CN114591930A

CN114591930A - Enhanced reverse transcriptase, coding DNA, kit and application of enhanced reverse transcriptase in construction of RNA library

Info

Publication number: CN114591930A
Application number: CN202210500793.8A
Authority: CN
Inventors: 宋东亮; 刘娜; 曹振; 戴广伟; 柴常升
Original assignee: Yisheng Biotechnology Shanghai Co ltd
Current assignee: Yisheng Biotechnology Shanghai Co ltd
Priority date: 2022-05-10
Filing date: 2022-05-10
Publication date: 2022-06-07
Anticipated expiration: 2042-05-10
Also published as: CN114591930B

Abstract

The invention provides an enhanced reverse transcriptase, which is characterized in that: on the basis of mmlv wild-type reverse transcriptase, L22V; D145Q; C240G; T288E; L319A; L385K; T431Q; H595I; Q563A nine amino acid site mutation, its amino acid sequence is shown in SEQ ID NO: 1 is shown. And discloses a coding DNA, a kit and application thereof in RNA library construction. Compared with the conventional RNA library construction kit, the rapid RNA library construction kit containing the enhanced reverse transcriptase omits the step of synthesizing second strand cDNA, shortens the library construction time, obviously reduces the cycle number of library amplification, shortens the library construction time, and can finish the RNA library construction of 500pg level within 3.5 h.

Description

Enhanced reverse transcriptase, coding DNA, kit and application of enhanced reverse transcriptase in construction of RNA library

Technical Field

The invention relates to an enhanced reverse transcriptase, coding DNA, a kit and application thereof in RNA library construction, and belongs to the technical field of biology.

Background

Second generation sequencing has been widely used in various fields of biological research. NGS has high flux, low cost and sensitivity, can detect millions or even hundreds of millions of genetic information at one time, and accelerates the whole genome sequencing speed, thereby obviously reducing the sequencing cost. The NGS brings great convenience to genomics, transcriptomics, metagenome and the like of human beings, animals and plants. Especially in the field of pathogen detection in recent years, the second-generation sequencing shows more obvious advantages. The conventional pathogen detection needs pathogen culture, and has the problems of low positive rate, long detection period and the like under the influence of factors such as a culture medium and the like. The second-generation sequencing can directly obtain the genetic information of the pathogen from the sample without depending on culture, can quickly assemble the genetic information for unknown pathogen, and plays an important role in identifying infection of new pathogen.

The pathogen sample has the problems of small nucleic acid extraction amount, nucleic acid pollution and the like. Especially, the extraction of RNA pathogens has risks of host gDNA residue and the like, and can seriously affect the detection rate of the RNA pathogens. In addition, the RNA pathogen has less nucleic acid, and is easy to have the risk of library establishment failure and the like. Therefore, there is a need for an efficient RNA reverse transcriptase that can perform efficient reverse transcription on trace RNA samples, complete the construction of sequencing libraries, and facilitate the rapid and accurate diagnosis of physicians.

Disclosure of Invention

The invention aims to provide an enhanced reverse transcriptase, which can improve the reverse transcription capability of RNA and ensure the success rate of constructing an RNA library.

The technical scheme adopted by the invention is as follows: an enhanced reverse transcriptase characterized by: on the basis of mmlv wild-type reverse transcriptase, L22V; D145Q; C240G; T288E; L319A; L385K; T431Q; H595I; Q563A nine amino acid site mutation, the amino acid sequence is shown in SEQ ID NO: 1 is shown.

The invention also discloses the coding DNA of the enhanced reverse transcriptase, which is characterized in that: the sequence is shown as SEQ ID NO: 2, respectively.

The invention also discloses application of the enhanced reverse transcriptase in RNA library construction.

The invention also discloses a rapid RNA library construction kit which comprises the enhanced reverse transcriptase.

Preferably, the rapid RNA library construction kit also comprises the following reagent components:

1st cDNA synthetase reaction buffer solution and random primers;

fragmenting enzyme and reaction buffer;

ligase and reaction buffer

A library amplification reagent.

Preferably, the using amount of the enhanced reverse transcriptase is 100-300U, and more preferably 200U; .

Preferably, the microbial inoculum further comprises Murine RNase Inhibitor, and the using amount is preferably 20-60U, and more preferably 40U.

Preferably, the 1st cDNA synthetase reaction buffer comprises: 100-200 mM Tris-HcL, pH 8.3, 10-20 mM MgcL2, 200-300 mM KcL, 20-40 mM DTT, 1-2 mM dNTPs, preferably the buffer is: 150 mM Tris-HcL (pH 8.3), 15 mM MgcL2, 250 mM KcL, 30 mM DTT, 1.5 mM dNTPs.

Preferably, the random primer is an oligonucleotide fragment of 4 to 10 nucleotides, preferably an oligonucleotide fragment of 6 to 8 nucleotides.

In the rapid RNA Library building Kit, the Hieff NGS DNA & RNA Library Co-Prep Kit for Illumina (Cat.12299) Library building Kit components of Hieff NGS DNA & RNA Library Co-Prep Kit of the next holy organism can be adopted as the fragmenting enzyme, the reaction buffer, the ligase, the reaction buffer and the Library amplification reagent. The rapid RNA library construction kit of the invention does not need the step of synthesizing second strand cDNA, and can directly segment the cDNA after reverse transcription. Compared with the conventional RNA library construction kit, the second strand cDNA synthesis step is omitted, the library construction time is shortened, the cycle number of library amplification is obviously reduced, the library construction time is shortened, and the RNA library construction of 500pg level can be completed within 3.5 h.

The beneficial effects of the invention are as follows: the enhanced reverse transcription of the invention has high-efficiency reverse transcription capability on RNA, can obviously reduce the initial quantity off-line of RNA library construction, and ensures the library construction success rate of RNA samples.

The enhanced reverse transcriptase has stronger reverse transcription capability, compared with wild type enzyme, the amplification cycle number of the library can be reduced by 2-4, and the library can be successfully established for samples lower than the detection limit.

Drawings

FIG. 1 shows the rapid RNA library construction process and the conventional RNA library construction process analysis.

FIG. 2 shows the rapid RNA library construction process and library construction library quality test analysis of conventional RNA library construction.

FIG. 3 shows the rapid RNA library construction process and quality control analysis of conventional RNA library construction with different input amounts.

Detailed Description

The embodiments of the present invention will be further described with reference to the accompanying drawings, which are provided for illustration purposes.

Example 1: obtaining a mutated enhanced reverse transcriptase

For the pathogenic field, the problems of low sample concentration and poor reservoir building power exist. We performed site-directed mutagenesis of the reverse transcriptase of the full-length cDNA synthesis module in the present invention. Nine site-directed mutagenesis was performed on the murine leukemia virus reverse transcriptase mmlv wild type: L22V; D145Q; C240G; T288E; L319A; L385K; T431Q; H595I; Q563A. The coding DNA of the reverse transcriptase mutant is synthesized by the corporation of Venezuelan engineering bioengineering (Shanghai), and the sequence of the coding DNA is shown as SEQ ID NO: 2. and (3) carrying out mutation on the coding gene of the enhanced reverse transcriptase SEQ ID NO: 2, carrying out protein expression to obtain the enhanced reverse transcriptase.

Example 2: application of enhanced reverse transcriptase in RNA library construction

This example was subjected to the library construction procedure shown below using total RNA from the 293 cell line. The conventional RNA Library building process used in the invention refers to Hieff NGS DNA & RNA Library Co-Prep Kit for Illumina (Cat.12299) Library building Kit specifications of the next holy organisms to build a Library of total RNA. Referring to FIG. 1, a rapid RNA library construction procedure using the enhanced reverse transcriptase described in example 1 was performed:

1) taking 10ng of total RNA for RNA denaturation treatment:

table 1: RNA denaturation System and denaturation conditions

2) Synthesis of 1st cDNA Using enhanced reverse transcriptase:

table 2: reaction System and reaction conditions for 1st cDNA

3) Synthesis of 2nd cDNA Using DNA polymerase:

table 3: reaction system and reaction conditions of 2nd cDNA

4) Fragmentation of Full Length-cDNA was performed using the enzyme digestion system of the 12299 library construction kit:

table 4: fragmentation reaction system and reaction condition of Full Length-cDNA

5) Performing a joint connection reaction of fragmented cDNA by using a joint connection system of an 12299 library construction kit:

table 5: linker ligation reaction system and reaction conditions for fragmented cDNA

6) Reference 12299 library construction reagentPurification System after ligation of cassette, ligation product was purified using 0.6 Xmagnetic beads, and finally 22. mu.L of ddH was used₂O, eluting, and finally taking 20 mu L of the solution to carry out the amplification reaction of the library.

7) And (3) carrying out library amplification reaction by using an amplification system of the 12299 library construction kit:

table 6: library amplification reaction system and reaction conditions

8) After completion of amplification, 0.9 Xmagnetic bead purification (Yeasen, 12601) was performed using 35. mu.L ddH₂O, elution was performed, and 32. mu.L of the eluate was subjected to library quality inspection, as shown in FIG. 2, whereby the library sizes were substantially the same. As shown in Table 7, 1# and 2# are parallel experiments, and 3# and 4# are parallel experiments. Compared with the conventional RNA library construction kit, the rapid RNA library construction kit using the enhanced reverse transcriptase can reduce the number of cycles by about 3 under the condition of equivalent yield. Sequencing data appeared to be essentially consistent.

Table 7: library yield and sequencing data analysis

Example 3: library test of mutant enhanced reverse transcriptase at different input amounts of RNA

This example was subjected to the library construction procedure shown below using total RNA from the 293 cell line. The conventional RNA Library building process used in the invention refers to Hieff NGS DNA & RNA Library Co-Prep Kit for Illumina (Cat.12299) Library building Kit specifications of the next holy organisms to build a Library of total RNA. Rapid RNA library construction procedure Using enhanced reverse transcriptase RNA library construction tests at different input levels were performed as follows with reference to FIG. 1:

1) firstly, carrying out RNA denaturation treatment on total RNA with different input amounts:

table 8: RNA denaturation System and denaturation conditions

2) Synthesis of 1st cDNA Using enhanced reverse transcriptase:

table 9: reaction System and reaction conditions for 1st cDNA

3) Synthesis of 2nd cDNA Using DNA polymerase:

table 10: reaction system and reaction conditions of 2nd cDNA

table 11: fragmentation reaction system and reaction condition of Full Length-cDNA

table 12: linker ligation reaction system and reaction conditions for fragmented cDNA

6) Referring to the purification system after ligation of 12299 library construction kit, the ligation product was purified using 0.6 Xmagnetic beads, and 22. mu.L of ddH was used₂O, eluting, and finally taking 20 mu L of the solution to carry out the amplification reaction of the library.

7) Library amplification, namely performing library amplification reaction by using an amplification system of the 12299 library construction kit:

table 13: library amplification reaction system and reaction conditions

After completion of amplification, 0.9 Xmagnetic bead purification (Yeasen, 12601) was performed using 35. mu.L ddH₂O, elution was performed, and 32. mu.L of the eluate was subjected to library quality inspection, as shown in FIG. 3, whereby the library sizes were substantially the same. As shown in tables 14-15, the rapid RNA library construction kit using the enhanced reverse transcriptase can reduce the number of cycles by about 2-4 compared with the conventional RNA library construction kit under the condition of equivalent yield. The enhanced reverse transcriptase has stronger reverse transcription capability, compared with wild type enzyme, the amplification cycle number of the library can be reduced by 2-4, and the library can be successfully established for samples lower than the detection limit.

Table 14: rapid RNA library construction process library yield analysis

Table 15: library yield analysis for routine RNA library construction process

Sequence listing

<110> Histo Histoste of next (Shanghai) Ltd

<120> enhanced reverse transcriptase, coding DNA, kit and application thereof in RNA library construction

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 683

<212> PRT

<213> Artifical Sequence

<400> 1

Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys

1 5 10 15

Glu Pro Asp Leu Ser Val Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln

20 25 30

Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro

35 40 45

Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln

50 55 60

Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln

65 70 75 80

Arg Leu Leu Asp Gln Leu Ile Gly Val Pro Cys Gln Ser Pro Trp Asn

85 90 95

Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro

100 105 110

Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro

115 120 125

Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His

130 135 140

Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Phe Phe Ala Cys Leu Arg

145 150 155 160

Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro

165 170 175

Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly

180 185 190

Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu

195 200 205

Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Val Tyr Gln

210 215 220

Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly

225 230 235 240

Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser

245 250 255

Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr

260 265 270

Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Glu

275 280 285

Val Met Gly Gln Pro Thr Pro Lys Pro Arg Leu Gln Leu Arg Glu Phe

290 295 300

Leu Gly Thr Ala Gly Phe Cys Arg Pro Ile Trp Leu Gly Phe Ala Glu

305 310 315 320

Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn

325 330 335

Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu

340 345 350

Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu

355 360 365

Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln

370 375 380

Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Asp Leu Lys

385 390 395 400

Lys Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala

405 410 415

Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro

420 425 430

Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro

435 440 445

Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu

450 455 460

Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn

465 470 475 480

Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys

485 490 495

Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp

500 505 510

Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser

515 520 525

Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu

530 535 540

Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln

545 550 555 560

Arg Ala Ala Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly

565 570 575

Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala

580 585 590

His Ile Ile Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu

595 600 605

Gly Lys Asn Glu Ile Lys Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala

610 615 620

Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln

625 630 635 640

Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala

645 650 655

Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu

660 665 670

Ile Glu Asn Ser Ser Pro Asn Ser Arg Leu Ile

675 680

<210> 2

<211> 2049

<212> DNA

<213> Artifical Sequence

<400> 2

atgaccctga acattgaaga tgaacatcgc ctgcatgaaa ccagcaaaga accggatctg 60

agcgtgggca gcacctggct gagcgatttt ccgcaggcgt gggcggaaac cggcggcatg 120

ggcctggcgg tgcgccaggc gccgctgatt attccgctga aagcgaccag caccccggtg 180

agcattaaac agtatccgat gagccaggaa gcgcgcctgg gcattaaacc gcatattcag 240

cgcctgctgg atcagctgat tggcgtgccg tgccagagcc cgtggaacac cccgctgctg 300

ccggtgaaaa aaccgggcac caacgattat cgcccggtgc aggatctgcg cgaagtgaac 360

aaacgcgtgg aagatattca tccgaccgtg ccgaacccgt ataacctgct gagcggcctg 420

ccgccgagcc atcagtggta taccgtgctg gatctgaaag atttttttgc gtgcctgcgc 480

ctgcatccga ccagccagcc gctgtttgcg tttgaatggc gcgatccgga aatgggcatt 540

agcggccagc tgacctggac ccgcctgccg cagggcttta aaaacagccc gaccctgttt 600

gatgaagcgc tgcatcgcga tctggcggat tttcgcattc agcatccgga tctgattctg 660

ctggtgtatc aggatgatct gctgctggcg gcgaccagcg aactggattg ccagcagggc 720

acccgcgcgc tgctgcagac cctgggcaac ctgggctatc gcgcgagcgc gaaaaaagcg 780

cagatttgcc agaaacaggt gaaatatctg ggctatctgc tgaaagaagg ccagcgctgg 840

ctgaccgaag cgcgcaaaga agaagtgatg ggccagccga ccccgaaacc gcgcctgcag 900

ctgcgcgaat ttctgggcac cgcgggcttt tgccgcccga tttggctggg ctttgcggaa 960

atggcggcgc cgctgtatcc gctgaccaaa accggcaccc tgtttaactg gggcccggat 1020

cagcagaaag cgtatcagga aattaaacag gcgctgctga ccgcgccggc gctgggcctg 1080

ccggatctga ccaaaccgtt tgaactgttt gtggatgaaa aacagggcta tgcgaaaggc 1140

gtgctgaccc agaaactggg cccgtggcgc cgcccggtgg cgtatctgag cgatctgaaa 1200

aaaccggtgg cggcgggctg gccgccgtgc ctgcgcatgg tggcggcgat tgcggtgctg 1260

accaaagatg cgggcaaact gaccatgggc cagccgctgg tgattctggc gccgcatgcg 1320

gtggaagcgc tggtgaaaca gccgccggat cgctggctga gcaacgcgcg catgacccat 1380

tatcaggcgc tgctgctgga taccgatcgc gtgcagtttg gcccggtggt ggcgctgaac 1440

ccggcgaccc tgctgccgct gccggaagaa ggcctgcagc ataactgcct ggatattctg 1500

gcggaagcgc atggcacccg cccggatctg accgatcagc cgctgccgga tgcggatcat 1560

acctggtata ccggcggcag cagcctgctg caggaaggcc agcgcaaagc gggcgcggcg 1620

gtgaccaccg aaaccgaagt gatttgggcg aaagcgctgc cggcgggcac cagcgcgcag 1680

cgcgcggcgc tgattgcgct gacccaggcg ctgaaaatgg cggaaggcaa aaaactgaac 1740

gtgtatacca acagccgcta tgcgtttgcg accgcgcata ttattggcga aatttatcgc 1800

cgccgcggcc tgctgaccag cgaaggcaaa aacgaaatta aaaaagatga aattctggcg 1860

ctgctgaaag cgctgtttct gccgaaacgc ctgagcatta ttcattgccc gggccatcag 1920

aaaggccata gcgcggaagc gcgcggcaac cgcatggcgg atcaggcggc gcgcaaagcg 1980

gcgattaccg aaaccccgga taccagcacc ctgctgattg aaaacagcag cccgaacagc 2040

cgcctgatt 2049

Claims

1. An enhanced reverse transcriptase characterized by: on the basis of mmlv wild-type reverse transcriptase, L22V; D145Q; C240G; T288E; L319A; L385K; T431Q; H595I; Q563A nine amino acid site mutation, its amino acid sequence is shown in SEQ ID NO: 1 is shown.

2. The enhanced reverse transcriptase-encoding DNA of claim 1, wherein: the sequence is shown as SEQ ID NO: 2, respectively.

3. Use of the enhanced reverse transcriptase of claim 1 in the construction of an RNA library.

4. A rapid RNA banking kit comprising the enhanced reverse transcriptase of claim 1.

5. The rapid RNA banking kit according to claim 4, characterized in that the reagent components further comprise:

1st cDNA synthetase reaction buffer solution and random primers;

fragmenting enzyme and reaction buffer;

ligase and reaction buffer

A library amplification reagent.

6. The kit for rapid RNA library construction according to claim 5, wherein the amount of the enhanced reverse transcriptase used is 100 to 300U.

7. The kit for rapid RNA banking according to claim 5, which is characterized by further comprising a Murine RNase Inhibitor in an amount of 20-60U.

8. The rapid RNA pooling kit of claim 5 wherein said 1st cDNA synthetase reaction buffer comprises: 100-200 mM Tris-HcL, pH 8.3, 10-20 mM MgcL₂、200~300 mM KcL、20~40 mM DTT、1~2 mM dNTPs。

9. The rapid RNA library construction kit according to claim 5, wherein the random primer is an oligonucleotide fragment of 4-10 nucleotides.