阅读说明：本技术 一种痕量单链核酸样本制备方法 (Preparation method of trace single-stranded nucleic acid sample ) 是由 王永利 贺田芬 鞠巍 汤链 徐根明 谢玲灵 于 2021-10-11 设计创作，主要内容包括：本发明公开了一种痕量单链核酸样本制备方法。包括以下步骤：(1)获取碎片化的,且经过5’端磷酸化和3’端羟基化处理的DNA单链或者cDNA；(2)步骤(1)得到的DNA片段进行3’端辅助连接,延伸成DNA双链；(3)步骤(2)得到的DNA双链的5’端连接上接头,然后进行双模板底物扩增。本发明提高了制备痕量核酸样本的效率,并且减少了纯化步骤,对痕量的DNA起始原料测序前处理意义重大。(The invention discloses a preparation method of a trace single-stranded nucleic acid sample. The method comprises the following steps: (1) obtaining fragmented DNA single strands or cDNA which are subjected to 5 'end phosphorylation and 3' end hydroxylation treatment; (2) carrying out 3' end auxiliary connection on the DNA fragment obtained in the step (1) and extending into a DNA double strand; (3) and (3) connecting a joint to the 5' end of the DNA double strand obtained in the step (2), and then carrying out double-template substrate amplification. The method improves the efficiency of preparing the trace nucleic acid sample, reduces the purification steps, and has great significance for the pretreatment of the trace DNA starting material before sequencing.)

1. A method for preparing a trace single-stranded nucleic acid sample, which is characterized by comprising the following steps:

(1) obtaining fragmented DNA single strands or cDNA which are subjected to 5 'end phosphorylation and 3' end hydroxylation treatment;

(2) carrying out 3' end auxiliary connection on the DNA fragment obtained in the step (1) and extending into a DNA double strand; the method comprises the following specific steps:

an artificially synthesized nucleotide fragment helper and dNTP are used in the system, the dNTP is a mixture of common dNTP and hot-start dNTP, the common dNTP selects any one, two or three of the four dNTPs, and the hot-start dNTP corresponds to the remaining three, two or one of the selected common dNTPs; carrying out tailing reaction of the DNA fragment by using the common dNTP at a lower temperature; then, a higher temperature is given to the system, so that the hot-started dNTP enters an activated working state; then a relatively low temperature is given, the helper and the tail sequence at the 3 'end of the DNA fragment are complementarily combined, finally, the DNA polymerase respectively takes the helper and the DNA fragment as templates to extend forwards until the complementary strand of the helper extends out by taking the helper as the template and the complementary strand of the DNA fragment extends out by taking the DNA fragment as the template, the 3' end of the helper contains the sequence which is complementary with the tail of the DNA fragment, and the 3 'end needs to be sealed, so that the 3' tail of the helper is prevented from being 3 'tailed by the tail-adding enzyme in the system, and the 3' end is removed during extension;

(3) and (3) connecting a joint to the 5' end of the DNA double strand obtained in the step (2), and then carrying out double-template substrate amplification.

2. The method according to claim 1, wherein the DNA fragment obtained in step (1) has a length of 10bp to 1000 bp.

3. The method of claim 1, wherein in step (2): the length of the auxiliary is 15nt-120nt, and at least the 1 st base at the 3' end of the auxiliary is closed by adopting an RNA base; the RNA basic group is rA, rG, rC or rU.

4. The method of claim 3, wherein in step (2): the length of the auxiliary sub-unit is 30nt-60 nt.

5. The method of claim 1, wherein in step (2): the length of the DNA fragment tail-added sequence at the 3 ' end is 1-20 bases, and the base sequence of the 3 ' end of the helper and the DNA fragment tail-added sequence which is complementarily combined is any part of the RNA bases after the 3 ' end of the helper and up to the 14 th base.

6. The method of claim 5, wherein in step (2): the length of the tail sequence added at the 3' end of the DNA fragment is 3-10 bases.

7. The method of claim 5 or 6, wherein the number of tailed bases at the 3 'end of the DNA fragment is controlled by limiting the amount of common dNTPs and/or increasing the amount of helpers, and the number of tailed bases at the 3' end of the DNA fragment decreases as the concentration of the common dNTPs decreases; as the concentration of the helper increases, the number of tailing bases at the 3' end of the DNA fragment decreases.

8. The method of claim 1, wherein the concentration of the helper molecule in the system is between 10nM and 20 uM.

9. The method of claim 8, wherein the concentration of the helper molecule in the system is between 50nM and 10 uM.

10. The method of claim 9, wherein the concentration of the helper molecule in the system is between 100nM and 5 uM.

11. The method according to claim 1, 8,9 or 10, wherein the amount of the DNA fragment in the reaction system is 1fmol to 1nmol per 50 uL.

12. The method according to claim 1, wherein the concentration of common dNTPs in the system is 2 uM-2 mM; the concentration of common dNTP in the system is 1-3 times of that of each hot start dNTP.

13. The method according to claim 12, wherein the concentration of common dNTPs in the system is 10uM to 1 mM.

14. The method according to claim 13, wherein the concentration of common dNTPs in the system is 30uM to 0.5 mM.

15. The method of claim 1, wherein in step (2): firstly, carrying out tailing reaction on a DNA fragment at the temperature of 20-37 ℃ for 5-30 minutes; then reacting for 1-5 minutes at 90-95 ℃ to enable the hot-started dNTP to enter an activation working state, and simultaneously enabling the RNA base at the 3' end of the auxiliary son to drop from the auxiliary son to activate the extension function of the auxiliary son; and reacting at 45-72 ℃ for 2-30 minutes to make the helper and the tail sequence at the 3' end of the DNA fragment complementarily combine, and then respectively extending forwards by using the DNA polymerase with the helper and the DNA fragment as templates until the complementary strand of the helper extends out by using the helper as the template and the complementary strand of the DNA fragment extends out by using the DNA fragment as the template.

16. The method of claim 1, wherein the systematic tailing of the DNA fragments is achieved by: tailing the 3' end of the DNA fragment by using tailing enzyme and taking any one, two or three of common dATP, dTTP, dGTP and dCTP added into the system and 4 dNTPs as materials; the polymerase used for polymerization in the system comprises at least one of Taq polymerase or high-fidelity polymerase; taq polymerase includes: 2G Robust DNA polymerase, rTaq DNA polymerase and TaqB DNA polymerase, wherein the high-fidelity polymerase comprises: the kappa HIFI hot start high-fidelity polymerase, pfu DNA polymerase and phusion DNA polymerase.

17. The method according to claim 16, wherein the concentration of the tailgating enzyme in the system is 0.15U/uL to 15U/uL; the concentration of the polymerase in the system is 0.01U/uL-2U/uL.

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a preparation method of a trace single-stranded nucleic acid sample.

Background

Nucleic acid is a generic term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and is a biological macromolecular compound synthesized by polymerizing many nucleotide monomers, which is one of the most basic substances of life. Among naturally occurring nucleic acids, ribonucleic acid (RNA) is mostly present in single-stranded and hairpin forms, and is also found in small amounts in circular forms. Deoxyribonucleic acid (DNA) exists mainly in a double-stranded form of double helix, and also exists partially in a single-stranded form. In the fields of biotechnology such as nucleic acid detection, in vitro diagnosis, gene sequencing, and the like, nucleic acids are also produced in a single-stranded form during various treatments of the nucleic acids. For example, organisms with single-stranded DNA or single-stranded RNA as genetic material such as human transfusion virus (TTV), parvovirus, and the like exist in nature. Sequencing studies of the genetic material of these organisms are not easily accomplished using traditional NGS pre-sequencing sample handling methods. In the forensic field, DNA or RNA extracted from rotten various human-derived materials such as bone, nails, hair, etc. has often been highly degraded into single-stranded fragmented nucleic acids and is already in trace amounts (e.g., pg or fg scale). There is currently no efficient means for sample preparation for these trace amounts of fragmented nucleic acids. In addition, the DNA methylation detection also involves a step of heavy salt conversion of the DNA, and most of the DNA after heavy salt conversion becomes relatively short fragmented single-stranded DNA. A particularly efficient technique is also lacking for the treatment of these single-stranded nucleic acids.

Disclosure of Invention

The invention aims to provide a method for preparing a sample for trace nucleic acid (DNA or RNA). The method is independent of the double-stranded structure of the DNA. The method has high sample preparation efficiency, and is widely suitable for trace single-stranded or double-stranded DNA and RNA with various lengths.

A preparation method of a trace single-stranded nucleic acid sample comprises the following steps:

(1) obtaining fragmented DNA single strands or cDNA which are subjected to 5 'end phosphorylation and 3' end hydroxylation treatment;

(2) carrying out 3' end auxiliary connection on the DNA fragment obtained in the step (1) and extending into a DNA double strand; the method comprises the following specific steps:

an artificially synthesized nucleotide fragment helper and dNTP are used in the system, the dNTP is a mixture of common dNTP and hot-start dNTP, the common dNTP selects any one, two or three of the four dNTPs, and the hot-start dNTP corresponds to the remaining three, two or one of the selected common dNTPs; carrying out tailing reaction of the DNA fragment by using the common dNTP at a lower temperature; then, a higher temperature is given to the system, so that the hot-started dNTP enters an activated working state; then a relatively low temperature is given, the helper and the tail sequence at the 3 'end of the DNA fragment are complementarily combined, finally, the DNA polymerase respectively takes the helper and the DNA fragment as templates to extend forwards until the complementary strand of the helper extends out by taking the helper as the template and the complementary strand of the DNA fragment extends out by taking the DNA fragment as the template, the 3' end of the helper contains the sequence which is complementary with the tail of the DNA fragment, and the 3 'end needs to be sealed, so that the 3' tail of the helper is prevented from being 3 'tailed by the tail-adding enzyme in the system, and the 3' end is removed during extension;

(3) and (3) connecting a joint to the 5' end of the DNA double strand obtained in the step (2), and then carrying out double-template substrate amplification.

Fragmenting nucleic acid: the starting template of the present invention may be double-stranded or single-stranded DNA, or may be RNA. Can be various lengths from 10bp to 1000 bp. Preferably 50bp to 800bp, and more preferably 100bp to 300 bp. If the DNA or RNA is too long, the fragmentation process is first required. The fragmentation protocol may be one commonly used in the genetic testing industry. For example, the long double-stranded DNA can be subjected to ultrasonication, endonuclease cleavage, or the like. The long-chain RNA may be treated with divalent metal ions at a high temperature of 95 ℃ or higher. Not only the treatment method exemplified above but also any method capable of efficiently fragmenting nucleic acids may be used. Fragmentation is not necessary if the nucleic acid itself is already a shorter fragment.

Phosphorylation and hydroxylation treatment of the 3 'end of cDNA 5': for RNA molecules, reverse transcription may be performed using a reverse transcription primer phosphorylated at the 5 ' end, so that the product of reverse transcription is a cDNA phosphorylated at the 5 ' end and hydroxylated at the 3 ' end, which satisfies the conditions. Modification of 5' end phosphorylation may also be performed during reverse transcription or at the same time during reverse transcription using an enzyme with similar effect as the T4 PNK enzyme. For DNA molecules, 5 '-end phosphorylation modification and 3' -end hydroxylation treatment can be performed using an enzyme having the same effect as the T4 PNK enzyme. The processing method of the step is not limited to the described scheme, and any processing scheme capable of achieving the same effect can be implemented in the invention.

In the step (2): the length of the auxiliary sub-unit is 15nt-120nt, preferably 36-60 nt; at least the 1 st base (the 1 base has the best effect, and 2-3 bases can also be used) at the 3' end of the helper is closed by adopting RNA base; the RNA basic group is rA, rG, rC or rU.

In the step (2): the length of the 3 'end tailing sequence of the DNA fragment ranges from 1 to 20 bases, preferably from 3 to 10 bases, and the base sequence of the 3' end of the helper to which the 3 'end tailing sequence of the DNA fragment complementarily binds is any part of the 3' end RNA base of the helper up to the 14 th base.

The 3 'end of the helper of the present invention has partial base sequence complementary to the 3' end of DNA. Complementary means that if the common dNTP added to the system is dATP, the complementary sequence corresponding to the 3' end of the helper should be T bases. The complementary nucleotide sequence may be any portion of nucleotides 2 to 15 of the 3' end of the helper. For example, the nucleotide sequence may be 2 to 8 th, 2 to 9 th, 2 to 10 th, 2 to 11 th, 2 to 12 th, 2 to 13 th, 2 to 14 th, or 2 to 15 th bases at the 3' -end of the helper. The 1 st base at the 3' end of the helper is an RNA base. The base may be rA, rG, rC or rU to prevent tailgating of the helper or 3' tailgating by the tailgating enzyme in the system. This is critical to prevent the formation of adaptor dimers from the nucleic acid sample.

Further, the number of the tailing bases at the 3 'end of the DNA fragment is controlled by limiting the amount of the common dNTPs and/or increasing the amount of the helper molecules, and the number of the tailing bases at the 3' end of the DNA fragment is reduced as the concentration of the common dNTPs is reduced; as the concentration of the helper increases, the number of tailing bases at the 3' end of the DNA fragment decreases.

Further, the concentration of the helper molecule in the system is between 10nM and 20uM, preferably between 50nM and 10uM, and more preferably between 100nM and 5 uM.

Further, the amount of DNA fragments in the reaction system was 1fmol to 1nmol/50 uL.

The common dNTP selects any one, two or three of the four dNTPs, and the hot-started dNTP corresponds to the remaining three, two or one of the selected common dNTPs; for example: it can be understood that: one common dNTP is selected, and the other three kinds of hot start dNTPs are selected; two common dNTPs are selected, and the other two hot start dNTPs are selected; three kinds of common dNTPs are selected, and the other one is selected as the hot start dNTP.

Further, the concentration of the common dNTP in the system is 2uM to 2mM, preferably 10uM to 1mM, and further preferably 30uM to 0.5mM, and the concentration of the common dNTP in the system is 1-fold to 3-fold that of each hot-start dNTP monomer.

Further, in the step (2): firstly, carrying out tailing reaction on a DNA fragment at the temperature of 20-37 ℃ for 5-30 minutes; then reacting for 1-5 minutes at 90-95 ℃ to enable the hot-started dNTP to enter an activation working state, and simultaneously enabling the RNA base at the 3' end of the auxiliary son to drop from the auxiliary son to activate the extension function of the auxiliary son; and reacting at 45-72 ℃ for 2-30 minutes to make the helper and the tail sequence at the 3' end of the DNA fragment complementarily combine, and then respectively extending forwards by using the DNA polymerase with the helper and the DNA fragment as templates until the complementary strand of the helper extends out by using the helper as the template and the complementary strand of the DNA fragment extends out by using the DNA fragment as the template.

Further, the implementation manner of tailing the DNA fragments in the system comprises the following steps: tailing the 3' end of the DNA fragment by using tailing enzyme and taking any one, two or three of common dATP, dTTP, dGTP and dCTP added into the system and 4 dNTPs as materials; preferably: the concentration of the tailgating enzyme (terminal transferase) in the system is 0.15U/uL to 15U/uL, preferably 0.4U/uL to 5U/uL, and more preferably 0.6U/uL to 3U/uL.

Further, the polymerase used for polymerization in the system comprises at least one of Taq polymerase or high fidelity polymerase; taq polymerase includes: 2G Robust DNA polymerase, rTaq DNA polymerase and TaqB DNA polymerase, wherein the high-fidelity polymerase comprises: one or more of a high fidelity polymerase (Roche, # kk 2602), pfu DNA polymerase, phusion DNA polymerase with kappa HIFI hot start; preferably Taq DNA polymerase;

preferably: the concentration of polymerase in the system is 0.01U/uL-2U/uL; preferably 0.05U/uL-1U/uL, and more preferably 0.08U/uL-0.5U/uL.

Further, if the high fidelity polymerase is used for the extension in step (2) and the extension product is blunt-ended, the ligation linker used correspondingly is a blunt-ended linker; if Taq polymerase is used for extension and the extension product is a sticky end with 3 'A bases, the adaptor used correspondingly is a sticky end adaptor with 3' T bases.

The invention preferably comprises the following components: the extension uses a polymerase which does not have exo activity in the 3 'to 5' direction, such as Taq DNA polymerase, because this scheme can ensure that the double-stranded extension products and the double-stranded linker are not self-ligated, i.e., can ensure that only the double-stranded extension products and the double-stranded linker are ligated.

The invention has the advantages that:

the dNTPs in the system of the invention are a mixture of ordinary dNTPs and hot-start dNTPs. For example: the dNTPs are ordinary dATP and hot-start dTTP, dCTP, dGTP (e.g., hot-start dNTPs from TriLINK). The method is suitable for DNA tailing reaction at a lower temperature, and the suitable tailing enzyme can only add common dATP to the 3' end of DNA. The donor system is then brought to a higher temperature to activate the hot-started dTTP, dCTP, dGTP. The helper is complementarily bound to the DNA3 'tail by a relatively low temperature, and then extended forward by DNA polymerase along the 3' tail of the DNA until the helper is used as a template to extend the complementary strand of the helper. The advantage of this scheme is that DNA tailing, DNA 3' end auxiliary connection and double strand extension can be performed in one reaction system. The efficiency of preparing trace nucleic acid samples is improved, and the purification steps are reduced. This is significant for trace amounts of DNA starting material sample handling.

The 3' end of the helper in the scheme needs to be closed so as to avoid the DNA tailing reaction from mistakenly considering the helper as the template DNA for carrying out the false tailing operation. The last base at the 3 'end of the helper is RNA, and the blocked RNA base is dropped under high temperature (e.g., 95 ℃) to expose the hydroxyl at the 3' end of the helper, so that polymerase in a donor system polymerizes to generate a template complementary strand.

According to the invention, since the nucleic acid template and the extension complementary strand thereof are connected with the adaptor sequence, the universal primer can be used for amplification, and finally, compared with a scheme of only connecting one strand (the nucleic acid template or the complementary strand thereof), the amplification yield can be doubled in a PCR amplification exponential phase.

Drawings

FIG. 1 is a flow chart of the RNA-based technique of the present invention.

Fig. 2 is a schematic diagram of the present invention.

FIG. 3 is a gel diagram of an amplification product of example 1 of the present invention.

FIG. 4 is a graph showing the results of system S1 in example 2 of the present invention.

FIG. 5 is a graph showing the results of system S2 in example 2 of the present invention.

FIG. 6 is a graph showing the results of system S3 in example 2 of the present invention.

FIG. 7 is a graph showing the results of system S4 in example 2 of the present invention.

FIG. 8 is a graph showing the results of system S5 in example 2 of the present invention.

FIG. 9 is a graph showing the results of system S1 in example 3 of the present invention.

FIG. 10 is a graph showing the results of system S2 in example 3 of the present invention.

FIG. 11 is a graph showing the results of system S3 in example 3 of the present invention.

FIG. 12 is a gel diagram of an amplification product of example 4 of the present invention.

FIG. 13 is a graph showing the sequencing result of the amplified product in example 4 of the present invention.

Detailed Description

The following examples are intended to further illustrate the invention without limiting it.

Example 1

Long-chain RNA molecule sample preparation

Arabidopsis mRNA was extracted using a commercial kit. Taking 500pg mRNA for sample treatment;

reverse transcription primer P1 sequence:

5’P-NNNNNNNN-3’。

1. fragmentation of nucleic acids

The reaction system is as follows:

reacting at 70 ℃ for 1.5 minutes;

qiagen column purification.

2. Reverse transcription of RNA

The reaction system is as follows:

reacting at 65 ℃ for 5 minutes, and immediately placing on ice;

the following reaction reagents are prepared:

adding the 12uL RNA-containing reaction system in the last step into 7.5uL reagent;

after 2 minutes of reaction at 25 ℃ Superscript III reverse transcriptase 1uL was added immediately. After mixing uniformly, the following reactions are carried out:

storing at 25 deg.C for 10 min, 42 deg.C for 50 min, and 4 deg.C;

qiagen column purification after completion of the reaction.

Tailing and assisted production of cDNA3

Helper a1 sequence:

GTGACTGGAGTTCAGAGGTCTCTTCTCTCTCTCTGATCTTTTrG, shown in SEQ ID NO.1, and the 3' end of the helper is RNA base G;

the reaction system is as follows:

firstly, storing 30uL of cDNA (with the concentration of 200 pM) on a PCR instrument at 95 ℃ for 1 minute and at 4 ℃;

adding the 20uL reaction mixed solution into the cDNA, and uniformly mixing;

the samples were stored in a PCR apparatus at 37 ℃ for 10 minutes, 95 ℃ for 2 minutes, 72 ℃ for 5 minutes, and 4 ℃.

4.5' end fitting connection

5' end fitting ADT1 preparation:

ADT1-U:

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT, see SEQ ID NO.2

ADT1-D:

p-GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG, see SEQ ID NO.3

Mixing ADT1-U and ADT1-D, etc., placing in PCR instrument at 98 deg.C for 2 min, slowly cooling to room temperature to anneal into double chains to obtain 5' end connector ADT 1;

the following reaction system was prepared:

storing at 25 deg.C for 15 min, 65 deg.C for 20 min, and 4 deg.C on PCR instrument;

2x Ampure xp beads.

5. Dual template substrate amplification

The amplification primer sequences are as follows:

PCR-F1:

AATGATACGGCGACCACCGA, see SEQ ID NO.4

PCR-R1:

CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGT, see SEQ ID NO.5

The amplification reaction system is as follows:

the amplification procedure was as follows:

5uL of the final amplification product was subjected to 2% agarose gel electrophoresis.

The experimental results of example 1 are shown in fig. 3.

Example 2

Effect of mononucleotide concentration on the number of 3' -tailed bases

A30 nt section of artificially synthesized single-stranded DNA oligonucleotide was taken.

The following experiment was performed depending on the concentration of mononucleotide added;

the reaction system is as follows:

firstly, storing 30uL (concentration 10 uM) of artificial synthetic single-stranded DNA oligonucleotide of 30nt at 95 ℃ for 1 minute and 4 ℃ on a PCR instrument;

adding the 20uL reaction mixed solution into the DNA, and uniformly mixing;

storing at 37 deg.C for 10 min, 95 deg.C for 2 min, 72 deg.C for 5 min, and 4 deg.C on PCR instrument;

the product was run through a capillary electrophoresis and the effect of different single nucleotide (dATP) concentrations on the number of tailed bases was observed by observing the length and size of the single stranded DNA.

The experimental results of example 2 are shown in fig. 4-8.

The results of example 2 show that the number of tailed bases is related to the concentration of single nucleotides in the system, with higher concentrations giving more tailed; too much tailing takes up a large amount of data in NGS sequencing; these data belong to meaningless data; therefore, the selection of an appropriate amount of mononucleotide is important for an efficient reaction.

Example 3

Effect of the amount of the helper on the number of tailed bases at the 3' end

Taking a section of artificially synthesized single-stranded DNA oligonucleotide of 30 nt;

the following experiments were performed according to different ratios of the added accessory seeds;

the reaction system is as follows:

firstly, storing 30uL (concentration 10 uM) of artificial synthetic single-stranded DNA oligonucleotide of 30nt at 95 ℃ for 1 minute and 4 ℃ on a PCR instrument;

adding the 20uL reaction mixed solution into the DNA, and uniformly mixing;

storing at 37 deg.C for 10 min, 95 deg.C for 2 min, 72 deg.C for 5 min, and 4 deg.C on PCR instrument;

the product is run on a capillary electrophoresis, and the influence of different ratios of the auxiliary molecules and the connecting molecules on the number of the added tail base can be observed by observing the length and the size of the single-stranded DNA.

Example 3 the results of the experiment are shown in fig. 9-11.

The results of example 3 show that the number of tailed bases is related to the amount of helper in the system; the higher the concentration of the auxiliary seeds in a certain range, the less the amount of tailing added; therefore, in the practical application of the present invention, the selection of the proper concentration of the auxiliary is important for the efficient reaction; this helps to terminate the 3' end tailing reaction prematurely.

Example 4

Circulating blood free DNA (cfDNA) methylated sample treatment

Methylation conversion treatment was performed using ZYMO EZ DNA Methylation-Gold Kit Methylation Kit (ZYMO RESEARCH, # D5005);

the methyl converted cfDNA was subjected to the following procedure.

DNA 3' end tailing and helper generation

Helper a1 sequence:

GTGACTGGAGTTCAGAGGTCTCTCTTCTCTCTCTCCGATCTTTTrG, and the 3' end of the auxiliary is RNA base G;

the reaction system is as follows:

the T4 polynucleotide kinase can sufficiently phosphorylate the 5' end of the reaction material cfDNA;

30uL of DNA (concentration 3 nM) was stored in a PCR instrument at 95 ℃ for 1 minute and 4 ℃;

adding the 20uL reaction mixed solution into the cDNA, and uniformly mixing;

the samples were stored in a PCR apparatus at 37 ℃ for 10 minutes, 95 ℃ for 2 minutes, 72 ℃ for 5 minutes, and 4 ℃.

2.5' end fitting connection

5' end fitting ADT1 preparation:

ADT1-U:

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

ADT1-D:

p-GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG

mixing ADT1-U and ADT1-D, etc., placing in PCR instrument at 98 deg.C for 2 min, slowly cooling to room temperature to anneal into double chains to obtain 5' end connector ADT 1;

the following reaction system was prepared:

storing at 25 deg.C for 15 min, 65 deg.C for 20 min, and 4 deg.C on PCR instrument;

2x Ampure xp beads.

3. Dual template substrate amplification

The amplification primer sequences are as follows:

PCR-F1:

AATGATACGGCGACCACCGA

PCR-R1:

CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGT

the amplification reaction system is as follows:

the amplification procedure was as follows:

the final amplification product was electrophoresed on 2% agarose gel (5 uL) as shown in FIG. 12.

4. The amplification products after methylation treatment of cfDNA were subjected to WGBS (whole genome methylation sequencing) double-ended 150bp sequencing on the illumina sequencing platform. The unique alignment efficiency of the data reaches 83.9%. See fig. 13.

Sequence listing

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