CN112176420A - Method and kit for co-building DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) - Google Patents
Method and kit for co-building DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) Download PDFInfo
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Abstract
The invention discloses a method and a kit for co-establishing a DNA and RNA library, and relates to the technical field of biology. The method comprises the following steps: 1) fragmentation of RNA and denaturation of DNA; 2) synthesizing single-strand cDNA and performing strand displacement reaction; 3) DNA3' adaptor connection; 4) and (4) amplifying the library. The kit comprises: a tool enzyme, a tool enzyme reaction system, a linker system, a PCR reaction system and Low-EDTA-TE. The invention realizes the library construction for simultaneously completing sequencing on the DNA/RNA samples, breaks through the conventional library construction means, avoids the complex operation of respectively constructing the DNA and the RNA, and effectively saves the cost.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for simultaneously constructing a sequencing library by DNA and RNA.
Background
Different pooling strategies are currently used for different nucleic acid samples. The common DNA sample is firstly subjected to end repair and A addition, then is subjected to adaptor connection, and finally the construction of a DNA sequencing library is completed through PCR amplification, which takes about 3 hours. The common RNA sample firstly needs to be captured by Poly (A) or removed by ribosomal RNA, then cDNA is synthesized, and finally the construction of an RNA sequencing library is completed by a DNA sample library construction strategy, which takes 8 h. Metagenomic sequencing plays a key role in the diagnosis, detection, tracking and tracing of new pathogens. The traditional library construction method cannot really realize the common library construction of DNA and RNA samples, and the currently existing method for constructing the DNA/RNA common library is as follows: after RNA is subjected to reverse transcription by an oligo-dT primer, Tn5 transposase randomly cuts an RNA/DNA hybrid strand, after fragmentation, the transposase adds joints at two ends of the hybrid strand, and then PCR amplification is carried out. The core point of the method is that Tn5 transposase is used for breaking and adding partial joints to RNA/cDNA complex double strands and DNA double strands, although the method can greatly optimize the library building step and save the library building time, the Tn5 transposase breaking the library building still has some inevitable defects: 1) tn5 transposase disruption recognition is specific, the first 9 bPs sequenced have obvious preference, even mutated Tn5 transposase cannot completely eliminate bais; 2) tn5 library construction is enzyme quantity dependent, the requirement for DNA quantification is high, and the interruption can be influenced by inaccurate ratio of enzyme and DNA; 3) intolerance to DNA solution impurities, which can affect the breaking effect; 4) tn5 libraries are not suitable for degradable samples or nucleic acids with small molecular fragments.
Therefore, it is necessary to provide a novel efficient co-library construction method for DNA/RNA co-library construction different from Tn5 transposase library construction by using the principle of single-strand library construction to overcome the defects of the prior art, thereby simplifying the library construction process, reducing the library construction time, solving the problem of sequencing preference, and being suitable for nucleic acid samples with degraded samples and different molecular fragments.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for constructing a DNA and RNA library together based on the principle of single-strand library construction, so as to effectively simplify the library construction process, reduce the library construction time, solve the problem of sequencing preference and also be suitable for the purpose of degrading samples and nucleic acid samples with different molecular fragment sizes.
In order to achieve the above object, the present invention provides a method for co-pooling DNA and RNA, comprising the steps of:
1. performing RNA fragmentation and DNA denaturation on the mixed sample of RNA and DNA;
2. carrying out a DNA strand displacement reaction by using a random primer based on the S1 product, and carrying out 5 'joint on a DNA fragment to form 5' Trunctedadapter-DNA; reverse transcribing the RNA to a single-stranded cDNA with a 5 'linker to form a 5' Trunctedadapter-cDNA;
3. digesting redundant primers by using exonuclease, and purifying products by using magnetic beads;
4. adding a section of poly-oligonucleotide PloyC to the 3' end of the DNA with the 5' joint and the 3' end of the cDNA with the 5' joint to obtain a 5' rounded adapter-DNA-poly-oligonucleotide structure, and connecting the 3' end of the 5' rounded adapter-DNA-poly-oligonucleotide with a P7 joint by using DNA repair enzyme, DNA polymerase and DNA ligase;
5. and amplifying the PCR library to obtain the on-machine sequencing library.
Further, the step 1 comprises fragmenting the DNA in the mixed sample.
Further, the random primer is T5 TrunctedAdapter + N6, and the nucleotide sequence of the random primer is shown as SEQ ID NO: 1 is shown.
Further, the step S2 is to perform a single strand cDNA synthesis and strand displacement reaction under the conditions of Klenow (exo-) polymerase, reverse transcriptase and RNAINHIBITOR.
Further, the reverse transcriptase is one of Hiscript II reverse transcriptase, Hiscript III reverse transcriptase and Hiscript IV reverse transcriptase.
Further, the step 3 digests the residual primers using Exo I exonuclease.
Further, the DNA polymerase is selected from one of Klenow (exo-) polymerase, Bsu DNA polymerase, phi29 polymerase, Taq polymerase, A family DNA polymerase and B family high fidelity polymerase.
The invention also aims to provide a kit for co-construction of a DNA and RNA library, which comprises the following components:
1) tool enzyme: the tool enzyme comprises reverse transcriptase, terminal transferase, DNA repair enzyme, DNA polymerase and DNA ligase;
2) tool enzyme reaction system: buffer solution required by the reaction corresponding to each tool enzyme;
3) a linker system: random primers and T7 TruncatedAdap;
4) an exonuclease digestion system;
5) a PCR reaction system;
6)Low-EDTA-TE。
further, the nucleotide sequence of the random primer is shown as SEQ ID NO: 1 is shown.
Further, the reverse transcriptase is one of Hiscript II reverse transcriptase, Hiscript III reverse transcriptase and Hiscript IV reverse transcriptase;
further, the DNA ligase is one of T4 DNA ligase, Taq DNA ligase and E.coli DNA ligase;
further, the polymerase is one of Klenow (exo-) polymerase, Bsu DNA polymerase, phi29 polymerase, Taq polymerase, A family DNA polymerase and B family high fidelity polymerase.
Compared with the prior art, the invention has the beneficial effects that:
1) the invention realizes the simultaneous completion of the library construction of the sequencing library by the DNA/RNA sample. Breaks through the conventional library construction strategy, does not depend on the integrity of the sample, and is suitable for constructing various DNA and RNA samples. The invention can be used for macro gene sequencing and has good practicability for virus microorganisms and the like;
2) the method has the advantages of simple experimental steps, short time and capability of effectively reducing the cost.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a flow chart of a method for DNA/RNA co-pooling of the present invention;
FIG. 2 is a schematic diagram of a DNA/RNA co-constructed library of the present invention;
FIG. 3 is a schematic diagram of a T7 Truncatedadapter structure;
FIG. 4 is a graph showing the analysis of the efficiency of single-strand cDNA synthesis in the pooling of RNA samples in example 1;
FIG. 5 is a schematic representation of library preparation of the RNA sample of example 1;
FIG. 6 is a schematic diagram of library preparation of the DNA sample in example 2;
FIG. 7 is a schematic diagram of library preparation of a mixed sample of DNA and RNA in example 3.
Detailed Description
The following examples are intended to illustrate the invention without limiting its scope. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
The library building principle of the invention is as follows: the construction of a mixed sample of RNA and DNA is rapidly realized through the breaking of RNA and the denaturation of DNA in a sample, the synthesis of one-strand cDNA and strand displacement reaction, the connection of a DNA3' joint and the formation of a single strand and the amplification of PCR. So as to effectively simplify the library construction process, reduce the library construction time, solve the problem of sequencing preference, and also adapt to the purpose of degrading a sample and nucleic acid samples with different molecular fragment sizes, the flow chart of the library construction method is shown in figure 1, and the schematic diagram of the library construction is shown in figure 2.
EXAMPLE 1 pooling of RNA samples
In this example, 50ng of mouse mRNA and 50ng of rat mRNA were used for the library construction test, and RNA library construction was performed by RNA fragmentation, one-strand cDNA synthesis and strand displacement reaction, DNA3' linker ligation, and library amplification, with two replicates per sample set for reliability of detection.
1) Fragmentation of RNA:
the reaction system is prepared as follows:
the composition of the fragment buffer is: 320mM Tris-HCl, 800mM KCl, 240mM MgCl2The pH was 7.9. And (3) running a reaction program: 94 ℃/10min, 4 ℃/hold.
2) Strand displacement reaction:
the fragmented RNA was subjected to one-strand cDNA synthesis by random primers. The random primer is obtained by adding a T5 Trunctedadapter to the 5' end of N6, and the primer sequence is as follows:
5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNN-3', the mixed Enzyme 1st cDNA/DNA Synthesis Enzyme Mix used was: 20U Klenow (exo-), 200U Script II RT, 20U RNase Inhibitor. The control was no T5 TrunctedAdapter added to the 5' end of N6, and the primer sequences were: 5 '-NNNN-3'.
The reaction system is prepared as follows:
and (3) running a reaction program: 25 ℃/10min, 37 ℃/30min, 42 ℃/30min, 70 ℃/10min, 4 ℃/hold. After the reaction is finished, the synthesis efficiency of the single-strand cDNA is quantitatively detected by using the internal reference genes MuGAPDH, Mu beta-actin, RnGAPDH and Rn beta-actin of mice and rats. The primer sequences are as follows:
MuGAPDHF：5'-AGGTCGGTGTGAACGGATTTG-3'
MuGAPDHR：5'-TGTAGACCATGTAGTTGAGGTCA-3'
Muβ-actinF：5'-GGAGATTACTGCCCTGGCTCCTA-3'
Muβ-actinR：5'-GACTCATCGTACTCCTGCTTGCTG-3'
RnGAPDHF：5'-AGTGCCAGCCTCGTCTCATA-3'
RnGAPDHR：5'-AGAGAAGGCAGCCCTGGTAA-3'
Rnβ-actinF：5'-GCAGTTGGTTGGAGCAA-3'
Rnβ-actinR：5'-ATGCCGTGGATACTTGGA-3'
the reaction mixture for single-strand cDNA synthesis was diluted 200-fold, and 1. mu.L of the reaction mixture was taken in combination with 199. mu. L H2And O, and then carrying out quantitative PCR detection. TB Green Using TakaraTMPremix Ex TaqTM(Tli RNaseH Plus) (RR420A) the reaction system was configured as follows:
and (3) running a reaction program: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 10s, extension at 72 ℃ for 20s, temperature from 65 ℃ to 95 ℃, 0.5 ℃/s, and hold at 4 ℃.
The experimental results are: the synthesis efficiency of one-strand cDNA using random primers was consistent with that of N6. FIG. 3 is an analysis of the synthesis efficiency of single-stranded cDNA, in which the synthesis efficiency of single-stranded cDNA of random primers was not changed compared with that of N6, and is shown in FIG. A as MuGAPDH of mouse, in FIG. B as Mu β -actin of mouse, in FIG. C as RnGAPDH of rat, and in FIG. D as Rn β -actin of rat.
3) Purification of Single Strand cDNA
DNA was purified according to the 1.0 XAMPure XP magnetic bead ratio. 21 μ L of Low EDTATE eluted beads. mu.L of DNA supernatant was taken, purified once with 1.0 × AMPure XP magnetic beads, eluted with 17. mu.L of Low EDTATE, and 15. mu.L was taken for DNA3' linker ligation.
4) DNA3' linker ligation
First, 15. mu.L of single-stranded cDNA was denatured at 95 ℃ for 2min, immediately placed on ice, and the DNA3' linker ligation reaction solution was added. Reagent T7 Buffer used: 700mM Tris-HCl, 100mM Mg (Ac)2，260uM NAD+，10mM DTT，10mMATP，500mM NaCl，500mM KAc； T7 Enzyme mix：10U TdT(NEB，M0315)，60U E.coli DNA ligase(NEB， M0205)，10U T4 PNK(T4 Polynucleotide Kinase，NEB，M0201)，10U Klenow(NEB, M0212); the T7 truncatadedpater linker structure is shown in fig. 3, and adopts T7 truncatadedpater linker in patent publication CN 110607353A.
The reaction system is configured as follows:
and (3) running a reaction program: pre-denaturation at 37 ℃ for 15 min; hold at 95 deg.C, 2min, 4 deg.C.
5) Library amplification
Amplification by PCR was performed using a commercial reagent 2 XKAPA HiFi HotStart ReadyMix (Kapa, KM 2618). The amplification primers were Universal PCR primers (Universal PCR Primer, RM20248, Abclonal) and Index primers (Index, RM22201, Abclonal).
The reaction system is prepared as follows:
the reaction conditions were as follows:
after 11cycles of reaction, 1.0 × AMPure XP magnetic bead capture, using 21 μ L of Low-EDTA-TE elution;
the yield of the constructed library is as follows:
the experimental results are as follows: as shown in FIG. 4, wherein A is the peak diagram of the PCR library of mouse RNA, 11cycles, the library yield is 23ng/ul,21ul water elution, the main peak is about 500 bp; b is the peak image of the PCR library of RNA of rat, 11cycles, the library yield is 17.2ng/ul,21ul water elution, the main peak is about 500 bp. It can be seen that this method can complete the construction of a sequencing library for RNA.
EXAMPLE 2DNA sample pooling
In this example, a library construction test was performed using non-fragmented genomic DNA of human and 200bp fragmented DNA, respectively, and DNA library construction was completed by DNA denaturation, one-strand cDNA synthesis, ligation of DNA3' adapters, and library amplification, with two replicates per sample set for detection reliability.
1) Denaturation of DNA:
the reaction system is prepared as follows:
the composition of the fragment buffer is: 320mM Tris-HCl, 800mM KCl, 240mM MgCl2pH 7.9. And (3) running a reaction program: 94 ℃/10min, 4 ℃/hold.
2) Strand displacement reaction:
the DNA was subjected to strand displacement reaction by random primers. The random primer is obtained by adding a T5 Trunctedadapter to the 5' end of N6, and the primer sequence is as follows:
5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNN-3', the mixed Enzyme 1st cDNA/DNA Synthesis Enzyme Mix used was: 20U Klenow (exo-), 200U Script II RT, 20U RNase Inhibitor.
The reaction system is prepared as follows:
and (3) running a reaction program: 25 ℃/10min, 37 ℃/30min, 42 ℃/30min, 70 ℃/10min, 4 ℃/hold.
3) Purification of Strand Displacement DNA
DNA was purified according to the 1.0 XAMPure XP magnetic bead ratio. 21 μ L of Low EDTATE eluted beads. mu.L of DNA supernatant was taken, purified once with 1.0 × AMPure XP magnetic beads, eluted with 17. mu.L of Low EDTATE, and 15. mu.L was taken for DNA3' linker ligation.
4) DNA3' linker ligation
First, 15. mu.L of single-stranded cDNA was denatured at 95 ℃ for 2min, immediately placed on ice, and the DNA3' linker ligation reaction solution was added. Reagent T7 Buffer used: 700mM Tris-HCl, 100mM Mg (Ac)2260uM NAD +, 10mM DTT, 10mM MATP, 500mM NaCl, 500mM KAc; t7 Enzyme mix: 10U TdT (NEB, M0315), 60U e.coli DNA ligase (NEB, M0205), 10U T4 PNK (T4 polynuceotide Kinase, NEB, M0201), 10U Klenow (NEB, M0212); the T7 Truncatedadapter adopts a T7 Truncatedadapter linker in the patent publication CN 110607353A.
The reaction system is configured as follows:
and (3) running a reaction program: pre-denaturation at 37 ℃ for 15 min; hold at 95 deg.C, 2min, 4 deg.C.
5) Library amplification
Amplification by PCR was performed using a commercial reagent 2 XKAPA HiFi HotStart ReadyMix (Kapa, KM 2618). The amplification primers were Universal PCR primers (Universal PCR Primer, RM20248, Abclonal) and Index primers (Index, RM22201, Abclonal).
The reaction system is prepared as follows:
the reaction conditions were as follows:
8/9 cycles of reaction were completed, and 1.0 × AMPure XP magnetic beads were captured and eluted with 21 μ L of Low-EDTA-TE;
the yield of the constructed library is as follows:
the experimental results are as follows: as shown in FIG. 6, wherein A is PCR library amplification of non-fragmented DNA of human, B is PCR library amplification of fragmented DNA of human, A is PCR library peak pattern of non-fragmented DNA sample of human, 9cycles, library yield 25ng/ul 21ul water elution, main peak is about 350bp, B is PCR library peak pattern of fragmented DNA sample of human, 8cycles, library yield 24.8ng/ul 21ul water elution, main peak is about 350bp, the method can complete the construction of DNA sequencing library.
Example 3: construction of DNA/RNA samples
In this embodiment, the common DNA/RNA mixed sample is subjected to library construction test by using the DNA/RNA co-library construction method described above. We tested two samples: including pooled samples of human fragmented DNA and Rat RNA and non-fragmented human DNA and Rat RNA samples. The following is a basic DNA/RNA library construction procedure, with two replicates per sample set up for assay reliability.
1) Fragmentation of RNA and denaturation of DNA
The reaction system is prepared as follows:
the composition of the fragment buffer is: 320mM Tris-HCl, 800mM KCl, 240mM MgCl2pH 7.9. And (3) running a reaction program: 94 ℃/10min, 4 ℃/Hold. And (3) running a reaction program: 94 ℃/10min, 4 ℃/Hold.
2) Synthesis of single-stranded cDNA and strand displacement reaction
The reaction system is prepared as follows:
the random primer is formed by adding a T5 truncated Adapter to the 5' end of N6, and the primer sequence is as follows: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNN-3', the mixed Enzyme 1st cDNA/DNA Synthesis Enzyme Mix used was: 20U Klenow (exo-), 200U Script II RT, 20U RNase Inhibitor. The control was no T5 TrunctedAdapter added to the 5' end of N6, and the primer sequences were: 5 '-NNNN-3'.
The 1st cDNA/DNA Synthesis Enzyme Mix is Klenow (exo-) polymerase, reverse transcriptase and RNAinhibitor, wherein the reverse transcriptase can be selected from Hiscript II reverse transcriptase, Hiscript III reverse transcriptase and Hiscript IV reverse transcriptase in commercial products.
And (3) running a reaction program: 25 ℃/10min, 37 ℃/30min, 42 ℃/30min, 70 ℃/10min, 4 ℃/hold. After the reaction is finished, purifying twice according to the proportion of 1.0 multiplied by AMPure XP magnetic beads, and digesting the residual primer by selecting Exo I exonuclease. mu.L of Low EDTA TE eluted magnetic beads, and 15. mu.L of the eluted magnetic beads were used for DNA3' linker ligation.
3) DNA3' linker ligation
mu.L of single-stranded cDNA was first denatured at 95 ℃ for 2min, immediately placed on ice, and ligated to the 3' linker of DNA using T7 Truncated Adapter, T7 Truncated Adapter using the T7 Truncated Adapter linker of patent publication CN 110607353A.
The DNA3' adaptor ligation reaction solution is added.
The reaction system is configured as follows:
reagent T7 Buffer used: 700mM Tris-HCl, 100mM Mg (Ac)2，260uM NAD+，10mM DTT，10mMATP，500mM NaCl，500mM KAc；
T7 Enzyme mix contains DNA polymerase, DNA ligase, DNA repair enzymes. The polymerase may be selected from one of the following commercially available: klenow (exo-) polymerase, Bsu DNA polymerase, phi29 polymerase, Taq polymerase, A family DNA polymerase, B family high fidelity polymerase; the ligase may be selected from one of the following commercially available: t4 DNA ligase, Taq DNA ligase and e.
In this embodiment, the T7 Enzyme mix comprises: 10U TdT (NEB, M0315), 60U E.coli DNA ligase (NEB, M0205), 10U T4 PNK (T4 polynuceotide Kinase, NEB, M0201), 10U Klenow (NEB, M0212).
And (3) running a reaction program: pre-denaturation at 37 ℃ for 15 min; hold at 95 deg.C, 2min, 4 deg.C.
3.4) library amplification
The reaction system is prepared as follows:
the reaction conditions were as follows:
7/8 cycles of reaction were completed, and 1.0 × AMPure XP magnetic beads were captured and eluted with 21 μ L of Low-EDTA-TE;
the yield of the constructed library is as follows:
the experimental results are as follows: as shown in fig. 7, wherein a is PCR library amplification of the non-fragmented DNA and RNA co-building library of human, and B is PCR library amplification of the fragmented DNA and RNA co-building library of human, wherein a is PCR library peak diagram of the non-fragmented DNA and RNA co-building library of human, 7cycles, library yield 31.3ng/ul, 21ul water elution, main peak is around 400 bp; b is PCR library peak image of segmented DNA and RNA co-constructed library of human, 8cycles, library peak type 28.5ng/ul 21ul water elution, main peak is about 400 bp. From the established library, the method can simultaneously complete the construction of the DNA/RNA sequencing library, can realize the fragmentation of DNA in a mixed sample, and only needs 3.5 hours in the library construction process.
From the above embodiments, the method for co-building a library of DNA and RNA provided by the present invention can effectively combine a DNA sample and an RNA sample together to build a library, has important significance for simplifying a library building process and reducing library building time, and can solve the problem of sequencing preference, and the method has no requirement on the input amount of the sample, can adapt to various environmental samples, degrades the sample and nucleic acid samples with different molecular fragment sizes, and is a novel efficient co-building library method.
The invention is not limited solely to that described in the specification and embodiments, and additional advantages and modifications will readily occur to those skilled in the art, so that the invention is not limited to the specific details, representative embodiments, and illustrative examples shown and described herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.
Sequence listing
<110> Shanghai Engyi Biotech Co., Ltd
<120> method for co-building DNA and RNA library
<141> 2020-09-15
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 39
<212> DNA
<213> Artificial Sequence
<400> 1
acactctttc cctacacgac gctcttccga tctnnnnnn 39
Claims (10)
1. A method for co-building a library of DNA and RNA is characterized by comprising the following steps:
s1, carrying out RNA fragmentation and DNA denaturation on a mixed sample of RNA and DNA;
s2, based on the product of S1, carrying out DNA strand displacement reaction by using a random primer, and carrying out 5 'joint on a DNA fragment to form 5' rounded Adapter-DNA; reverse transcribing the RNA to a single-stranded cDNA with a 5 'linker to form a 5' truncated Adapter-cDNA;
s3, digesting redundant primers by using exonuclease, and purifying products by using magnetic beads;
s4, adding a section of poly oligonucleotide PloyC to the 3' end of the DNA with the 5' joint and the 3' end of the single-strand cDNA with the 5' joint to obtain a 5' rounded Adapter-DNA-poly oligonucleotide structure, and connecting the 3' end of the 5' rounded Adapter-DNA-poly oligonucleotide with a P7 joint by using DNA repair enzyme, DNA polymerase and DNA ligase;
and S5, amplifying the PCR library to obtain an on-machine sequencing library.
2. The method for co-pooling DNA and RNA according to claim 1, wherein the step S1 comprises fragmenting DNA in the mixed sample.
3. The method for co-pooling of DNA and RNA according to claim 1, wherein the random primer is T5 truncated Adapter + N6, and the nucleotide sequence of the random primer is as shown in SEQ ID NO: 1 is shown.
4. The method for co-pooling of DNA and RNA according to claim 1, wherein the step S2 comprises performing a single-strand cDNA synthesis and strand displacement reaction under Klenow (exo-) polymerase, reverse transcriptase and RNA Inhibitor conditions.
5. The method for co-pooling DNA and RNA according to claim 1, wherein the step S3 is performed by using Exo I exonuclease to digest the residual primers.
6. The method for co-pooling DNA and RNA of claim 1 wherein said P7 linker is T7 Truncated Adapter.
7. The method for co-pooling of DNA and RNA according to claim 1, wherein the reverse transcriptase is one of Hiscript II reverse transcriptase, Hiscript III reverse transcriptase, and Hiscript IV reverse transcriptase; the DNA polymerase is selected from one of Klenow (exo-) polymerase, Bsu DNA polymerase, phi29 polymerase, Taq polymerase, A family DNA polymerase and B family high fidelity polymerase.
8. A kit for co-building a library of DNA and RNA is characterized by comprising the following components:
1) tool enzyme: the tool enzyme comprises reverse transcriptase, terminal transferase, DNA repair enzyme, DNA polymerase and DNA ligase;
2) tool enzyme reaction system: buffer solution required by the reaction corresponding to each tool enzyme;
3) a linker system: random primers and T7 Truncated Adapter;
4) an exonuclease digestion system;
5) a PCR reaction system;
6)Low-EDTA-TE。
9. the kit of claim 8, wherein the random primer nucleotide sequence is as set forth in SEQ ID NO: 1 is shown.
10. The kit of claim 8, wherein:
the reverse transcriptase is one of Hiscript II reverse transcriptase, Hiscript III reverse transcriptase and Hiscript IV reverse transcriptase;
the DNA ligase is one of T4 DNA ligase, Taq DNA ligase and E.coli DNA ligase;
the polymerase is one of Klenow (exo-) polymerase, Bsu DNA polymerase, phi29 polymerase, Taq polymerase, A family DNA polymerase and B family high fidelity polymerase.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113718343A (en) * | 2021-09-03 | 2021-11-30 | 翌圣生物科技(上海)股份有限公司 | Rapid RNA library building method and kit |
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