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WO2024124400A1 - Système de construction de banque de méthylation ciblée fondé sur la pcr multiplex, procédé et son utilisation - Google Patents

Système de construction de banque de méthylation ciblée fondé sur la pcr multiplex, procédé et son utilisation Download PDF

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Publication number
WO2024124400A1
WO2024124400A1 PCT/CN2022/138685 CN2022138685W WO2024124400A1 WO 2024124400 A1 WO2024124400 A1 WO 2024124400A1 CN 2022138685 W CN2022138685 W CN 2022138685W WO 2024124400 A1 WO2024124400 A1 WO 2024124400A1
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primer
universal
dna
round
sequence
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Chinese (zh)
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杨林
张艳艳
陈芳
蒋慧
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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Priority to PCT/CN2022/138685 priority Critical patent/WO2024124400A1/fr
Priority to CN202280100895.9A priority patent/CN120077148A/zh
Publication of WO2024124400A1 publication Critical patent/WO2024124400A1/fr
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a targeted methylation library construction system based on multiplex PCR, a method and an application thereof.
  • DNA methylation is an epigenetic regulatory modification that participates in regulating the amount of protein synthesis without changing the base sequence.
  • DNA methylation is a very wonderful chemical modification. The care of relatives, aging of the body, smoking, alcoholism and even obesity will be recorded faithfully on the genome by methylation. The genome is like a diary, and methylation is used as text to record the experience of the human body.
  • DNA methylation is an important epigenetic marker information. Obtaining methylation level data for all C sites in the whole genome is of great significance for the study of spatiotemporal specificity of epigenetics.
  • mapping of DNA methylation levels in the whole genome and the analysis of high-precision methylation modification patterns of specific species will surely have a milestone significance in epigenomic research, and lay the foundation for the study of basic mechanisms such as cell differentiation and tissue development, as well as animal and plant breeding, human health and disease research.
  • WGBS Whole Genome Bisulfite Sequencing
  • Targeted methylation sequencing technology can be divided into probe capture and multiplex PCR-based sequencing technologies.
  • probe capture the required starting amount is high, and it is difficult to capture some trace samples such as plasma free DNA.
  • design and operation process of the probe capture probe is too complicated, the detection cycle is long, and the cost is high.
  • the biggest problems encountered by the current multiplex PCR based on bisulfite are the primer dimers between primers and the specificity of primer amplification.
  • the multiplex PCR after conventional bisulfite treatment after the DNA is treated with bisulfite, the unmethylated cytosine is converted to uracil, and most (99%) cytosines on the genome are unmethylated, so the bases of most sequences are changed from the previous four compositions of A/T/C/G to A/T/G.
  • one primer is designed for the positive strand and the other is designed for the complementary strand. Therefore, one strand used for PCR is a sequence rich in ATG, and the other strand is a sequence rich in ATC.
  • This three-base complementary primer sequence is easy to form primer dimers.
  • the formation of primer dimers also increases sharply.
  • too many primers are consumed due to the generation of primer dimers, resulting in the failure of multiplex PCR. Therefore, to solve the problem of multiplex bisulfite multiplex PCR, the problem of primers easily forming primer dimers must be solved first.
  • the complexity of the genome is reduced and the base sequence on the genome becomes simple, resulting in a decrease in the specificity of primer binding on the genome, leading to the existence of non-specific products and affecting the amplification effect.
  • the purpose of the present invention is to solve at least one of the above-mentioned technical defects, especially the problems of non-specific amplification of primers and primer dimers in the multiplex PCR targeted detection of methylation sites in the prior art.
  • the inventors studied a semi-nested multiple amplification method with universal sequence introduction.
  • the enrichment of target methylation regions was integrated into the two-step semi-nested PCR, reducing primer dimers and improving primer amplification specificity, achieving amplification of tens of thousands of target methylation sites in one tube.
  • This method can be compatible with multiple target methylation sites in one tube, or multiple target methylation sites and unmethylated sites can be captured simultaneously in one tube, achieving DNA methylation detection, or DNA and DNA methylation detection at the same time, improving the accuracy and efficiency of genomic methylation site detection.
  • the present invention provides a method for multiplex PCR.
  • the method comprises: a first round of amplification reaction 1, wherein the system of the first round of amplification reaction 1 comprises: conversion DNA and one or more conversion DNA primer pairs, wherein each of the conversion DNA primer pairs comprises a forward primer and a reverse primer, and the 5' end of the forward primer or the reverse primer in each of the conversion DNA primer pairs has a universal sequence 1; a second round of amplification reaction 1, wherein the system of the second round of amplification reaction 1 comprises: the product of the first round of amplification reaction 1 and one or more primer sets, wherein each of the primer sets comprises: 1) a semi-nested primer 1 having a 3' end that binds to one or more target regions of the product of the first round of amplification reaction 1 and a 5' end that has a universal sequence 2, wherein the semi-nested primer 1 is arranged at a position close to the target region of the forward primer or the reverse
  • the inventors reduce the proportion of dimers by introducing a round of semi-nested amplification. During this round of semi-nested PCR, a semi-nested specific primer and one or two universal primers are included.
  • the above method further includes at least one of the following additional technical features:
  • the conversion DNA is DNA after cytosine methylation conversion treatment.
  • the method according to a specific embodiment of the present invention can effectively detect the methylation sites in the target region of the genome.
  • the methylation conversion treated DNA is obtained by subjecting the DNA to a bisulfite or enzyme-assisted treatment.
  • the enzyme includes at least one of the following: TET methylcytosine dioxygenase 2 (TET2).
  • the universal sequence 1 and the universal sequence 2 are the same or different.
  • the T base content in the conversion DNA primer pair is 10%-70%.
  • At least one of the universal sequence 1, universal sequence 2, universal primer 1 and universal primer 2 includes a sequencing adapter sequence, a sample tag sequence or a molecular tag sequence.
  • CG sites and SNP sites should be avoided as much as possible, and continuous polyT structures should be avoided.
  • the primer length is 15-50 bp, and the TM value is 50-65.
  • the molar ratio of the primer pair of the conversion DNA can be adjusted according to the target fragment to be detected and the primer requirements.
  • the conditions of the first round of amplification reaction 1 are: 90°C-97°C, 0.5-5min, 1 cycle; 90°C-97°C, 25-35s, 55-65°C, 1.5-2.5min, 65°C-78°C, 25-35s, 10-30 cycles; 65°C-78°C, 4min-6min, 1 cycle.
  • the conditions of the second round of amplification reaction 1 are 90°C-97°C, 0.5-5min, 1 cycle; 90°C-97°C, 25-35s, 55-65°C, 1.5-2.5min, 65°C-78°C, 25-35s, 10-30 cycles; 65°C-78°C, 4min-6min, 1 cycle.
  • the present invention provides a multiplex PCR method.
  • the method comprises the following steps: a first round of amplification reaction 2, wherein the system of the first round of amplification reaction 2 comprises: original DNA, conversion DNA, one or more original DNA primer pairs and one or more conversion DNA primer pairs, wherein each of the original DNA primer pairs and conversion DNA primer pairs comprises a forward primer and a reverse primer, and the 5' end of the forward primer or the reverse primer in each of the original DNA primer pairs and conversion DNA primer pairs has a universal sequence 3;
  • the second round of amplification reaction 2 includes: the product of the first round of amplification reaction 2 and the second round of amplification primer set, wherein the second round of amplification primer set includes: i) a semi-nested primer 2, having a 3' end that binds to one or more target regions in the product of the first round of amplification reaction 2 and a 5' end that has a universal sequence 4, the semi-nested primer 2 is arranged downstream of the forward primer or reverse primer that does not have the universal sequence 3 in the first round of amplification system 2, and ii) a universal primer 3 having a 3' end that is the same as the universal sequence 3 and/or a universal primer 4 having a 3' end that is the same as the universal sequence 4.
  • multiple target methylation sites and unmethylated sites can be effectively captured simultaneously in one tube, and the untreated original DNA and the methylated converted DNA can be detected simultaneously, thereby improving the accuracy and efficiency of the detection of genomic methylation sites.
  • the original DNA is untreated DNA.
  • the conversion DNA is a DNA subjected to cytosine methylation conversion treatment.
  • the methylation conversion treated DNA is obtained by subjecting the DNA to bisulfite or enzyme treatment.
  • the methylase comprises at least one of the following: TET methylcytosine dioxygenase 2 (TET2).
  • the universal sequence 3 and the universal sequence 4 are the same or different.
  • the T base content of the conversion DNA primer pair is 10%-70%.
  • At least one of the universal sequence 3, the universal sequence 4, the universal primer 3 and the universal primer 4 includes a sequencing adapter sequence, a sample tag sequence or a molecular tag sequence.
  • CG sites and SNP sites should be avoided as much as possible, continuous polyT structures should be avoided, and the TM value should be 50-65.
  • a person skilled in the art can adjust the molar ratio of the original DNA, the converted DNA, one or more original DNA primer pairs and one or more converted DNA primer pairs included in the system of the first round of amplification reaction 2 as required.
  • the molar ratio of the first round of amplification reaction 2 product and the second round of amplification primer group included in the system of the second round of amplification reaction 2 can also be adjusted.
  • the conditions of the first round of PCR amplification reaction 2 are 90°C-97°C, 0.5-5min, 1 cycle; 90°C-97°C, 25-35s, 55-65°C, 1.5-2.5min, 65°C-78°C, 25-35s, 12-18 cycles; 65°C-78°C, 4min-6min, 1 cycle.
  • the conditions of the second round of PCR amplification reaction 2 are: 90°C-97°C, 0.5-5min, 1 cycle; 90°C-97°C, 25-35s, 55-65°C, 1.5-2.5min, 65°C-78°C, 25-35s, 18-22 cycles; 65°C-78°C, 4min-6min, 1 cycle.
  • the present invention provides a method for preparing a sequencing library.
  • the method comprises the step of constructing a sequencing library using the method described in the first aspect or the second aspect.
  • the above method further includes at least one of the following additional technical features:
  • the method further includes purifying the product of the second round of PCR amplification system 1 or 2 in the aforementioned multiplex PCR method.
  • the purification treatment is a magnetic bead purification treatment, an ethanol precipitation treatment or a tubular kit purification treatment.
  • the present invention provides a kit.
  • the kit comprises at least one of the following: one or more first-round conversion DNA primer pairs, the first-round conversion DNA primer pairs having a forward primer and a reverse primer, wherein the 5' end of the forward primer or the reverse primer has a universal sequence 1;
  • One or more second-round conversion DNA primer sets comprising: 1) a semi-nested primer 1 having a 3' end that binds to a conversion DNA target region and a 5' end that has a universal sequence 2, and 2) a universal primer 1 having a 3' end that is identical to the universal sequence 1 and/or a universal primer 2 having a 3' end that is identical to the universal sequence 2.
  • the kit further comprises one or more first-round original DNA primer pairs, wherein the first-round original DNA primer pair comprises a forward primer and a reverse primer, wherein the 5' end of the forward primer or the reverse primer comprises a universal sequence 3; and
  • One or more second-round original DNA primer sets include: 3) semi-nested primer 2, having a 3' end binding to the original DNA target region and a 5' end having a universal sequence 4, and 4) universal primer 3 having a 3' end identical to the universal sequence 3 and/or universal primer 4 having a 3' end identical to the universal sequence 4.
  • the kit includes a set of upstream and downstream specific primers consisting of 3'-end specific sequences designed for multiple target segment DNA and/or methylated DNA sequences and different universal sequences added to their 5' ends, and a set of upstream and downstream universal primers whose 3' ends are complementary to the universal sequences and whose 5' ends are sequencing adapter sequences.
  • the kit can amplify thousands of target methylation sites in one tube, and can also capture DNA and methylated DNA in one tube simultaneously during the detection process, thereby realizing the simultaneous amplification of DNA and DNA methylation.
  • the amplified library can be used for the simultaneous detection of DNA and DNA methylation sites.
  • the sequence length of the forward primer and the reverse primer in the first round conversion DNA primer pair is 15-50 bases.
  • the first round conversion DNA primer pair is a primer pair designed for the converted DNA, and the primer design principle follows the basic design principle of primers.
  • the sequence length of the universal sequence 1 and the universal sequence 2 is 10-100 bases.
  • the universal sequence 1 and the universal sequence 2 are not particularly limited and can be any fixed sequence or functional sequence, including but not limited to sequencing adapter sequence, sample tag sequence, molecular tag sequence, etc.
  • the sequence length of the semi-nested primer 1 and the semi-nested primer 2 is 15-50 bases.
  • the semi-nested primer 1 and the semi-nested primer 2 are designed for the transformed DNA, and the primer design principle follows the basic design principle of the primer.
  • the sequences of the universal primer 1 and the universal primer 2 may be the same or different.
  • the sequence length of the universal primer 1 and the universal primer 2 is 10-100 bases.
  • the universal primer 1 and the universal primer 2 can be any fixed sequence or functional sequence, including but not limited to a sequencing adapter sequence, a sample tag sequence, a molecular tag sequence, and the like.
  • the kit may further include one or more of a DNA sample extraction reagent, a DNA methylation conversion reagent, a Taq enzyme, dNTPs, a divalent magnesium ion, and a PCR system buffer.
  • the present invention proposes the use of the above-mentioned kit in preparing a sequencing library.
  • the kit can amplify methylated DNA, or methylated and unmethylated mixed DNA, and the library obtained after amplification meets the sequencing requirements.
  • the present invention provides a sequencing library.
  • the library is obtained using the method described in the third aspect.
  • the present invention proposes a method for sequencing a target nucleic acid molecule and/or detecting methylation sites.
  • the method comprises: 1) constructing a sequencing library for the target nucleic acid molecule according to the method described in the third aspect; 2) sequencing the sequencing library to obtain sequencing results; and 3) based on the sequencing results, determining the nucleic acid sequence of the target nucleic acid molecule and/or the methylation sites of the nucleic acid molecule.
  • the method according to an embodiment of the present invention can effectively sequence the original DNA and/or detect the methylation sites of the methylated modified DNA.
  • the present invention proposes a method for detecting methylation-related diseases.
  • the method comprises the following steps: i) constructing a subject sequencing library using the method described in the third aspect for a nucleic acid molecule derived from a subject; ii) sequencing the subject sequencing library to obtain a sequencing result; iii) determining the methylation site of the nucleic acid molecule derived from the subject based on the sequencing result; and iv) judging whether the subject suffers from a methylation-related disease based on the methylation site.
  • the above detection method may further include at least one of the following additional technical features:
  • the methylation-related disease includes at least one selected from the following: cardiovascular disease, cerebrovascular disease, autoimmune disease, metabolic disease and cancer.
  • the cardiovascular disease is ischemic cardiomyopathy, atherosclerosis, hypertension or heart failure.
  • the cerebrovascular disease is cerebral hemorrhage or cerebral stroke.
  • the autoimmune disease is psoriasis, lupus erythematosus or psoriasis.
  • the metabolic disease is obesity, type 2 diabetes, non-alcoholic fatty liver disease or osteoporosis.
  • the cancer is colorectal cancer, lung cancer, liver cancer, gastric cancer, pancreatic cancer or brain glioma.
  • the methylation-specific primer pair F/R is rich in ATG and ATC and is easy to form dimers, so the inventors introduced a round of semi-nested amplification to reduce the proportion of dimers.
  • this round of semi-nested PCR 1) all semi-nested specific PCR primers are rich in ATG bases or rich in ATC bases, and all specific ATG-ATG or ATC-ATC are difficult to form dimers with each other, and 2) during the second round of amplification, the semi-nested specific primers (ATG or ATC) and the universal primer (ATCG) are also difficult to form dimers, so the proportion of dimers in amplification can also be effectively reduced in the second round of amplification.
  • two rounds of PCR are used to improve the specificity of targeted amplification, and another round of semi-nested amplification is performed on the basis of the first round of PCR amplification to improve the specificity of target amplification.
  • the sequencing library prepared by the multiplex PCR method described in the present application is compatible with the simultaneous capture of DNA and DNA methylation, and can achieve simultaneous detection of DNA and DNA methylation.
  • Fig. 1 is a schematic diagram of the design of semi-nested PCR amplification primers introduced by the universal sequence (fixed tag) T1 or T2 provided in an embodiment of the present invention, wherein three primers are designed corresponding to each target region, namely, the outer specific primer pair F and R, and the semi-nested primer F1 or R1, the 5' end of one of the primers F or R in the outer specific primer pair is connected to the universal sequence T1, the 5' end of the semi-nested primer is connected to the universal sequence T2, and the semi-nested primer is arranged downstream of the F or R primer whose 5' end is not connected to the universal sequence T1;
  • FIG2 is a schematic diagram of nested PCR amplification according to an embodiment of the present invention, wherein the fixed tag is introduced;
  • FIG3 is a schematic diagram of library preparation based on fixed tag introduction according to an embodiment of the present invention.
  • FIG4 is a fragment distribution diagram of a methylation library based on fixed tag introduction according to an embodiment of the present invention, with a fragment size of 150-180 bp;
  • FIG. 5 is a graph showing the stability (Pearson coefficient) of methylated DNA detection with different initial input amounts according to an embodiment of the present invention
  • FIG6 is a schematic diagram of preparing a mixed library of DNA and methylated DNA based on fixed tag introduction according to an embodiment of the present invention
  • FIG. 7 is a fragment distribution diagram of a mixed library of DNA and methylated DNA based on fixed tag introduction according to an embodiment of the present invention, wherein the fragment size of the methylated library is 150-180 bp, and the fragment size of the DNA library is 340-390 bp.
  • the present application proposes a method for preparing a targeted methylation sequencing library based on multiplex PCR, wherein a semi-nested multiplex amplification method for introducing universal sequences is invented.
  • a semi-nested multiplex amplification method for introducing universal sequences is invented.
  • the enrichment of the target methylation region is integrated into the two-step semi-nested PCR, reducing primer dimers and improving primer amplification specificity, thereby achieving one-tube amplification of tens of thousands of target methylation sites.
  • this method is compatible with two schemes, namely, methylated DNA is captured in one tube, or unmethylated DNA and methylated DNA are captured simultaneously in one tube, the latter of which can achieve simultaneous detection of DNA and DNA methylation.
  • the specific technical scheme is as follows:
  • This example mainly includes the following experimental contents: gDNA is chemically converted (bisulfite) or enzymatically converted (TET enzyme-assisted conversion) to convert unmethylated cytosine into uracil.
  • sample NA12878 gDNA is treated with bisulfite, and detection primers are designed. Then, the DNA and primers are used to prepare a DNA targeted methylation library, and the obtained library is placed on an MGISEQ-2000 sequencer for on-machine sequencing, with the sequencing type being PE100, and then data analysis is performed, including data utilization, alignment rate, amplicon specificity, uniformity and other performance.
  • the specific experimental operations are as follows:
  • the experiment was conducted using EZ DNA Methylation-Gold Kit TM (ZYMO), and the sample NA12878 gDNA was set to 4 mass gradients of 0.5, 1, 5, and 10 ng, with 3 replicates for each gradient.
  • the gDNA was co-treated with bisulfite, and the specific steps were as follows:
  • CT conversion reagent solution Take out CT conversion reagent (solid mixture) from the above kit, add 900 ⁇ L of water, 50 ⁇ L of M-dissolving buffer and 300 ⁇ L of M-dilution buffer to the CT conversion reagent, dissolve and shake at room temperature for 10 minutes or shake on a shaker for 10 minutes.
  • the PCR product can be immediately used for the next step or stored at 4°C (up to 20 hours) for future use.
  • step 11 Place the Zymo-Spin IC TM Column obtained in step 10) in a new 1.5 mL EP tube, add 40 ⁇ L of M-elution buffer r to the column matrix, place at room temperature for 2 minutes, and centrifuge at full speed (>10,000 x g) to elute the target DNA.
  • each pair of primers is responsible for amplifying a target region, and a fixed universal sequence T1 is added to the 5' end of one primer (forward primer F or reverse primer R) in each pair of primers (as shown in FIG. 1 );
  • step ii) designing a semi-nested specific primer F1 or R1 based on the first-round specific primer, the binding position of which is set inside the first-round specific amplification primer described in step i) (as shown in FIG1 ), wherein another universal sequence (T2, the sequences of T2 and T1 may be the same or different) is introduced into the 5′ end of the semi-nested specific primer;
  • reaction product was purified using 1.5X AMPure magnetic beads and then dissolved in 22 ⁇ L elution buffer.
  • the obtained library was subjected to high-throughput sequencing, the sequencing platform was MGISEQ-2000, and the sequencing type was PE100. After sequencing, the data were aligned and various basic parameters were statistically analyzed, including offline data, available data, alignment rate, specificity, uniformity, etc.
  • NA12878 gDNA is used for the experiment.
  • the gDNA is converted into uracil by chemical conversion (bisulfite) or enzymatic conversion (TET enzyme-assisted conversion) to convert unmethylated cytosine.
  • a methylation capture panel containing 24 methylation sites is designed; at the same time, a DNA capture panel without methylation is designed, containing 20 DNA capture regions.
  • the gDNA of the NA12878 standard is treated with bisulfite, and then mixed with genomic DNA.
  • the mixed DNA is subjected to DNA and DNA methylation mixed library preparation according to the steps of the invention.
  • the sample is repeated 3 times, and the obtained library is placed on the MGISEQ-2000 sequencer for on-machine sequencing.
  • the sequencing type is PE100, and then data analysis is performed, including data utilization, alignment rate, amplicon specificity, uniformity and other performances.
  • the specific experimental operation is as follows:
  • CT conversion reagent solution Take out CT conversion reagent (solid mixture) from the above kit, add 900 ⁇ L of water, 50 ⁇ L of M-dissolving buffer and 300 ⁇ L of M-dilution buffer to the CT conversion reagent, dissolve and shake at room temperature for 10 minutes or shake on a shaker for 10 minutes.
  • the PCR product can be immediately used for the next step or stored at 4°C (up to 20 hours) for future use.
  • step 11 Place the Zymo-Spin IC TM Column obtained in step 10) in a new 1.5 mL EP tube, add 40 ⁇ L of M-elution buffer r to the column matrix, place at room temperature for 2 minutes, and centrifuge at full speed (>10,000 x g) to elute the methylated target DNA.
  • step 12 The methylated DNA obtained in step 11) was mixed with 5 ng of genomic DNA, and the mixed DNA was used as a template for subsequent library preparation.
  • First round of specific primer design One or more pairs of specific primers (F/R) are designed for the original genomic DNA sequence or the methylated genomic DNA sequence. Each pair of primers is responsible for the amplification of a target region of the original DNA or methylated DNA genome. A fixed universal sequence T1 is added to the 5' end of one primer (F or R) in each pair of primers. All primers with T1 sequence and all primers without T1 are amplified by single or multiplex PCR in one tube to obtain the target product with T1 adapter;
  • U1 Design a universal primer U1, the 3' end sequence of U1 contains all the sequences of T1 (U1 ⁇ T1). All specific primers with T2 sequence and a universal primer (U1) are used to perform multiple amplification on the first round of PCR products to obtain the target fragment with T2 sequence and U1 universal primer; sequencing adapter sequence, sample tag sequence, molecular tag sequence, chemical modification (phosphorylation, amination, etc.), enzyme cutting site (USER enzyme, restriction endonuclease), etc. can be introduced into the T1, T2 and U1 primer sequences for subsequent different molecular experiments.
  • the obtained library was subjected to high-throughput sequencing using the sequencing platform MGISEQ-2000 and the sequencing type PE100.
  • the sequencing data was aligned and the basic parameters were statistically analyzed, including offline data, available data, alignment rate, GC content, etc.
  • the basic performance parameters of DNA sequencing obtained by the hybrid sequencing scheme are shown in Table 12.
  • the data utilization rate is 0.99 ⁇ 0.00
  • the overall alignment rate is 0.98 ⁇ 0.02
  • the specificity is 0.98 ⁇ 0.00
  • the uniformity is 1.00 ⁇ 0.00, which can achieve effective detection of DNA targeting.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of “plurality” is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

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Abstract

La présente invention concerne un système de construction de banque de méthylation ciblée fondé sur la PCR multiplex, un procédé et son utilisation. Le procédé comprend : un premier cycle de réaction d'amplification (1), le système du premier cycle de réaction d'amplification (1) comprenant : un ADN matrice et une ou plusieurs paires d'amorces, chaque paire d'amorces comprenant une amorce sens et une amorce antisens, et l'extrémité 5' de l'amorce sens ou de l'amorce antisens de chaque paire d'amorces étant pourvue d'une séquence universelle (1) ; et un deuxième cycle de réaction d'amplification (1), le système du deuxième cycle de réaction d'amplification (1) comprenant un produit du premier cycle de réaction d'amplification (1) et un ou plusieurs ensembles d'amorces, et chaque ensemble d'amorces comprenant : 1) une amorce semi-nichée (1) dont l'extrémité 3' est liée à une ou plusieurs régions cibles dans le produit du premier cycle de réaction d'amplification (1) et dont l'extrémité 5' présente une séquence universelle (2), et 2) une amorce universelle (1) dont l'extrémité 3' est identique à celle de la séquence universelle (1) et/ou une amorce universelle (2) dont l'extrémité 3' est identique à celle de la séquence universelle (2).
PCT/CN2022/138685 2022-12-13 2022-12-13 Système de construction de banque de méthylation ciblée fondé sur la pcr multiplex, procédé et son utilisation Ceased WO2024124400A1 (fr)

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PCT/CN2022/138685 WO2024124400A1 (fr) 2022-12-13 2022-12-13 Système de construction de banque de méthylation ciblée fondé sur la pcr multiplex, procédé et son utilisation
CN202280100895.9A CN120077148A (zh) 2022-12-13 2022-12-13 一种基于多重pcr的靶向甲基化建库体系、方法及其应用

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