' end can act synergistically, thereby enabling the efficiency of second strand synthesis, end repair and end A addition of cDNA to be improved. In addition, the use of the Y-type linker in the linker ligation reaction can improve the ligation efficiency, thereby contributing to the improvement of the efficiency of mRNA library construction.
In addition, in the method for constructing an mRNA library according to one aspect of the present disclosure, optionally, in the first reaction system, the working concentration of the MMLV reverse transcriptase is 8U/μ L to 12U/μ L, and the working concentration of the RNase inhibitor is 1.0U/μ L to 1.5U/μ L. This can effectively improve the efficiency of first strand cDNA synthesis.
In addition, in the method for constructing an mRNA library according to an aspect of the present disclosure, optionally, in the second reaction system, the DNA polymerase I has a working concentration of 0.6U/μ L to 1U/μ L, the glycosylnuclease H has a working concentration of 0.1U/μ L to 0.2U/μ L, the T4DNA polymerase has a working concentration of 0.05U/μ L to 0.1U/μ L, the T4 polynucleotide kinase has a working concentration of 0.3U/μ L to 0.5U/μ L, the Taq-B DNA polymerase has a working concentration of 0.04U/μ L to 0.08U/μ L, and the Klenow fragment has a working concentration of 0.08U/μ L to 0.12U/μ L. This can effectively improve the reaction efficiency in the second reaction system.
In addition, in the method for constructing an mRNA library according to an aspect of the present disclosure, optionally, the first buffer further includes Tris-HCl, magnesium chloride, potassium chloride, and dithiothreitol, and in the first reaction system, the working concentration of dNTP is 0.3mM to 0.7mM, the working concentration of Tris-HCl is 0.03M to 0.06M, the working concentration of magnesium chloride is 2.0mM to 2.5mM, the working concentration of potassium chloride is 0.03M to 0.08M, and the working concentration of dithiothreitol is 6mM to 10 mM. In this case, the first buffer solution can maintain the pH of the first reaction system and the stability of the enzyme.
In addition, in the method for constructing an mRNA library according to an aspect of the present disclosure, optionally, the second buffer further includes Tris-HCl, magnesium chloride, sodium chloride, dithiothreitol, dATP, and ATP; in the second reaction system, the working concentration of dNTP is 0.3mM to 0.6mM, the working concentration of Tris-HCl is 6mM to 10mM, the working concentration of magnesium chloride is 0.2mM to 0.5mM, the working concentration of sodium chloride is 0.03M to 0.06M, the working concentration of dithiothreitol is 3mM to 6mM, the working concentration of dATP is 1.2mM to 1.4mM, and the working concentration of ATP is 0.7mM to 1 mM. In this case, the second buffer solution can maintain the pH of the second reaction system and the stability of the enzyme.
In addition, in a method of constructing an mRNA library according to an aspect of the present disclosure, optionally, the mRNA fragments are subjected to a fragmentation process, and the fragmentation process is performed in a fragmentation reaction system in which a fragmentation buffer including Tris-HCl, magnesium ions, and random hexamer primers is mixed with an mRNA sample. Thus, a suitable length of mRNA fragment can be obtained and the random hexamer primer required for subsequent synthesis of the first strand of cDNA can be provided.
In addition, in the method for constructing an mRNA library according to an aspect of the present disclosure, optionally, the third buffer further includes ATP, Tris-HCl, magnesium chloride, dithiothreitol, and polyethylene glycol 8000, and in the third reaction system, the working concentration of the T4DNA ligase is 10U/. mu.l to 30U/. mu.l; the working concentration of the Y-shaped joint is 1 multiplied by 10-7M to 3X 10-7M; the working concentration of the dimethyl sulfoxide is 1-1.5%, the working concentration of the ATP is 1.5-3 mM, the working concentration of the Tris-HCl is 0.04-0.08M, the working concentration of the magnesium chloride is 0.03-0.05M, the working concentration of the dithiothreitol is 1-3 mM, and the working concentration of the polyethylene glycol 8000 is 5-8 wt%. In this case, the third buffer solution can maintain the pH of the third reaction system and the stability of the enzyme, and can enhance the linker ligation reaction.
In addition, in the method for constructing an mRNA library according to an aspect of the present disclosure, optionally, the first sequence of the Y-linker is 5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC-3', and the second sequence is 5 '-gatcgaagcacacgtctgaatccagtcacxxxxxsattcgtatgccgtcttctgcttg-3', wherein the 3 'end of the first sequence is modified by thio, the 5' end of the second sequence is modified by phosphorylation, nnnn is a molecular tag sequence, and XXXXXXXX is a sample tag sequence. Therefore, the connection efficiency of the Y-shaped joint can be improved, and the accuracy of subsequent sequencing can also be improved.
Another aspect of the present disclosure provides a kit for constructing an mRNA library, which includes: a first mixed enzyme solution having an rnase inhibitor and a reverse transcriptase and a first buffer solution having dntps, and a second mixed enzyme solution having DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase and Klenow fragment and a second buffer solution having dntps, wherein the reverse transcriptase is MMLV reverse transcriptase lacking ribonuclease H activity, the Klenow fragment lacks nuclease activity for nick translation from 5 'end to 3' end and nuclease activity for correction from 3 'end to 5' end, the first mixed enzyme solution and the first buffer solution are used to form a first reaction system for first strand cDNA synthesis, and the second mixed enzyme solution and the second buffer solution are used to form a second reaction system for second strand cDNA synthesis, end repair and end-plus-a.
In the present disclosure, rnase inhibitors can prevent mRNA from being degraded, and MMLV reverse transcriptase lacking rnase H activity can increase the yield of first strand cDNA synthesis, thereby increasing the efficiency of first strand cDNA synthesis. In addition, DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase and Klenow fragment lacking 5 'to 3' nick translation activity and 3 'to 5' correction can have a synergistic effect, and can improve the efficiency of second strand cDNA synthesis, end repair and end-to-A addition, thus contributing to the improvement of the efficiency of mRNA library construction.
In addition, in the kit for constructing an mRNA library according to another aspect of the present disclosure, optionally, in the first reaction system, the working concentration of the MMLV reverse transcriptase is 8U/μ L to 12U/μ L, and the working concentration of the RNase inhibitor is 1.0U/μ L to 1.5U/μ L; in the second reaction system, the working concentration of the DNA polymerase I is 0.6U/muL to 1U/muL, the working concentration of the glyconuclease H is 0.1U/muL to 0.2U/muL, the working concentration of the T4DNA polymerase is 0.05U/muL to 0.1U/muL, the working concentration of the T4 polynucleotide kinase is 0.3U/muL to 0.5U/muL, the working concentration of the Taq-BDNA polymerase is 0.04U/muL to 0.08U/muL, and the working concentration of the Klenow fragment is 0.08U/muL to 0.12U/muL. Therefore, the reaction efficiency of each reaction can be effectively improved.
In addition, in a kit for constructing an mRNA library according to another aspect of the present disclosure, optionally, the first buffer further includes Tris-HCl, magnesium chloride, potassium chloride, and dithiothreitol, and in the first reaction system, the working concentration of dNTP is 0.3mM to 0.7mM, the working concentration of Tris-HCl is 0.03M to 0.06M, the working concentration of magnesium chloride is 2.0mM to 2.5mM, the working concentration of potassium chloride is 0.03M to 0.08M, and the working concentration of dithiothreitol is 6mM to 10 mM. In this case, the first buffer solution can maintain the pH of the first reaction system and the stability of the enzyme.
In addition, in the kit for constructing an mRNA library according to another aspect of the present disclosure, optionally, the second buffer further includes Tris-HCl, magnesium chloride, sodium chloride, dithiothreitol, dATP, and ATP, and in the second reaction system, the working concentration of the dNTP is 0.3mM to 0.6mM, the working concentration of the Tris-HCl is 6mM to 10mM, the working concentration of the magnesium chloride is 0.2mM to 0.5mM, the working concentration of the sodium chloride is 0.03M to 0.06M, the working concentration of the dithiothreitol is 3mM to 6mM, the working concentration of the dATP is 1.2mM to 1.4mM, and the working concentration of the ATP is 0.7mM to 1 mM. In this case, the second buffer solution can maintain the pH of the second reaction system and the stability of the enzyme.
In addition, in a kit for constructing an mRNA library according to another aspect of the present disclosure, optionally, a fragmentation buffer for fragmentation, T4DNA ligase, a third buffer with dimethyl sulfoxide, and a Y-type adaptor, and a pre-mixed solution with an amplification enzyme and an amplification primer, wherein the T4DNA ligase, the third buffer, and the Y-type adaptor are used to form a third reaction system for ligation of the adaptor, and the pre-mixed solution and the amplification primer are used to form a fourth reaction system for amplification reaction. Thus, the kit can be used for mRNA fragmentation processing, and can amplify an mRNA library.
According to the present disclosure, a method and a kit for constructing an mRNA library, which are advantageous for improving library construction efficiency, can be provided.
Drawings
Figure 1 is a flow diagram illustrating a method of constructing an mRNA library according to examples of the present disclosure.
Fig. 2 is a flow diagram illustrating a method of constructing an mRNA library in the comparative example of the present disclosure.
Detailed Description
Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same components are denoted by the same reference numerals, and redundant description thereof is omitted. The drawings are schematic and the ratio of the dimensions of the components and the shapes of the components may be different from the actual ones.
In the present disclosure, the unit "M" may be abbreviated as mol/L, the unit "mM" may be abbreviated as mmol/L, and the unit "μ M" may be abbreviated as μmol/L in the present disclosure.
Figure 1 is a flow diagram illustrating a method of constructing an mRNA library according to examples of the present disclosure.
As shown in fig. 1, the method for constructing an mRNA library according to the present embodiment may include: first strand cDNA synthesis (step S10), second strand cDNA synthesis, end repair and end addition of A (step S20), linker ligation (step S30) and library amplification (step S40).
In some examples, step S10 may include preparing and mixing an mRNA fragment, a first mixed enzyme solution having an rnase inhibitor and a reverse transcriptase, and a first buffer solution having dntps as a first reaction system. Wherein the reverse transcriptase is MMLV reverse transcriptase lacking ribonuclease H activity. In addition, the first reaction system may be subjected to a first heat treatment in which a first strand of cDNA is synthesized using the mRNA fragment as a template to obtain a first reaction solution, and in some examples, the step S20 may include mixing the first reaction solution with a second mixed enzyme solution having DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase, and Klenow fragment, and a second buffer solution having dNTP as a second reaction system. Wherein the Klenow fragment lacks nuclease activity for nick translation from 5 'to 3' and nuclease activity for correction from 3 'to 5'. Alternatively, the second reaction system may be subjected to a second heat treatment in which a second strand of cDNA is synthesized using the first strand of cDNA as a template, and then the cDNA consisting of the first strand of cDNA and the second strand of cDNA is subjected to end repair and end addition of A to form a second reaction solution.
In some examples, step S30 may include mixing the second reaction solution with T4DNA ligase, a third buffer solution with dimethyl sulfoxide, and a Y-junction as a third reaction system. Further, the third reaction system may be subjected to a third heat treatment in which the cDNA is ligated to the Y-junction, and then purified to obtain a third reaction solution.
In some examples, step S40 may include adding a premix solution with an amplification enzyme and an amplification primer mixture as a fourth reaction system to the third reaction solution. Alternatively, the fourth reaction system may be subjected to a fourth heat treatment in which the cDNA is subjected to an amplification reaction and then purified to obtain an mRNA library.
In this embodiment, in the first reaction system, rnase inhibitor can prevent mRNA from being degraded, and MMLV reverse transcriptase lacking rnase H activity can increase the yield of first strand cDNA synthesis, thereby increasing the efficiency of first strand cDNA synthesis. In addition, the second strand synthesis, end repair and end A addition of cDNA are all performed in the second reaction system, and in the second reaction system, DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-BDNA polymerase, and nuclease activity for nick translation lacking from 5 'end to 3' end and Klenow fragment for correction from 3 'end to 5' end can act synergistically, thereby enabling the efficiency of second strand synthesis, end repair and end A addition of cDNA to be improved. In addition, the use of the Y-type linker in the linker ligation reaction can improve the ligation efficiency, thereby contributing to the improvement of the efficiency of mRNA library construction.
In addition, the second strand synthesis of cDNA, end repair, and end addition of A are all performed in the second reaction system, so that the number of steps (e.g., sample addition, lid closing, machine reaction, lid opening, etc.) for end repair and end addition of A can be reduced, and the purification process after cDNA is formed by second strand synthesis of cDNA can be omitted, whereby the reaction steps can be simplified and the time for library construction can be shortened, and the steps can be omitted, so that the use of reagents can be reduced, and the cost can be reduced.
In some examples, the method of constructing an mRNA library may include a preceding step. Among other things, a pre-step can be used to prepare mRNA fragments. Additionally, in some examples, the pre-step may include extracting RNA and isolating mRNA.
In some examples, the sample source from which the RNA is extracted may be a eukaryote. Additionally, in some examples, fungi, blood, paraffin embedded tissue, animal tissue, plant tissue, cultured cells, cell lines, and the like. For example, human renal cortex tissue, mouse liver, young leaves, mature roots, stems, yeast, human oral epithelial cells, and the like.
In some examples, RNA can be extracted and purified using precipitation, column chromatography, or RNA extraction kits to obtain RNA samples. In addition, in some examples, preferably, the RNA integrity value (RIN) of the RNA sample may be greater than 7. In other words, high quality RNA samples are preferred.
In some examples, mRNA samples can be obtained from RNA samples by isolation and purification. In addition, in some examples, mRNA samples can be obtained by capturing ploy a. Thus, an mRNA sample having a poly a tail (ploy a tail) can be isolated from an RNA sample.
In some examples, mRNA may be isolated and purified using oligo (dT) cellulose or oligo (U) agarose affinity chromatography.
In some examples, mRNA samples can be obtained using magnetic beads (i.e., oligo (dt) magnetic beads) with poly-thymidylate (poly-t) modified surfaces. In this case, Poly T on the surface of the magnetic beads can bind to Poly a at the 3' end of mRNA through hydrogen bonding, thereby enabling specific capture of mRNA.
In some examples, mRNA samples can be obtained by an mRNA capture kit. Additionally, in some examples, the mRNA capture kit can include mRNA capture magnetic beads (e.g., oligo (dt) magnetic beads), a magnetic bead binding buffer, a magnetic bead wash, and a Tris buffer.
In some examples, the pre-step may also be a fragmentation process. In addition, in some examples, the mRNA sample may be subjected to a fragmentation process to obtain mRNA fragments (fragmentation reaction solution). In other words, the mRNA fragments may be subjected to a fragmentation process. Therefore, mRNA fragments with proper length can be obtained, and further, the subsequent sequencing of the mRNA library can be facilitated.
In some examples, the fragmentation process can be performed in a fragmentation reaction system in which a fragmentation buffer is mixed with an mRNA sample. In addition, in some examples, the fragmentation buffer can include Tris-HCl, magnesium ions, and random hexamer primers. In this case, Tris-HCl can be used to maintain the pH stability of the fragmentation reaction system, magnesium ions can be used to break down mRNA, and random hexamer primers required for the subsequent synthesis of the first strand of cDNA can be provided. In addition, a random hexamer primer is added to form a fragmentation buffer solution, and a fragmentation reaction system mixed with the mRNA sample is subjected to fragmentation treatment, so that the random hexamer primer can be better combined with the mRNA fragment.
In some examples, the working concentration of Tris-HCl may be 5mM to 20mM in the fragmentation reaction system. For example, the working concentration of Tris-HCl may be 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM or 20 mM.
In some examples, the working concentration of magnesium ions in the fragmentation reaction system may be 0.3mM to 1.0 mM. For example, the working concentration of magnesium ions may be 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, or 1.0mM, for example.
In some examples, the random hexamer primer can be a mixture of random sequence primers (with 4) comprising 6 bases6Seed possible sequence). In addition, in some examples, the 5' end of the random hexamer primer can have a phosphorylation modification.
In some examples, the working concentration of random hexamer primers in the fragmentation reaction system is 30 μ M to 60 μ M. For example, working concentrations of random hexamer primers can be 30. mu.M, 33. mu.M, 35. mu.M, 38. mu.M, 40. mu.M, 43. mu.M, 45. mu.M, 48. mu.M, 50. mu.M, 53. mu.M, 55. mu.M, 58. mu.M, or 60. mu.M.
In some examples, the fragmentation reaction system can perform the fragmentation reaction at 80 ℃ to 95 ℃. This enables cleavage of mRNA by magnesium ions at high temperature. In addition, in some examples, the fragmentation reaction system can perform the fragmentation reaction at 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃, 91 ℃, 92 ℃, 93 ℃, 94 ℃ or 95 ℃. In other examples, the fragmentation process may be performed in a PCR instrument.
In some examples, in step S10, the mRNA fragment may be mixed with a first mixed enzyme solution and a first buffer solution into a first reaction system for synthesizing a first strand of cDNA. Specifically, the fragmentation reaction solution may be mixed with a first mixed enzyme solution and a first buffer solution to form a first reaction system for synthesizing a first strand of cDNA.
In some examples, the first mixed enzyme solution may include an rnase inhibitor and a reverse transcriptase. Thus, rnase inhibitors can inhibit mRNA degradation and reverse transcriptase can synthesize the first strand of cDNA. In addition, in some examples, the reverse transcriptase is an MMLV reverse transcriptase lacking rnase H activity. This enables the first strand cDNA to be synthesized more efficiently.
In some examples, the working concentration of MMLV reverse transcriptase in the first reaction system is 8U/. mu.L to 12U/. mu.L and the working concentration of RNase inhibitor is 1U/. mu.L to 1.5U/. mu.L. This can effectively improve the efficiency of first strand cDNA synthesis.
In some examples, the working concentration of MMLV reverse transcriptase in the first reaction system can be 8U/. mu.L, 8.5U/. mu.L, 9U/. mu.L, 9.5U/. mu.L, 10U/. mu.L, 10.5U/. mu.L, 11U/. mu.L, 11.5U/. mu.L, or 12U/. mu.L. In addition, in some examples, the working concentration of the RNase inhibitor in the first reaction system may be 1U/. mu.L, 1.1U/. mu.L, 1.2U/. mu.L, 1.3U/. mu.L, 1.4U/. mu.L, or 1.5U/. mu.L.
In some examples, the working concentration of RNase inhibitor in the first reaction system may be 0.05U/. mu.L, 0.06U/. mu.L, 0.07U/. mu.L, 0.08U/. mu.L, 0.09U/. mu.L, 0.1U/. mu.L, 0.11U/. mu.L, 0.12U/. mu.L, 0.13U/. mu.L, 0.14U/. mu.L, 0.15U/. mu.L, 0.16U/. mu.L, 0.17U/. mu.L, 0.18U/. mu.L, 0.19U/. mu.L, or 0.2U/. mu.L.
In some examples, the first buffer can include dntps. This provides a starting material for first strand cDNA synthesis. In the present disclosure, dntps may refer to a mixture of dATP, dTTP, dCTP and dGTP. In addition, in some examples, the dntps can be an equal mixture of dATP, dTTP, dCTP, and dGTP.
In some examples, the first buffer may also include Tris-HCl, magnesium chloride (MgCl)2) Potassium chloride (KCl) and Dithiothreitol (DTT). In this case, Tris-HCl is used to maintain the stability of the pH of the first reaction system, magnesium chloride can provide magnesium ions to increase the activity of the enzyme, potassium chloride is used to adjust the ionic strength, dithiothreitol can maintain the stability of the enzyme, in other words, the first buffer can maintain the pH of the first reaction system and the stability of the enzyme, and can provide substances (e.g., dntps, magnesium ions) and conditions (e.g., pH, ionic strength) required for the reaction.
In some examples, in the first reaction system, the working concentration of dNTPs may be from 0.3mM to 0.7mM, the working concentration of Tris-HCl may be from 0.03M to 0.06M, the working concentration of magnesium chloride may be from 2.0mM to 2.5mM, the working concentration of potassium chloride may be from 0.03M to 0.08M, and the working concentration of dithiothreitol may be from 6mM to 10 mM. This provides an environment favorable for first strand cDNA synthesis, and contributes to an improvement in the efficiency of first strand cDNA synthesis.
In some examples, the working concentration of dNTPs in the first reaction system can be 0.3mM, 0.4mM, 0.5mM, 0.6mM, or 0.7 mM. In addition, in some examples, the working concentration of Tris-HCl may be 0.03M, 0.035M, 0.04M, 0.045M, 0.05M, 0.055M, or 0.06M in the first reaction system.
In some examples, the working concentration of magnesium chloride in the first reaction system may be 2.0mM, 2.1mM, 2.2mM, 2.3mM, 2.4mM, or 2.5 mM. In addition, in some examples, the working concentration of potassium chloride in the first reaction system may be 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M.
In some examples, the working concentration of dithiothreitol in the first reaction system can be 6mM, 6.5mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5mM, 10 mM.
In some examples, the fragmentation reaction is mixed with a first mixed enzyme solution and a first buffer solution in a first reaction system, where the fragmentation reaction may comprise random hexamer primers. In addition, in some examples, the working concentration of random hexamer primers in the first reaction system is 15 μ M to 30 μ M. For example, the working concentration of random hexamer primers can be 15. mu.M, 16. mu.M, 17. mu.M, 18. mu.M, 19. mu.M, 20. mu.M, 21. mu.M, 22. mu.M, 23. mu.M, 24. mu.M, 25. mu.M, 26. mu.M, 27. mu.M, 28. mu.M, 29. mu.M, or 30. mu.M.
In some examples, in step S10, the first reaction system may be subjected to a first heat treatment. Thus, the first reaction system can produce a first strand cDNA synthesis reaction. In other examples, in the first heat treatment, a first strand of cDNA may be synthesized using the mRNA fragment as a template and a first reaction solution obtained. In other examples, the first reaction solution may comprise a first strand of cDNA hybridized to a double strand of mRNA (cDNA-mRNA double strand).
In some examples, the first heat treatment may be performed according to a first predetermined procedure. In other examples, the first heat treatment may be performed in a PCR instrument. Additionally, in some examples, the first predetermined procedure may be: random hexamer primer binding was performed by heating to 25 ℃ for 10min, followed by first strand cDNA synthesis by heating to 42 ℃ for 15min, followed by enzyme inactivation by heating to 70 ℃ for 15 min.
In some examples, in step S20, the first reaction solution may be mixed with a second mixed enzyme solution and a second buffer solution into a second reaction system for synthesizing a second strand of cDNA, end repair, and end-plus-a.
In some examples, the second mixed enzyme solution can include DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase, and Klenow fragment. In this case, ribonuclease H can remove mRNA before second strand synthesis of cDNA, DNA polymerase I and Klenow fragment can be used for second strand synthesis of cDNA, T4DNA polymerase and T4 polynucleotide kinase can be used for end repair, Taq-B DNA polymerase can be used for end addition of A, and DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase and Klenow fragment can have a synergistic effect between them, which can improve the reaction efficiency of the individual reactions in the second reaction system.
In some examples, the Klenow fragment can lack nuclease activity for nick translation from 5 'to 3' and nuclease activity for correction from 3 'to 5'.
In some examples, in the second reaction system, the working concentration of DNA polymerase I may be 0.6U/μ L to 1U/μ L, the working concentration of glyconuclease H may be 0.1U/μ L to 0.2U/μ L, the working concentration of T4DNA polymerase may be 0.05U/μ L to 0.1U/μ L, the working concentration of T4 polynucleotide kinase may be 0.3U/μ L to 0.5U/μ L, the working concentration of Taq-BDNA polymerase may be 0.04U/μ L to 0.08U/μ L, and the working concentration of Klenow fragment may be 0.08U/μ L to 0.12U/μ L. This can effectively improve the reaction efficiency in the second reaction system.
In some examples, the working concentration of DNA polymerase I in the second reaction system may be 0.6U/. mu.L, 0.65U/. mu.L, 0.7U/. mu.L, 0.75U/. mu.L, 0.8U/. mu.L, 0.85U/. mu.L, 0.9U/. mu.L, 0.95U/. mu.L, or 1U/. mu.L.
In some examples, the working concentration of glycosylnuclease H in the second reaction system can be 0.1U/. mu.L, 0.11U/. mu.L, 0.12U/. mu.L, 0.13U/. mu.L, 0.14U/. mu.L, 0.15U/. mu.L, 0.16U/. mu.L, 0.17U/. mu.L, 0.18U/. mu.L, 0.19U/. mu.L, or 0.2U/. mu.L.
In some examples, the working concentration of T4DNA polymerase in the second reaction system may be 0.05U/. mu.L, 0.06U/. mu.L, 0.07U/. mu.L, 0.08U/. mu.L, 0.09U/. mu.L, or 0.1U/. mu.L. In addition, in some examples, the working concentration of T4 polynucleotide kinase in the second reaction system can be 0.3U/. mu.L, 0.35U/. mu.L, 0.4U/. mu.L, 0.45U/. mu.L, or 0.5U/. mu.L.
In some examples, the working concentration of Taq-BDNA polymerase in the second reaction system may be 0.04U/. mu.L, 0.05U/. mu.L, 0.06U/. mu.L, 0.07U/. mu.L, or 0.08U/. mu.L. In addition, in some examples, the working concentration of Klenow fragment in the second reaction system can be 0.08U/. mu.L, 0.09U/. mu.L, 0.10U/. mu.L, 0.11U/. mu.L, or 0.12U/. mu.L.
In some examples, the second buffer can include dntps. This provides a starting material for second strand cDNA synthesis.
In some examples, the second buffer can further include Tris-HCl, magnesium chloride, sodium chloride, dithiothreitol, dATP, and ATP. In this case, Tris-HCl is used to maintain the stability of pH of the second reaction system, magnesium chloride is able to provide magnesium ions to increase the activity of the enzyme, sodium chloride is used to adjust the ionic strength, dithiothreitol is able to maintain the stability of the enzyme, dATP is able to provide a raw material of terminal addition a, ATP is used to provide energy for the reaction in the second reaction system, in other words, the first buffer is able to maintain the pH of the first reaction system and the stability of the enzyme, and is able to provide substances (e.g., dNTP, magnesium ions, dATP, ATP) and conditions (e.g., pH, ionic strength) required for the reaction.
In some examples, in the second reaction system, the working concentration of dNTPs may be from 0.3mM to 0.6mM, the working concentration of Tris-HCl may be from 6mM to 10mM, the working concentration of magnesium chloride may be from 0.2mM to 0.5mM, the working concentration of sodium chloride may be from 0.03M to 0.06M, the working concentration of dithiothreitol may be from 3mM to 6mM, the working concentration of dATP may be from 1.2mM to 1.4mM, and the working concentration of dATP may be from 0.7mM to 1 mM. This can provide an environment favorable for the reaction in the second reaction system, and can contribute to an improvement in the reaction efficiency of each reaction in the second reaction system. In other examples, the working concentration of dATP can be the working concentration of total dATP that comprises dATP of the dntps.
In some examples, the working concentration of dNTPs in the second reaction system can be 0.3mM, 0.35mM, 0.4mM, 0.45mM, 0.5mM, 0.55mM, or 0.6 mM. In addition, in some examples, the working concentration of Tris-HCl in the second reaction system may be 6mM, 6.5mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5mM, 10 mM.
In some examples, the working concentration of magnesium chloride in the second reaction system may be 0.2mM, 0.25mM, 0.3mM, 0.35mM, 0.4mM, 0.45mM, or 0.5 mM. In some examples, the working concentration of sodium chloride in the second reaction system may be 0.03M, 0.035M, 0.04M, 0.045M, 0.05M, 0.055M, 0.06M.
In some examples, the working concentration of dithiothreitol in the second reaction system can be 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, or 6 mM. In addition, in some examples, the working concentration of dATP in the second reaction system can be 1.2mM, 1.25mM, 1.3mM, 1.35mM, or 1.4 mM. In other examples, the working concentration of ATP may be 0.7mM, 0.8mM, 0.9mM, or 1mM in the second reaction system.
In some examples, the second reaction system may be subjected to a second heat treatment in step S20. Thus, second strand cDNA synthesis, end repair and end addition of A are carried out in the second reaction system.
In some examples, in the second heat treatment, optionally, a second strand of cDNA is synthesized using the first strand of cDNA as a template, followed by end repair and end addition of a to the cDNA composed of the first strand of cDNA and the second strand of cDNA to form a second reaction solution. In other examples, the second reaction solution may comprise a cDNA fragment consisting of a first strand of cDNA and a second strand of cDNA, end-repaired and end-added with a.
In some examples, the second heat treatment may be performed according to a second predetermined procedure. In other examples, the second heat treatment may be performed in a PCR instrument. Additionally, in some examples, the second predetermined procedure may be heating to 16 ℃ for 30min for second strand cDNA synthesis, end repair, and end addition a.
In some examples, in step S30, the second reaction solution may be mixed with T4DNA ligase, a third buffer solution, and a Y-linker into a third reaction system.
In some examples, the working concentration of T4DNA ligase in the third reaction system may be from 10U/. mu.L to 30U/. mu.L. For example, the working concentration of T4DNA ligase may be 10U/. mu.L, 12U/. mu.L, 14U/. mu.L, 15U/. mu.L, 18U/. mu.L, 20U/. mu.L, 22U/. mu.L, 24U/. mu.L, 25U/. mu.L, 26U/. mu.L, 28U/. mu.L, or 30U/. mu.L.
In some examples, the working concentration of the Y-linker in the third reaction system may be 1 × 10-7M to 3X 10-7And M. For example, the Y-junction may have a working concentration of 1X 10-7M、1.2×10-7M、1.4×10-7M、1.5×10-7M、1.7×10-7M、2×10-7M、2.5×10-7M、2.7×10-7M or 3X 10-7M。
In some examples, the third buffer may include dimethyl sulfoxide (DMSO). This enhances the ligation reaction between the Y-shaped linker and the cDNA. In some examples, the third buffer may also include ATP, Tris-HCl, magnesium chloride, dithiothreitol, and polyethylene glycol 8000(PEG 8000). In this case, Tris-HCl can maintain the pH stability of the third reaction system, magnesium ions in ATP and magnesium chloride are used to catalyze T4DNA ligase, dithiothreitol can maintain the stability of T4DNA ligase, polyethylene glycol 8000 can thicken the third reaction system to facilitate Y-type linker ligation to cDNA, in other words, the third buffer can maintain the pH of the third reaction system and the stability of the enzyme, and can enhance linker ligation reaction.
In some examples, in the third reaction system, the working concentration of dimethyl sulfoxide may be 1% to 1.5%, the working concentration of ATP may be 1.5mM to 3mM, the working concentration of Tris-HCl may be 0.04M to 0.08M, the working concentration of magnesium chloride may be 0.03M to 0.05M, the working concentration of dithiothreitol may be 1mM to 3mM, and the working concentration of polyethylene glycol 8000 may be 5 wt% to 8 wt%. This provides an environment favorable for the linker ligation reaction, and improves the reaction efficiency of the linker ligation reaction.
In some examples, the working concentration of dimethyl sulfoxide in the third reaction system may be 1%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%. In addition, the working concentration of dimethyl sulfoxide in the third reaction system is expressed in% by volume.
In some examples, the working concentration of ATP may be 1.5mM, 1.8mM, 2mM, 2.3mM, 2.5mM, 2.8mM, or 3mM in the third reaction system.
In some examples, the working concentration of Tris-HCl in the third reaction system may be 0.04M, 0.045M, 0.05M, 0.055M, 0.06M, 0.065M, 0.07M, 0.075M, or 0.08M. In other examples, the working concentration of magnesium chloride in the third reaction system may be 0.03M, 0.035M, 0.04M, 0.045M, or 0.05M.
In some examples, the working concentration of dithiothreitol in the third reaction system can be 1mM, 1.2mM, 1.5mM, 1.8mM, 2mM, 2.2mM, 2.5mM, 2.8mM, or 3 mM.
In some examples, the working concentration of polyethylene glycol 8000 can be 5 wt%, 5.5 wt%, 6 wt%, 6.5 wt%, 7 wt%, 7.5 wt%, or 8 wt% in the third reaction system. In addition, the working concentration of polyethylene glycol 8000 in the third reaction system is expressed in mass percent concentration wt%.
In some examples, the first sequence of the Y-type linker may be 5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC-3' and the second sequence is 5 '-GATCGGAAGAGCACACTCTGTCAACTCCAGTCACXXXXXATCTCGTATGCCGTCTTCTGCTTG-3', thereby improving the efficiency of ligation of the Y-type linker to the cDNA.
In some examples, nnnnnnnnnn may be a molecular tag sequence and XXXXXXXX may be a sample tag sequence. In this case, the molecular tag sequence can be used to identify the cDNA fragment and the sample tag sequence can be used to identify the sample, which can help to improve the accuracy of subsequent sequencing.
In some examples, nnnnnnnnn can be a random base sequence. In other examples, XXXXXXXX can be a random base sequence. In addition, the base sequence of NNNNNNNN can be different from the base sequence of XXXXXXXXX.
In some examples, the 3 'end of the first sequence is modified by thio and the 5' end of the second sequence is modified by phosphorylation. This can further improve the efficiency of ligation of the Y-type linker to cDNA.
In some examples, the third reaction system may be subjected to a third heat treatment in step S30. Thereby, a linker connecting reaction can occur in the third reaction system. In other examples, the cDNA may be ligated to the Y-type linker in the third heat treatment.
In some examples, the third heat treatment may be performed according to a third predetermined program. In other examples, the third heat treatment may be performed in a PCR instrument. Additionally, in some examples, the third predetermined procedure may be heating to 22 ℃ for 15min for joint connection.
In some examples, after the third heat treatment, the third reaction solution may be obtained by subjecting the third reaction system to a purification treatment. In addition, in some examples, the third reaction system after the third heat treatment is purified using magnetic beads. In other examples, the third reaction solution comprises cDNA with a Y-linker attached thereto.
In some examples, in step S40, the third reaction solution may be mixed with the premix and the amplification primers into a fourth reaction system. In other examples, the premix may include an amplification enzyme. In addition, in some examples, the premix may include all of the components necessary to perform PCR, except for the templates and primers.
In some examples, the premix may be a Taq PCR Mix premix, a KAPA HiFi PCR Mix premix, or a Q5 hotspot PCR Mix premix. For example, the premix may be a1 × KAPA HiFi PCR Mix premix. In other examples, the working concentration of the premix in the fourth reaction system is 1 ×.
In some examples, the amplification primer may be a combination of a forward primer and a reverse primer. In other examples, the sequence of the forward primer may be 5'-AATGATACGGCGACCACC GA-3' and the reverse primer may be 5'-CAAGCAGAAGACGGCATACGA-3'.
In some examples, the working concentration of amplification primers may be 2 × 10-7M to 6X 10-7And M. For example, the working concentration of amplification primers may be. 2X 10-7M、2.5×10-7M、2.8×10-7M、3×10-7M、3.5×10-7M、4.0×10-7M、4.5×10-7M、5.0×10-7M、5.5×10-7M、6×10-7M。
In some examples, in step S40, the fourth reaction system may be subjected to a fourth heat treatment. This enables the amplification reaction to proceed in the fourth reaction system. In other examples, the cDNA may undergo an amplification reaction in the fourth heat treatment.
In some examples, the fourth heat treatment may be performed according to a fourth predetermined program. In other examples, the fourth heat treatment may be performed in a PCR instrument. Additionally, in some examples, the fourth predetermined procedure may be a PCR amplification reaction procedure. For example, the fourth predetermined procedure may be: the pre-denaturation is first carried out by heating to 98 ℃ for 30 seconds, then the denaturation procedure is carried out by maintaining 98 ℃ for 10 seconds, then the annealing procedure is carried out by cooling to 60 ℃ for 30 seconds, then the extension procedure is carried out by heating to 72 ℃ for 30 seconds, and the cycle is 9 to 15 times in the manner of "denaturation → annealing → extension", then the extension is carried out for 5min at 72 ℃ to ensure that the reactants are fully extended.
In some examples, after the fourth heat treatment, a purification treatment of the fourth reaction system may obtain an mRNA library. In addition, in some examples, the fourth reaction system after the fourth heat treatment is purified using magnetic beads. In other examples, purification may be performed using a PCR product kit.
In some examples, the first reaction system, the second reaction system, and the third reaction system may be formed within one reaction vessel (e.g., reaction tube). This can simplify the operation process. Specifically, after a first reaction system is formed into a first reaction solution by a first heat treatment in a reaction tube, a second mixed enzyme solution and a second buffer solution are continuously added to the reaction tube to form a second reaction system, the second reaction system is formed into a second reaction solution by a second heat treatment in the reaction tube, then T4DNA ligase, a Y-linker and a third buffer solution are continuously added to the reaction tube to form a third reaction system, and then the third reaction system is formed into a third reaction solution by a third heat treatment in the reaction tube.
The kit for constructing an mRNA library according to the present embodiment may include a first mixed enzyme solution, a first buffer solution, a second mixed enzyme solution, and a second buffer solution. In some examples, the first mixed enzyme solution may have an rnase inhibitor and a reverse transcriptase. Wherein the reverse transcriptase is MMLV reverse transcriptase lacking ribonuclease H activity. In other examples, the second mixed enzyme solution can have DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase, and Klenow fragment. In addition, the Klenow fragment may lack nuclease activity for nick translation from the 5 'end to the 3' end and nuclease activity for correction from the 3 'end to the 5' end. In some examples, the first mixed enzyme solution and the first buffer solution may be used to form a first reaction system for first strand synthesis of cDNA. In addition, the second mixed enzyme solution and the second buffer solution can be used to form a second reaction system for second strand cDNA synthesis, end repair, and end-plus-A.
In this embodiment, rnase inhibitors can prevent mRNA degradation and MMLV reverse transcriptase, which lacks rnase H activity, can increase the yield of first strand cDNA synthesis, thereby increasing the efficiency of first strand cDNA synthesis. In addition, DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase and Klenow fragment lacking 5 'to 3' nick translation activity and 3 'to 5' correction can have a synergistic effect, and can improve the efficiency of second strand cDNA synthesis, end repair and end-to-A addition, thus contributing to the improvement of the efficiency of mRNA library construction.
In addition, the second strand synthesis of cDNA, end repair, and end addition of A are all performed in the second reaction system, so that the number of steps (e.g., sample addition, lid closing, machine reaction, lid opening, etc.) for end repair and end addition of A can be reduced, and the purification process after cDNA is formed by second strand synthesis of cDNA can be omitted, whereby the reaction steps can be simplified and the time for library construction can be shortened, and the steps can be omitted, so that the use of reagents can be reduced, and the cost can be reduced.
In some examples, optionally, in the first reaction system, the working concentration of MMLV reverse transcriptase is 8U/μ L to 12U/μ L, and the working concentration of rnase inhibitor is 1.0U/μ L to 1.5U/μ L; in the second reaction system, the working concentration of DNA polymerase I is 0.6U/μ L to 1U/μ L, the working concentration of glycosylnuclease H is 0.1U/μ L to 0.2U/μ L, the working concentration of T4DNA polymerase is 0.05U/μ L to 0.1U/μ L, the working concentration of T4 polynucleotide kinase is 0.3U/μ L to 0.5U/μ L, the working concentration of Taq-B DNA polymerase is 0.04U/μ L to 0.08U/μ L, and the working concentration of Klenow fragment is 0.08U/μ L to 0.12U/μ L. Therefore, the reaction efficiency of each reaction can be effectively improved.
In some examples, the first buffer may further include Tris-HCl, magnesium chloride, potassium chloride, and dithiothreitol, and the working concentration of dNTPs may be 0.3mM to 0.7mM, Tris-HCl may be 0.03M to 0.06M, magnesium chloride may be 2.0mM to 2.5mM, potassium chloride may be 0.03M to 0.08M, and dithiothreitol may be 6mM to 10mM in the first reaction system. In this case, the first buffer solution can maintain the pH of the first reaction system and the stability of the enzyme, and can provide substances and conditions required for the reaction.
In some examples, the second buffer may further include Tris-HCl, magnesium chloride, sodium chloride, dithiothreitol, dATP, and ATP, and in the second reaction system, the working concentration of dNTP may be from 0.3mM to 0.6mM, the working concentration of Tris-HCl may be from 6mM to 10mM, the working concentration of magnesium chloride may be from 0.2mM to 0.5mM, the working concentration of sodium chloride may be from 0.03M to 0.06M, the working concentration of dithiothreitol may be from 3mM to 6mM, the working concentration of dATP may be from 1.2mM to 1.4mM, and the working concentration of ATP may be from 0.7mM to 1 mM. In this case, the second buffer solution can maintain the pH of the second reaction system and the stability of the enzyme, and can provide substances and conditions required for the reaction.
In some examples, the first mixed enzyme solution, the first buffer solution, and the first reaction system may be as described in the method of constructing an mRNA library described above. In other examples, the second mixed enzyme solution, the second buffer solution, and the second reaction system may be as described in the method of constructing an mRNA library described above.
In some examples, the kit may include a fragmentation buffer. Among them, a fragmentation buffer may be used for the fragmentation treatment. In addition, in some examples, a fragmentation buffer can be used to form the fragmentation reaction system. Thus, the mRNA fragmentation treatment can be performed using the kit. In other examples, the fragmentation buffer can be as described in the methods of constructing an mRNA library described above.
In some examples, the kit may include T4DNA ligase, a third buffer, and a Y-linker. In addition, in some examples, T4DNA ligase, a third buffer, and a Y-linker may be used to form a third reaction system. Wherein the third reaction system may be used for ligation linker reactions. In other examples, T4DNA ligase, a third buffer, and a Y-linker may be as described above in the methods for constructing an mRNA library.
In some examples, the kit may include a premix and amplification primers. In other examples, the premix and the amplification primers may be used to form a fourth reaction system. In addition, the fourth reaction system may be used for an amplification reaction. Thus, an mRNA library can be amplified using the kit. In other examples, the premix and amplification primers can be as described above in the methods for constructing an mRNA library.
In some examples, the kit can further include mRNA capture magnetic beads (e.g., oligo (dt) magnetic beads), a magnetic bead binding buffer, a magnetic bead wash, and a Tris buffer. This enables capture of mRNA using the kit.
In some examples, the kit may include a fragmentation buffer for a fragmentation process, T4DNA ligase, a third buffer with dimethyl sulfoxide, and a Y-type linker, and a pre-mix with an amplification enzyme and an amplification primer, wherein the T4DNA ligase, the third buffer, and the Y-type linker may be used to form a third reaction system for the ligation linker, and the pre-mix and the amplification primer may be used to form a fourth reaction system for the amplification reaction. Thus, the kit can be used for mRNA fragmentation processing, and can amplify an mRNA library.
In some examples, the procedure for constructing an mRNA strand-specific library using the kit may be the same as that of any of the methods described above for constructing an mRNA strand-specific library. In other words, the method for constructing an mRNA chain-specific library using the kit can be the same as any of the above-described methods for constructing an mRNA chain-specific library.
According to the present disclosure, a method and a kit for constructing an mRNA library, which are advantageous for improving library construction efficiency, can be provided.
To further illustrate the present disclosure, the methods for constructing mRNA libraries provided by the present disclosure are described in detail below with reference to examples, and the beneficial effects achieved by the present disclosure are fully demonstrated with reference to comparative examples.
[ example 1]
In this example, total RNA of purified human leukocyte samples was extracted using a commercial kit as RNA samples, followed by quantification of RNA samples using a qubit3.0 fluorescence quantifier, detection of sample completion by an Agilent4200 bioanalyzer, and finally selection of RNA samples with a concentration of 216 ng/. mu.l and a RIN of 10 as RNA samples to be processed.
In this example, the formulations of the first buffer solution, the first mixed enzyme solution, the second buffer solution, and the third buffer solution are shown in table 1 below. Wherein the dNTPs are a mixture of equal amounts of dATP, dTTP, dCTP and dGTP. In addition, the first sequence of the Y-junction is 5'-AATGATACGGCGACCACCGAGATCTACACATATGCGCACACTCTTTCCCTACACGACGCTCTTCCGATC-3' and the second sequence is 5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTGATCGTATCTCGTATGCCGTCTTCTGCTTG-3'.
TABLE 1
(mRNA isolation and fragmentation)
(1) According to the concentration of the RNA sample of 216ng/ul, 1000ng of the RNA sample was taken into a 0.2ml PCR tube A, nuclease-free water was then added to a total volume of 50. mu.L and placed on ice for use, 50. mu.L of mRNA capture magnetic beads equilibrated to room temperature were then added, gently pipetted 10 times to mix well, and mRNA capture was performed in a PCR instrument according to the capture procedure shown in Table 2 below.
TABLE 2 Capture procedure
(2) After the program run was complete, PCR tube a was removed and placed on a magnetic stand, and after the solution was clarified, the supernatant was carefully removed.
(3) Taking the PCR tube A out of the magnetic frame, adding 200 mu L of magnetic bead washing liquid to resuspend the magnetic beads, then gently sucking by using a pipettor for 10 times, fully mixing, then placing on the magnetic frame, and carefully removing the supernatant after the solution is clarified.
(4) The PCR tube A was removed from the magnetic frame, 50. mu.L of Tris buffer was added to resuspend the magnetic beads, followed by gentle pipetting 10 times using a pipette and mixing well, followed by mRNA elution in a PCR machine according to the elution procedure shown in Table 3.
TABLE 3 elution procedure
(5) After the program was run, the PCR tube a was removed and 50 μ L of magnetic bead binding buffer was added, followed by gentle pipetting 10 times using a pipette and mixing well, followed by standing at room temperature for 5min to bind mRNA to the magnetic beads.
(6) PCR tube a was placed on a magnetic rack to separate mRNA from total RNA, and after the solution was clarified, the supernatant was carefully removed.
(7) Taking the PCR tube A out of the magnetic frame, adding 200 mu L of magnetic bead washing liquid to resuspend the magnetic beads, then gently sucking by using a pipettor for 10 times, fully mixing, then placing on the magnetic frame, and carefully removing the supernatant after the solution is clarified.
(8) The PCR tube A was taken out of the magnetic frame, 12. mu.L of fragmentation buffer was added to resuspend the magnetic beads, followed by gentle pipetting 10 times using a pipette and mixing well, and mRNA fragmentation was performed in a PCR instrument according to the fragmentation procedure shown in Table 4.
TABLE 4 fragmentation procedure
(9) After the program run was completed, the PCR tube A was immediately placed on a magnetic stand, after the solution was clarified, 10. mu.L of the supernatant (mRNA fragment) was pipetted into a new PCR tube (i.e., PCR tube B), and a first strand cDNA synthesis reaction was immediately performed.
(cDNA Synthesis, terminal repair and addition of A at the end)
First, in a PCR tube B, a first reaction system for synthesizing a first strand of cDNA is prepared according to the following table 5, a pipette is used for gently sucking and beating for 10 times to fully mix the first strand of cDNA, then a first strand cDNA synthesis reaction is carried out in a PCR instrument according to a first preset program shown in the table 6, wherein the temperature of a PCR hot cover is set to be 105 ℃, and the PCR tube B is immediately taken out after the first preset program is finished running.
TABLE 5 first reaction System
TABLE 6 first predetermined procedure
And (II) preparing a second reaction system for synthesizing a second strand of cDNA, repairing the tail end and adding A to the tail end of the PCR tube B according to the table 7, gently sucking and beating the second reaction system for 10 times by using a pipette, fully and uniformly mixing the second reaction system and the tail end, and then reacting in a PCR instrument according to a second preset program shown in the table 8, wherein the temperature of a PCR hot cover is set to be 105 ℃, and immediately taking out the PCR tube B after the second preset program is finished.
TABLE 7 second reaction System
TABLE 8 second Preset procedure
(Joint ligation and purification)
(a) Preparing a third reaction system for synthesizing a connecting joint in the PCR tube B according to the table 9, slightly sucking and beating the third reaction system for 10 times by using a pipette, fully and uniformly mixing the third reaction system and the PCR tube B, and then carrying out joint connection reaction in a PCR instrument according to a third preset program shown in the table 10, wherein the temperature of a PCR hot cover is set to be 105 ℃, and immediately taking out the PCR tube B after the third preset program is operated.
TABLE 9 third reaction System
TABLE 10 third Preset procedure
(b) Add 35. mu.L of water to PCR tube B to make up 100. mu.L, then aspirate 90. mu.L of the mixed magnetic beads (0.9X) and add to PCR tube B, and gently pipette the mixture, incubate at room temperature for 5min, place PCR tube B on a magnetic rack, and remove the supernatant after the solution is clear.
(c) Add 200. mu.L of freshly prepared 80% ethanol to PCR tube B to rinse the beads, incubate at room temperature for 30sec, carefully remove the supernatant.
(d) Repeating step (c) 1 time.
(e) And keeping the PCR tube B on the magnetic frame all the time, and uncapping and drying the magnetic beads at room temperature for about 8 min.
(f) Adding 22 mu L of nuclease-free water into the PCR tube B, gently sucking and beating the mixture by using a pipette, fully and uniformly mixing the mixture, standing the mixture at room temperature for 2min, placing the mixture on a magnetic frame, and carefully sucking 20 mu L of purified product into a new PCR tube (namely the PCR tube C) after the solution is clarified.
(library amplification and library quality control)
(i) In the PCR tube C, a fourth reaction system for amplification reaction was prepared according to table 11, and gently pipetted 10 times to mix well, and then PCR amplification reaction was performed in the PCR instrument according to a fourth predetermined program shown in table 12, where the temperature of the PCR hot lid was set to 105 ℃, and when the fourth predetermined program was finished, the PCR tube C was immediately taken out.
TABLE 11 fourth reaction System
TABLE 12 fourth Preset procedure
(ii) Pipette 40 μ L of the mixed magnetic beads (0.8 ×) into PCR tube C, gently pipette the mixture, incubate at room temperature for 5min, place the sample on a magnetic rack, and remove the supernatant after the solution is clear.
(iii) Add 200. mu.L of freshly prepared 80% ethanol to PCR tube C to rinse the beads, incubate at room temperature for 30sec, carefully remove the supernatant.
(iv) (iv) repeating step (iii) 1 time.
(v) And keeping the PCR tube C on the magnetic frame all the time, and uncapping and drying the magnetic beads at room temperature for about 8 min.
(vi) And adding 20 mu L of nuclease-free water into the PCR tube C, gently sucking and beating the mixture by using a pipette, fully and uniformly mixing the mixture, standing the mixture at room temperature for 2min, placing the mixture on a magnetic frame, and carefully sucking all supernate into a new PCR tube (namely the PCR tube D) after the solution is clarified to obtain the mRNA library.
(vii) The concentration of the mRNA library in the PCR tube D was measured using a Qubit3.0 fluorescence quantifier, and the size of the mRNA library was measured using an Agilent4200 bioanalyzer, and the results of the library quality test are shown in Table 14.
[ examples 2 to 4]
Examples 2 to 4 in comparison with example 1, the second mixed enzyme solutions prepared according to the formulations shown in Table 13 were used, and the remaining solutions were treated in the same manner as in example 1 until mRNA libraries and library quality control were obtained, and the library quality control results are shown in Table 14.
TABLE 13 second Mixed enzyme solution formulation
[ comparative example ]
In this comparative example, use was made of
UltraTMRNA Library Prep Kit for
The kit was used to construct an mRNA library from the same samples as in examples 1 to 4 (the library construction procedure is shown in fig. 2) according to the instructions and the library quality test was performed in the same manner as in example 1, and the library quality test results are shown in table 14.
TABLE 14 library quality test results
| Cost of | Application time (h) | Fragment size (bp) | Ex-warehouse concentration (ng/uL) | Efficiency of amplification | |
| Example 1 | 38.14 | 3.5h | 406 | 49.6 | 141％ |
| Example 2 | 39.62 | 3.5h | 413 | 39.2 | 111％ |
| Example 3 | 39.98 | 3.5h | 423 | 47.7 | 136％ |
| Example 4 | 40.45 | 3.5h | 432 | 40.6 | 115％ |
| Comparative example | 285.89 | 6.1h | 412 | 35.2 | 100％ |
As can be seen from Table 14, in examples 1 to 4, the cost is reduced, the amplification efficiency is improved, and the time is reduced by 2.6h compared with the comparative example, particularly, in the above examples, the cost of library construction in example 1 is reduced by about 86% compared with the comparative example, and the amplification efficiency is improved by 41%, therefore, the method for constructing the mRNA strand specific library used in the examples is beneficial to improving the library construction efficiency.
While the present disclosure has been described in detail above with reference to the drawings and the embodiments, it should be understood that the above description does not limit the present disclosure in any way. Those skilled in the art can make modifications and variations to the present disclosure as needed without departing from the true spirit and scope of the disclosure, which fall within the scope of the disclosure.
Claims (13)
1. A method of constructing an mRNA library, comprising:
the method comprises the following steps:
preparing and mixing an mRNA fragment, a first mixed enzyme solution having an RNase inhibitor and a reverse transcriptase, and a first buffer solution having dNTP as a first reaction system, and subjecting the first reaction system to a first heat treatment, wherein in the first heat treatment, a first cDNA strand is synthesized using the mRNA fragment as a template to obtain a first reaction solution, and the reverse transcriptase is an MMLV reverse transcriptase lacking in RNase H activity;
mixing the first reaction solution with a second mixed enzyme solution containing DNA polymerase I, ribonuclease H, T4, T4 polynucleotide kinase, Taq-B DNA polymerase, and Klenow fragments, which lack nuclease activity for nick translation from the 5 'end to the 3' end and nuclease activity for correction from the 3 'end to the 5' end, and a second buffer solution containing dntps as a second reaction system, and subjecting the second reaction system to a second heat treatment in which a second cDNA strand is synthesized using the first cDNA strand as a template, followed by end repair and end addition a of cDNA composed of the first cDNA strand and the second cDNA strand to form a second reaction solution;
mixing the second reaction solution with T4DNA ligase, a third buffer solution containing dimethyl sulfoxide, and a Y-shaped linker to obtain a third reaction system, subjecting the third reaction system to a third heat treatment in which cDNA is ligated to the Y-shaped linker, and purifying the third heat treatment to obtain a third reaction solution; and is
Adding a premixed solution containing an amplification enzyme and an amplification primer to the third reaction solution and mixing them to form a fourth reaction system, subjecting the fourth reaction system to a fourth heat treatment, subjecting cDNA to an amplification reaction in the fourth heat treatment, and purifying the cDNA after the fourth heat treatment to obtain an mRNA library.
2. The method of claim 1, wherein:
in the first reaction system, the working concentration of the MMLV reverse transcriptase is 8U/mu L to 12U/mu L, and the working concentration of the RNase inhibitor is 1.0U/mu L to 1.5U/mu L.
3. The method of claim 1, wherein:
in the second reaction system, the working concentration of the DNA polymerase I is 0.6U/muL to 1U/muL, the working concentration of the glyconuclease H is 0.1U/muL to 0.2U/muL, the working concentration of the T4DNA polymerase is 0.05U/muL to 0.1U/muL, the working concentration of the T4 polynucleotide kinase is 0.3U/muL to 0.5U/muL, the working concentration of the Taq-BDNA polymerase is 0.04U/muL to 0.08U/muL, and the working concentration of the Klenow fragment is 0.08U/muL to 0.12U/muL.
4. The method of claim 1 or 2, wherein:
the first buffer solution also comprises Tris-HCl, magnesium chloride, potassium chloride and dithiothreitol,
in the first reaction system, the working concentration of dNTP is 0.3mM to 0.7mM, the working concentration of Tris-HCl is 0.03M to 0.06M, the working concentration of magnesium chloride is 2.0mM to 2.5mM, the working concentration of potassium chloride is 0.03M to 0.08M, and the working concentration of dithiothreitol is 6mM to 10 mM.
5. A method according to claim 1 or 3, characterized by:
the second buffer further comprises Tris-HCl, magnesium chloride, sodium chloride, dithiothreitol, dATP, and ATP;
in the second reaction system, the working concentration of dNTP is 0.3mM to 0.6mM, the working concentration of Tris-HCl is 6mM to 10mM, the working concentration of magnesium chloride is 0.2mM to 0.5mM, the working concentration of sodium chloride is 0.03M to 0.06M, the working concentration of dithiothreitol is 3mM to 6mM, the working concentration of dATP is 1.2mM to 1.4mM, and the working concentration of ATP is 0.7mM to 1 mM. .
6. The method of claim 1, wherein:
the mRNA fragments are subjected to a fragmentation treatment, and the fragmentation treatment is carried out in a fragmentation reaction system formed by mixing a fragmentation buffer and an mRNA sample, wherein the fragmentation buffer comprises Tris-HCl, magnesium ions and random hexamer primers.
7. The method of claim 1, wherein:
the third buffer solution also comprises ATP, Tris-HCl, magnesium chloride, dithiothreitol and polyethylene glycol 8000,
in the third reaction system, the working concentration of the T4DNA ligase is 10U/. mu.L to 30U/. mu.L; the working concentration of the Y-shaped joint is 1 multiplied by 10-7M to 3X 10-7M; the working concentration of the dimethyl sulfoxide is 1-1.5%, the working concentration of the ATP is 1.5-3 mM, the working concentration of the Tris-HCl is 0.04-0.08M, the working concentration of the magnesium chloride is 0.03-0.05M, the working concentration of the dithiothreitol is 1-3 mM, and the working concentration of the polyethylene glycol 8000 is 5-8 wt%.
8. The method of claim 1 or 7, wherein:
the first sequence of the Y-shaped joint is 5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC-3', the second sequence is 5 '-GATCGGAAGAGCACACGTCTGAACCAGTCACXXXXXATCTCGTATGCCGTCTTCTGCTTG-3',
wherein the 3 'end of the first sequence is modified by thio, the 5' end of the second sequence is modified by phosphorylation, NNNNNNNN is a molecular tag sequence, and XXXXXXX is a sample tag sequence.
9. A kit for constructing an mRNA library, comprising:
the method comprises the following steps: a first mixed enzyme solution with an RNase inhibitor and a reverse transcriptase and a first buffer solution with dNTPs, and a second mixed enzyme solution with DNA polymerase I, ribonuclease H, T4DNA polymerase, T4 polynucleotide kinase, Taq-B DNA polymerase and Klenow fragment and a second buffer solution with dNTPs,
wherein the reverse transcriptase is an MMLV reverse transcriptase lacking ribonuclease H activity, the Klenow fragment lacks nuclease activity for nick translation from 5 'to 3' and nuclease activity for correction from 3 'to 5',
the first mixed enzyme solution and the first buffer solution are used for forming a first reaction system for cDNA first strand synthesis, and the second mixed enzyme solution and the second buffer solution are used for forming a second reaction system for cDNA second strand synthesis, end repair and end-plus-A.
10. The kit of claim 9, wherein:
in the first reaction system, the working concentration of the MMLV reverse transcriptase is 8U/mu L to 12U/mu L, and the working concentration of the RNase inhibitor is 1.0U/mu L to 1.5U/mu L;
in the second reaction system, the working concentration of the DNA polymerase I is 0.6U/muL to 1U/muL, the working concentration of the glyconuclease H is 0.1U/muL to 0.2U/muL, the working concentration of the T4DNA polymerase is 0.05U/muL to 0.1U/muL, the working concentration of the T4 polynucleotide kinase is 0.3U/muL to 0.5U/muL, the working concentration of the Taq-B DNA polymerase is 0.04U/muL to 0.08U/muL, and the working concentration of the Klenow fragment is 0.08U/muL to 0.12U/muL.
11. The kit of claim 9 or 10, wherein:
the first buffer solution also comprises Tris-HCl, magnesium chloride, potassium chloride and dithiothreitol,
in the first reaction system, the working concentration of dNTP is 0.3mM to 0.7mM, the working concentration of Tris-HCl is 0.03M to 0.06M, the working concentration of magnesium chloride is 2.0mM to 2.5mM, the working concentration of potassium chloride is 0.03M to 0.08M, and the working concentration of dithiothreitol is 6mM to 10 mM.
12. The kit of claim 9 or 10, wherein:
the second buffer further comprises Tris-HCl, magnesium chloride, sodium chloride, dithiothreitol, dATP, and ATP,
in the second reaction system, the working concentration of dNTP is 0.3mM to 0.6mM, the working concentration of Tris-HCl is 6mM to 10mM, the working concentration of magnesium chloride is 0.2mM to 0.5mM, the working concentration of sodium chloride is 0.03M to 0.06M, the working concentration of dithiothreitol is 3mM to 6mM, the working concentration of dATP is 1.2mM to 1.4mM, and the working concentration of ATP is 0.7mM to 1 mM.
13. The kit of claim 9, wherein:
also comprises a fragmentation buffer solution for fragmentation treatment, T4DNA ligase, a third buffer solution with dimethyl sulfoxide and a Y-shaped joint, as well as a premixed solution with an amplification enzyme and an amplification primer, wherein,
the T4DNA ligase, the third buffer solution and the Y-shaped joint are used for forming a third reaction system for connecting the joint, and the premixed solution and the amplification primer are used for forming a fourth reaction system for amplification reaction.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911222706.1A CN110846383B (en) | 2019-12-03 | 2019-12-03 | Method and kit for constructing mRNA library |
| CN202310879367.4A CN116926165A (en) | 2019-12-03 | 2019-12-03 | Method for constructing mRNA library for improving library construction efficiency |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911222706.1A CN110846383B (en) | 2019-12-03 | 2019-12-03 | Method and kit for constructing mRNA library |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310879367.4A Division CN116926165A (en) | 2019-12-03 | 2019-12-03 | Method for constructing mRNA library for improving library construction efficiency |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110846383A true CN110846383A (en) | 2020-02-28 |
| CN110846383B CN110846383B (en) | 2023-07-18 |
Family
ID=69607420
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911222706.1A Active CN110846383B (en) | 2019-12-03 | 2019-12-03 | Method and kit for constructing mRNA library |
| CN202310879367.4A Pending CN116926165A (en) | 2019-12-03 | 2019-12-03 | Method for constructing mRNA library for improving library construction efficiency |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310879367.4A Pending CN116926165A (en) | 2019-12-03 | 2019-12-03 | Method for constructing mRNA library for improving library construction efficiency |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN110846383B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111005074A (en) * | 2019-12-19 | 2020-04-14 | 江西海普洛斯医学检验实验室有限公司 | DNA library construction kit based on illumina sequencing platform, library construction method and application |
| CN114686453A (en) * | 2020-12-28 | 2022-07-01 | 广东菲鹏生物有限公司 | Method and kit for constructing transcriptome sequencing library |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105603535A (en) * | 2016-01-27 | 2016-05-25 | 北京诺禾致源生物信息科技有限公司 | Kit and method for constructing DNA library |
| US20180230527A1 (en) * | 2015-08-24 | 2018-08-16 | Qiagen Gmbh | Method for generating a rna-sequencing library |
| CN109957562A (en) * | 2019-03-06 | 2019-07-02 | 南京诺唯赞生物科技有限公司 | A kind of method and kit of rapid build transcript profile sequencing library |
-
2019
- 2019-12-03 CN CN201911222706.1A patent/CN110846383B/en active Active
- 2019-12-03 CN CN202310879367.4A patent/CN116926165A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180230527A1 (en) * | 2015-08-24 | 2018-08-16 | Qiagen Gmbh | Method for generating a rna-sequencing library |
| CN105603535A (en) * | 2016-01-27 | 2016-05-25 | 北京诺禾致源生物信息科技有限公司 | Kit and method for constructing DNA library |
| CN109957562A (en) * | 2019-03-06 | 2019-07-02 | 南京诺唯赞生物科技有限公司 | A kind of method and kit of rapid build transcript profile sequencing library |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111005074A (en) * | 2019-12-19 | 2020-04-14 | 江西海普洛斯医学检验实验室有限公司 | DNA library construction kit based on illumina sequencing platform, library construction method and application |
| CN114686453A (en) * | 2020-12-28 | 2022-07-01 | 广东菲鹏生物有限公司 | Method and kit for constructing transcriptome sequencing library |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110846383B (en) | 2023-07-18 |
| CN116926165A (en) | 2023-10-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11155813B2 (en) | Semi-random barcodes for nucleic acid analysis | |
| EP3247804B1 (en) | High multiplex pcr with molecular barcoding | |
| JP7514263B2 (en) | Method for attaching an adaptor to a sample nucleic acid | |
| JP2016511007A (en) | Methods, compositions and kits for generating stranded RNA or DNA libraries | |
| US20220389416A1 (en) | COMPOSITIONS AND METHODS FOR CONSTRUCTING STRAND SPECIFIC cDNA LIBRARIES | |
| USRE49207E1 (en) | Transposase-random priming DNA sample preparation | |
| US20190169603A1 (en) | Compositions and Methods for Labeling Target Nucleic Acid Molecules | |
| CN110846383A (en) | Method and kit for constructing mRNA library | |
| JP2023002557A (en) | Single primer to dual primer amplicon switching | |
| US20170175182A1 (en) | Transposase-mediated barcoding of fragmented dna | |
| CN112941635A (en) | Second-generation sequencing library building kit and method for improving library conversion rate | |
| CN107488655B (en) | Method for removing 5 'and 3' adaptor connection by-products in sequencing library construction | |
| CN110846724B (en) | Method and kit for constructing mRNA chain specific library | |
| EP3643787A1 (en) | Pcr primer pair and application thereof | |
| CN112176422B (en) | Method for constructing RNA library | |
| US20210301333A1 (en) | Methods and kits for highly multiplex single primer extension | |
| WO2021166989A1 (en) | Method for producing dna molecules having an adaptor sequence added thereto, and use thereof | |
| CN113278681A (en) | Depletion of large amounts of non-informative sequences | |
| JP2024512463A (en) | Blocking oligonucleotides for selective depletion of undesired fragments from amplified libraries | |
| WO2023172934A1 (en) | Target enrichment | |
| CN117343929A (en) | PCR random primer and method for enhancing targeted enrichment by using same |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |