WO2023159817A1

WO2023159817A1 - Genetic diagnosis probes and use thereof

Info

Publication number: WO2023159817A1
Application number: PCT/CN2022/100272
Authority: WO
Inventors: 李冰思; 宿静; 邱福俊; 王晨阳; 李晓玲; 张之宏; 汉雨生
Original assignee: 广州燃石医学检验所有限公司
Priority date: 2022-02-28
Filing date: 2022-06-22
Publication date: 2023-08-31
Also published as: CN114438080A

Abstract

Provided are genetic diagnosis probes and the use thereof. A probe is a nucleic acid molecule combination, the nucleic acid molecule combination comprising at least one nucleic acid probe group that covers a target area of a nucleic acid to be detected, and the nucleic acid probe group at least comprising nucleic acid probes that cover sense and antisense strands of the target area as well as respective complementary strands thereof.

Description

A gene diagnostic probe and its application

technical field

This application relates to the field of biomedicine, in particular to a gene diagnostic probe and its application.

Background technique

DNA methylation is an epigenetic modification, which is catalyzed by DNA methyl-transferase (DNA methyl-transferase, DNMT) to convert S-adenosylmethionine (SAM) into a methyl group. Donor, the cytosine of the CG two nucleotides of the DNA is selectively methylated, mainly forming 5-methylcytosine (5-mC) (commonly found in the 5'-CG-3' sequence of genes) And a small amount of N6-methylpurine (N6-mA) and 7-methylguanine (7-mG) structural genes contain many CpG structures, and the 5th carbon atom of two cytosines in 2CpG and 2GPC is usually methylated , and the two methyl groups present a specific three-dimensional structure in the DNA double-strand major groove.

DNA methylation plays an important role in the regulation of gene expression. Aberrant DNA methylation marks have been reported in the development of various diseases, including cancer. As a high-resolution, high-throughput technology, DNA methylation sequencing is increasingly recognized for its role in early cancer screening, diagnosis, and monitoring.

Whole Genome Bisulfite Sequencing (WGBS, Whole Genome Bisulfite Sequencing) is the gold standard for methylation sequencing, but it has become difficult for clinical application due to severe damage to DNA during processing and high sequencing costs. More importantly, most regions of the human genome are not active during the development of cancer, and cancer-related mutations are often concentrated in certain specific regions, such as CpG islands. CG dinucleotide is the most important methylation site, it is unevenly distributed in the genome, there are hypermethylation, hypomethylation and non-methylation regions, mC accounts for about the total C in mammals 2-7%.

CpG islands are abundant in the genome, and these detection and analysis can be greatly assisted by massively parallel nucleic acid sequencing (also known as "high-throughput sequencing" or "next-generation sequencing" (NGS)), making it possible to predict Where and where the cancer occurs becomes possible.

In addition, unmethylated cytosine (C) in DNA fragments after bisulfite treatment is converted to thymine (T), and the reduced C content leads to a stronger binding of cytosine (C)-guanine (G) has fewer binding sites, and the reduction of C content also reduces the complexity of the bases on the DNA, both of which increase the difficulty of hybrid capture.

At the same time, there is also a lack of a standard in the art that can directly reflect the level of DNA methylation for evaluating the capture performance of methylation capture probes. Embodiment 3 of Chinese Patent Announcement CN112646888B mentions a kind of utilization

Single Cell Kit (Qiagen, Cat#150343) and Mung Bean Nuclease (NEB, Cat#M0250L) were used to process NA12878 DNA to prepare 0% methylation standard. However, in the subsequent bisulfite conversion process of this actually prepared 0% methylation standard, almost all cytosine (C) will be converted into thymine (T), which greatly increases the complexity of the bases on the DNA. It is very difficult to capture, so it is not suitable as a standard for measuring the performance of methylated capture probes with high accuracy.

Therefore, there is a lack of a capture probe suitable for targeted sequencing of methylated DNA that meets the capture performance expectations and a standard for accurately measuring the capture accuracy of the probe.

Contents of the invention

The present application provides a high-precision gene hybridization capture probe, which can hybridize and capture methylation variation regions associated with various cancers, especially specific methylation characteristic regions. Through highly accurate methylation detection probes, human tumor gene detection preparations can be prepared to achieve early detection or early screening of cancers including but not limited to: brain cancer, lung cancer, skin cancer, nasopharyngeal cancer, throat cancer Cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, bowel cancer, rectal cancer, thyroid cancer, bladder cancer, kidney cancer, oral cancer, stomach cancer, solid tumors, ovarian cancer, esophagus cancer, gallbladder cancer, biliary tract cancer , breast cancer, cervical cancer, uterine cancer, prostate cancer, head and neck cancer, sarcoma, thoracic malignancy (except lung), melanoma, and testicular cancer.

The present application provides a combination of nucleic acid molecules, the combination of nucleic acid molecules comprising at least one nucleic acid probe set covering a target region of a nucleic acid to be detected, characterized in that the set of nucleic acid probes at least comprises: (1) and A first probe complementary to the first strand, the first strand being the sequence of the target region after base substitution; (2) a second probe complementary to the second strand, the second strand being the sequence of the target region The sequence of the complementary region of the target region after base substitution; and contains any one or both of the following two probes: (3) a third probe complementary to the third strand, and the third strand is complementary to the third strand The first strand is complementary; (4) a fourth probe complementary to the fourth strand, which is the complementary sequence of the second strand.

The application provides an application of the nucleic acid molecule combination described in the application in the preparation of human tumor gene detection preparations.

The present application provides a standard nucleic acid molecule used for assessing the accuracy of the degree of base modification detected in the application of the present application, the standard nucleic acid molecule comprises a candidate region where the degree of base modification is about 0%, and the candidate region The total length is about 1 bp to about 10000 bp.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.

Description of drawings

The particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:

Figure 1 shows the methylation measurement results of the "20% standard" and "50% standard" of the application, as well as the "zero methylation standard" and "full methylation standard" of the application ” methylation measurements.

Figures 2A-2C show the uniformity measurement results of the probes designed in this application.

Figure 3 shows the repeatability measurement results of the probes designed in this application.

Figures 4A-4C show the bias measurement results of the probes designed in this application.

Fig. 5 shows an exemplary reference schematic diagram of the capture probe design of the present application.

Fig. 6 shows an exemplary reference schematic diagram for calculating the methylation level in the present application.

Detailed ways

The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the terms "next-generation gene sequencing (NGS)", high-throughput sequencing" or "next-generation sequencing" generally refer to the second-generation high-throughput sequencing technology and higher-throughput sequencing methods developed thereafter. Next-generation sequencing platforms include but are not limited to existing sequencing platforms such as Illumina. With the continuous development of sequencing technology, those skilled in the art can understand that other sequencing methods and devices can also be used for this method. For example, two Generation gene sequencing can have the advantages of high sensitivity, high throughput, high sequencing depth, or low cost. According to the development history, influence, sequencing principles and technologies, there are mainly the following types: Massively Parallel Signature Sequencing (Massively Parallel Signature Sequencing, MPSS), Polony Sequencing, 454pyro sequencing, Illumina (Solexa) sequencing, Ion semi conductor sequencing, DNA nano-ball sequencing , Complete Genomics' DNA nanoarray and combined probe anchor ligation sequencing method, etc. The second-generation gene sequencing can make it possible to analyze the transcriptome and genome of a species in detail, so it is also called deep sequencing ( deep sequencing). For example, the method of the present application can also be applied to first-generation gene sequencing, second-generation gene sequencing, third-generation gene sequencing or single molecule sequencing (SMS).

In this application, the term "sample to be tested" generally refers to a sample that needs to be tested. For example, it is possible to detect whether one or more gene regions on the sample to be tested are modified.

In this application, the term "complementary region" generally refers to a region that is complementary to a reference nucleotide sequence. For example, a complementary nucleic acid can be a nucleic acid molecule that optionally has an opposite orientation. For example, the complementary may refer to having the following complementary associations: guanine and cytosine; adenine and thymine; adenine and uracil.

In this application, the term "hybridization" generally refers to a reaction in which one or more polynucleotides react to form a complex stabilized by hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonding can occur through Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner based on base complementarity. The complex may comprise two strands forming a double helix, three or more strands forming a multi-strand complex, self-hybridizing single strands, or any combination of these. The hybridization reaction may constitute a step in a wider method, such as the initiation of PCR or the enzymatic cleavage of polynucleotides by endonucleases. A second sequence that is completely complementary to a first sequence or that is polymerized by a polymerase using the first sequence as a template is said to be "complementary" to said first sequence. The term "hybridizable" as applied to a polynucleotide refers to the ability of a polynucleotide to form complexes that are stabilized by hydrogen bonds between the bases of the nucleotide residues in a hybridization reaction. In some embodiments, a hybridizable nucleotide sequence is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the sequence to which it hybridizes.

Terminology In this application, the terms "polynucleotide", "nucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably. They represent polymeric forms of nucleotides (deoxyribonucleotides or ribonucleotides) of any length, or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any function, whether known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene segments, loci (loci) defined by linkage analysis, exons, introns, messenger RNA (mRNA), translocation RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), microRNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, Plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers and linkers. A polynucleotide may include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if present, can be imparted either before or after assembly of the polymer. Nucleotide sequences can be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with labeling components.

In the present application, the term "modification state" generally refers to the modification state of the gene fragment, nucleotide or its base in the present application. For example, the modification state in the present application may refer to the modification state of cytosine. For example, a gene segment of the present application having a modified state may have altered gene expression activity. For example, the modification status of the present application may refer to the methylation modification of a base. For example, the modified state in this application may refer to the covalent bonding of a methyl group at the 5' carbon position of cytosine in the CpG region of genomic DNA, for example, it may become 5-methylcytosine (5mC). For example, a modification state can refer to the presence or absence of 5-methylcytosine ("5-mCyt") within the DNA sequence.

In this application, the term "methylation" generally refers to the methylation state of a gene fragment, nucleotide or its base in this application. For example, the DNA fragment where the gene in this application is located may have methylation on one strand or multiple strands. For example, the DNA fragment where the gene in this application is located may have methylation at one site or multiple sites.

In this application, the term "transformation" generally refers to the transformation of one or more structures into another structure. For example, the transformations of the present application can be specific. For example, cytosine without methylation modification can be converted into other structures (such as uracil), and cytosine with methylation modification can be substantially unchanged after conversion. For example, cytosine without methylation modification can be cleaved after conversion, and cytosine with methylation modification can be substantially unchanged after conversion.

In this application, the term "bisulfite", or "bisulfite" generally refers to a reagent that can distinguish DNA regions with and without modification states. For example, the bisulfite may include bisulfite, or an analog thereof, or a combination thereof. For example, bisulfite can deaminate the amino group of unmodified cytosine to distinguish it from modified cytosine. In this application, the term "analogue" generally refers to a substance having a similar structure and/or function. For example, analogs of bisulfite may have a similar structure to bisulfite. For example, an analog of bisulfite may refer to a reagent that can also distinguish between DNA regions that have a modified state and those that do not.

In this application, the term "comprising" generally means including specifically specified features, but not excluding other elements.

In this application, the term "about" generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

Detailed description of the invention

On the one hand, the present application provides a combination of nucleic acid molecules, the nucleic acid molecule in the combination of nucleic acid molecules has a binding free energy for a nucleic acid sequence derived from a target region and a binding free energy for a nucleic acid sequence derived from a non-target region differs by more than one specific threshold. For example, the specified threshold is from about 12 to about 50 kcal/mol. For example, the specific threshold is about 20 to 30 kcal/mol. For example, the specific threshold is about 20 kcal/mol.

For example, the combination of nucleic acid molecules of the present application is based on the screening of candidate target regions to determine suitable nucleic acid molecules. For example, the nucleic acid molecule combination designed for the candidate target region in the present application has a higher binding free energy for the nucleic acid sequence derived from the target region. For example, compared with the binding free energy of nucleic acid sequences derived from non-target regions, the combination of nucleic acid molecules designed in the present application for candidate target regions has higher binding free energy for nucleic acid sequences derived from target regions. For example, the nucleic acid molecules in the combination of nucleic acid molecules of the present application have a binding free energy for a nucleic acid sequence derived from a target region that differs from the binding free energy for a nucleic acid sequence derived from a non-target region by about 12 or more. For example, the nucleic acid molecules in the nucleic acid molecule combination of the present application have a binding free energy for the nucleic acid sequence derived from the target region, which is about 12 kcal/mol, about 13 kcal/mol higher than the binding free energy of the nucleic acid sequence derived from the non-target region.

kcal/mol, about 14 kcal/mol, about 15 kcal/mol, about 20 kcal/mol, about 25 kcal/mol, about 30 kcal/mol, about 40 kcal/mol, or about 50 kcal/mol.

In one aspect, the present application provides a combination of nucleic acid molecules, the combination of nucleic acid molecules comprising at least one nucleic acid probe set covering the target region of the nucleic acid to be tested, the set of nucleic acid probes at least comprising: (1) complementary to the first strand The first probe of the first strand is the sequence of the target region after base substitution; (2) the second probe complementary to the second strand, the second strand is the complementary sequence of the target region The sequence of the region after base substitution; and may contain any one of the following two probes or may contain two of the following two probes at the same time: (3) the third probe complementary to the third strand, so The third strand is complementary to the first strand; (4) a fourth probe complementary to the fourth strand, the fourth strand being the complementary sequence of the second strand. For example, the nucleic acid molecule combination of the present application is aimed at the target region assuming that the nucleic acid to be tested is zero methylation, the first strand (target upper strand, OT strand) of the region after base replacement, and the complementary region of the region The second strand after base substitution (the lower strand of the target, OB strand), and for the complementary strand of the first strand (the complementary strand of the upper strand of the target, CTOT strand), design a third probe complementary to the third strand ; Simultaneously design a fourth probe complementary to the fourth strand according to the complementary strand of the second strand (the complementary strand of the lower strand of the target, CTOB strand). For example, nucleic acid molecules of the present application are combined with capture probes for methylation detection. For example, capture probes for methylation detection in next-generation sequencing.

For example, the base-substituted site includes a site where cytosine is present. For example, the base substitution comprises a nucleic acid sequence in which cytosine is replaced by thymine or uracil through chemical and/or biological processes. For example, the base substitution includes obtaining a nucleic acid sequence in which all cytosines are replaced with thymine or uracil. The base replacement may include bisulfite conversion treatment, and the unmethylated C in the original upper chain and the original lower chain is converted into uracil

(U). Since uracil (U) is paired complementary with adenine (A), and the base paired with adenine (A) introduced in the PCR amplification of DNA is thymine (T), so the base substitution can be included in further During the PCR amplification of , the unmethylated C in the original upper strand and the original lower strand was replaced by T.

For example, in the nucleic acid molecule combination of the present application, the nucleic acid probe set further comprises: (1) a fifth probe complementary to the fifth strand, the fifth strand being the sequence of the target region without base substitution (2) a sixth probe complementary to the sixth strand, the sixth strand being a sequence in which the complementary region of the target region has not undergone base substitution; (3) a seventh probe complementary to the seventh strand, The seventh strand is complementary to the fifth strand; (4) an eighth probe complementary to the eighth strand, the eighth strand being the complementary sequence of the sixth strand. For example, for the nucleic acid molecule combination of the present application, four other probes are designed for the target region assumed to be fully methylated in the nucleic acid to be tested.

For example, the combination of nucleic acid molecules comprises a set of nucleic acid probes covering 10,000 or more different target regions of the nucleic acid to be tested. For example, the nucleic acid molecule combination of the present application is aimed at 10000 or more, 15000 or more, 20000 or more, 25000 or more, 30000 or more, 40000 or more, or 50,000 or more different target areas for design.

In one aspect, the present application provides a combination of nucleic acid molecules, in which standards for specific methylation levels, for example, the detection results of methylation standards for 20% and/or 50% methylation levels The index selected from the following group is met: the fluctuation of the detection result of the methylation level is 25% or lower, and the repeatability is 9E-05 or lower. Preferably, the fluctuation is the difference between the maximum value and the minimum value of the detection result, and the repeatability is the median value of the mean square error of the methylation level among multiple wells. For example, fluctuations in methylation levels are used to assess the accuracy of nucleic acid molecule combinations. For example, for methylation standards with 20% and/or 50% methylation levels, the detection result fluctuation of the nucleic acid molecule combination of the present application is 22% or lower, 23% or lower, 24% or lower, 25% or lower. % or less, 26% or less, or 27% or less. For example, for methylation standards with 20% and/or 50% methylation levels, the mean square error of the methylation levels detected by the candidate capture probe combinations for two or more repeated measurements of the nucleic acid molecule combinations of the present application Between about 1.3E-05 and about 2.7E-04, preferably 9E-05 or lower, more preferably about 8E-05 or lower, further preferably about 7E-05 or lower.

For example, the nucleic acid molecules in the combination of nucleic acid molecules are about 80 to about 120 bases in length. For example, the nucleic acid molecules in the combination of nucleic acid molecules are about 80, about 90, about 100, about 110, or about 120 bases in length.

For example, the region where any two nucleic acid molecules in the combination of nucleic acid molecules overlap comprises about 10 to about 110 bases. For example, the region where any two nucleic acid molecules in the combination of nucleic acid molecules overlap comprises about 10, about 20, about 50, about 70, about 80, about 90, about 100, or about 110 bases.

For example, the region to which the nucleic acid molecules in the combination of nucleic acid molecules are complementary does not contain 10 or more consecutive bases overlapping the repeat region. For example, the information of the repeating region can be described in what is known in the art, such as the repeating region (repeats) described in repeatmasker.org.

In one aspect, the present application provides a method for designing a combination of nucleic acid molecules, based on the first strand derived from the target region and subjected to base substitution and its complementary strand, and the second strand derived from the target region and subjected to base substitution and its complementary strand. Complementary chains, designing combinations of said nucleic acid molecules that are complementary to three or more of the above-mentioned chains.

On the one hand, the present application provides a method for designing a combination of nucleic acid molecules, which includes (1) screening target regions, and the combination of nucleic acid molecules designed for candidate target regions has a higher binding free energy for nucleic acid sequences derived from the target region (2) Design 4 probes for the candidate target region, the nucleic acid molecule combination of the present application is aimed at the target region assuming that the nucleic acid to be tested is fully methylated, and the first base substitution in the region One strand and its complementary strand, and four probes are designed for the second strand and its complementary strand after base substitution in the complementary region of the region; (3) screening nucleic acid molecule combinations, for the standard of specific methylation level As a product, a combination of nucleic acid molecules meeting the criteria selected from the following group is screened: the methylation level detection result fluctuation is 25% or lower, and the repeatability is 9E-05 or lower.

On the one hand, the present application provides a method for designing a combination of nucleic acid molecules, which includes (1) the combination of nucleic acid molecules designed for a candidate target region of the present application, the binding free energy of the nucleic acid sequence derived from the target region is higher than that of the source The binding free energy of the nucleic acid sequence in the non-target region is about 12 or higher; (2) the nucleic acid molecule combination of the present application is aimed at the target region assuming that the nucleic acid to be tested is fully methylated, and the base substitution of the region is The first strand (Top strand), the second strand (Bottom strand) after base substitution in the complementary region of this region, and the complementary strand (CTOT strand) to the first strand, the second strand complementary to the third strand is designed. Three probes; at the same time, according to the complementary strand (CTOB strand) of the second strand, the fourth probe complementary to the fourth strand is designed; (3) screening nucleic acid molecule combinations, for the standards of specific methylation levels, screening meets the selection A nucleic acid molecule combination of indicators from the following groups: a methylation level detection result fluctuation of 25% or less, and a repeatability of 9E-05 or less. Preferably, the fluctuation is the difference between the maximum value and the minimum value of the detection result, and the repeatability is the median value of the mean square error of the methylation level among multiple wells.

For example, in the design method of the present application, the nucleic acid molecule is combined with a capture probe for methylation detection. For example, the standard of specific methylation level used in the method of the present application, the standard of specific methylation level is prepared by the method of the present application.

In one aspect, the present application provides the nucleic acid molecule combination obtained by the design method of the nucleic acid molecule combination of the present application. For example, the nucleic acid molecule is combined with a capture probe for methylation detection.

In one aspect, the present application provides a kit comprising the nucleic acid molecule combination of the present application.

In one aspect, the present application provides the application of the nucleic acid molecule combination of the present application and/or the kit of the present application in the preparation of human tumor gene detection preparations. For example, the detection preparation is used to detect the base modification level of the target region. For example, the base modification includes methylation modification. For example, the human tumors are from homogenous tumors, heterogeneous tumors, hematological cancers and/or solid tumors. For example, the human tumor is from one or more of the following group of cancers: brain cancer, lung cancer, skin cancer, nasopharyngeal cancer, throat cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, intestinal Cancer, rectal cancer, thyroid cancer, bladder cancer, kidney cancer, oral cancer, stomach cancer, solid tumor, ovarian cancer, esophageal cancer, gallbladder cancer, biliary tract cancer, breast cancer, cervical cancer, uterine cancer, prostate cancer, head and neck cancer, sarcoma , Thoracic malignancy (except lung), melanoma, testicular cancer.

In one aspect, the present application provides a method for detecting the level of base modification, comprising providing the nucleic acid molecule combination of the present application and/or the kit of the present application. For example, the base modification includes methylation modification.

In one aspect, the present application provides a storage medium, which records a program capable of running the method of the present application. For example, the non-transitory computer readable storage medium may include a floppy disk, a flexible disk, a hard disk, a solid state storage (SSS) (such as a solid state drive (SSD)), a solid state card (SSC), a solid state module (SSM)), an enterprise high-grade flash drives, tape, or any other non-transitory magnetic media, etc. Non-transitory computer readable storage media may also include punched cards, paper tape, cursor sheets (or any other physical media having a pattern of holes or other optically identifiable markings), compact disc read only memory (CD-ROM) , Rewritable Disc (CD-RW), Digital Versatile Disc (DVD), Blu-ray Disc (BD) and/or any other non-transitory optical media.

In one aspect, the present application provides a device, and the device includes the storage medium of the present application. For example, the device further includes a processor coupled to the storage medium, and the processor is configured to execute based on a program stored in the storage medium to implement the method of the present application.

In one aspect, the present application provides a nucleic acid molecule used as a standard for detecting the degree of base modification, the nucleic acid molecule comprising a candidate region with a degree of base modification of about 0%. For example, the total length of the candidate region of the present application is about 1 bp to about 10000 bp. For example, the total length of the candidate region of the present application is about 1 bp, about 10 bp, about 100 bp, about 1000 bp, about 10000 bp, about 50000 bp, or about 100000 bp. For example, the nucleic acid molecule can be selected from one or more of the following cell lines: GM24385, GM12878, GM12877, GM24631.

In one aspect, the present application provides a method for preparing a base modification degree detection standard, the method comprising determining a candidate region in a nucleic acid molecule where the base modification degree is about 0%.

In one aspect, the present application provides the use of a nucleic acid molecule in the preparation of a base modification degree detection standard, the nucleic acid molecule comprising a candidate region with a base modification degree of about 0%.

For example, the degree of base modification includes the degree of methylation of cytosine in the candidate region.

In one aspect, the present application provides a set of candidate regions before base modification treatment as described in the nucleic acid molecule of the present application, which is used as a standard without base modification treatment. For example, the nucleic acid molecule can serve as a zero methylation standard.

In one aspect, the present application provides a collection of all base-modified candidate regions as described in the nucleic acid molecule of the present application, which is used as a base-modified standard. For example, the nucleic acid molecule can serve as a permethylation standard.

In one aspect, the present application provides a method for preparing a base modification degree detection standard, the method comprising determining a candidate region in a nucleic acid molecule where the base modification degree is about 0% before base modification treatment, and subjecting the nucleic acid molecule to Base modification treatment.

On the one hand, the present application provides the use of a nucleic acid molecule in the preparation of a base modification degree detection standard, the nucleic acid molecule comprises a candidate region with a base modification degree of about 0% before base modification treatment, and the nucleic acid molecule Perform base modification.

For example, by mixing the nucleic acid molecule before the base modification treatment and the nucleic acid molecule after the base modification treatment in a predetermined ratio, a methylation standard with a predetermined degree of base modification is obtained. For example, the base modification treatment comprises contacting the nucleic acid molecule with a methyltransferase. For example, if m% of the above-mentioned full methylation standard is mixed with 1-m% of the above-mentioned zero-methylation standard, the m% methylation standard can be obtained, and the m% methylation The degree of methylation of the standard in the candidate region is m%.

The present application provides a kit comprising the nucleic acid molecule of the present application. For example, the kit can be used as a standard for capture probes for methylation detection.

Not intending to be limited by any theory, the following examples are only for explaining the methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1

Probe Screening

The next-generation sequencing treated with bisulfite reduces the complexity of the library and poses a great challenge to the specificity of target capture. Compared with the methylated amplicon method (amplicon), the hybridization capture method is often suitable for long probes, thus providing Better specificity and tolerance to single-nucleotide polymorphism (SNP, single-nucleotide polymorphism). However, as the length of the probe increases, the melting temperature (Tm) further increases, causing some probes to easily form local secondary space structures, thereby limiting the capture ability. Therefore, the application provides a thermal solution for long-sequence probe design. Kinetic calculation method achieves highly uniform capture and good repeatability of genomic target regions.

The hybridization process realizes the specific binding of the target DNA (T, target): RNA (P, probe) complementary sequence. The equilibrium constant R _eq of this dynamic reaction can be calculated by the standard free energy ΔG ^o , and the latter can be calculated by bisulfite treatment All conversions, or all non-transformations are assumed to be calculated.

R _eq =[TP]/[T][P]

Hybridization yield (Ψ) can be calculated by forming DNA:RNA complementary binding or single-stranded morphology. Considering that P is far in excess in the system, in order to simplify the calculation,

Ψ＝[TP]/([Tp]+[T])

R _eq ′≡[c] ^-Δn *R _eq

Here [c] refers to the original concentration of the hybridization probe, Δn refers to the change of T and P species during the reaction, _Req ′ is used to evaluate and reflect the thermodynamic equilibrium: _Req ′>>1, then Ψ tends to 1; Similarly, R _eq ′<<1, then Ψ tends to 0. Standard free energy after introducing the concentration parameter

ΔG ^o ≡-RTlog( _Req ′)=ΔG ^o +(Δn)RTlog([c])

The specificity of probes in this hybridization capture system can be defined as

ΔΔG ^o ＝(ΔG ^o (T ^f P)-ΔG ^o (T ^f )ΔG ^o (P))-(ΔG ^o (T ⁿ P)-ΔG ^o (T ⁿ P)ΔG ^o (P))

Here T ⁿ refers to the hybridization product against the target sequence; T ^f refers to the non-specific hybridization product.

In order to obtain a probe combination with high precision, it is necessary to select an appropriate target region for designing candidate probe sequences. A suitable target region is such that the difference between the free energy of binding (ΔΔG ^o ) of the candidate probe for a nucleic acid sequence derived from the target region and the nucleic acid sequence derived from the non-target region is greater than a specified threshold value of about 12 to 50kcal/mol. Preferably, the difference (ΔΔG ^o ) between the free energy of binding of the candidate probes for the target region and the nucleic acid sequence derived from the non-target region is about 20 kcal/mol or higher, about 50 kcal/mol or higher.

According to previous test results, a 120nt probe only needs to have a 60nt similar sequence with the capture sequence to capture the sequence. For the pre-designed probe sequence, according to 60nt as the window and 1nt as the step size, the sliding window is performed to obtain the target subsequence set of each probe, and the length of each subsequence is 60nt, and the distance between each subsequence and the probe sequence is calculated. ΔΔG ^o . The calculation of ΔΔG ^o between two sequences can refer to calculation methods known in the art, such as Zhang, D et al. Nature Chemistry 4, 208-214 (2012). Combine the ΔΔG ^o results of all probe sequences and subsequences, and take the minimum value, which can be used as the ΔΔG ^o threshold. In the present application, the range of ΔΔG ^o that can be used to select suitable target regions is about 12 kcal/mol or higher. Preferably, the ΔΔG ^o range for selecting a suitable target region is about 20 kcal/mol or higher, about 50 kcal/mol or higher.

ΔΔG ^o can be calculated by the following example:

Example 1 (inappropriate probe region, that is, the probe has a similar sequence on the genome, and the ΔΔG ^o between the similar sequence and the probe is less than the threshold, and the probe is filtered):

Probe region: chr9:132331252-132331371:

Similar sequence region: chr22:23908697-23908816:

Compare two sequences:

The ΔΔG ^o value (full non-methylation state, all C converted to T) calculated by the similar sequence and the probe sequence was 11.27;

The ΔΔG ^o value (full methylation status, only non-CpG C converted to T) calculated from the similar sequence and the probe sequence was 11.55. Regions of interest with a ΔΔG ^o range of less than about 12 were discarded.

Example 2 (inappropriate probe region, the probe has a similar sequence on the genome, the ΔΔG ^o between the similar sequence and the probe is less than the threshold, and the probe is filtered):

Probe region: chr22:50176001-50176120:

Similar sequence region 1: chr22:50176480-50176599:

Comparison of Similar Sequence 1 and Probe Sequence:

The ΔΔG ^o value (full non-methylation state, all C converted to T) calculated by the similar sequence 1 and the probe sequence is 2.28;

The calculated ΔΔG ^o value (full methylation status, only non-CpG C converted to T) of the similar sequence 1 and the probe sequence was 2.28. Regions of interest with a ΔΔG ^o range of less than about 12 were discarded.

Similar sequence region 2: chr22:50176259-50176403:

Comparison of Similar Sequence 2 and Probe Sequence:

The ΔΔG ^o value (full non-methylation state, all C converted to T) calculated by the similar sequence 2 and the probe sequence was 4.67;

The calculated ΔΔG ^o value (full methylation status, only non-CpG C converted to T) of the similar sequence 2 and the probe sequence was 4.67. Regions of interest with a ΔΔG ^o range of less than about 12 were discarded.

Example 3 (appropriate probe region, the probe does not have a longer similar sequence on the genome, and the probe is retained):

Probe region: chr1:849521-849640:

Similar sequence region: chr10:133011469-133011488:

Similar sequences (similar sequences extend left and right until the length of the probe sequence is the same) compared with the probe sequence:

The ΔΔG ^o value calculated from the similar sequence and the probe sequence (full non-methylation state, all C converted to T) is 50.77;

The ΔΔG ^o value (full methylation status, only non-CpG C converted to T) calculated from the similar sequence and the probe sequence was 50.85. Target regions with a ΔΔG ^o range greater than about 12, the probe region is retained.

Example 2

capture probe design

Figure 5 provides a reference example for illustration only, a double-stranded DNA fragment expected to be detected for methylation shown above, sorted in the direction of the arrow, including the original upper strand (CCGGCATGTTTAAACGCT) and the original lower strand (AGCGTTTAAACATGCCGG), Some of them assume that the cytosine (C) in all CpGs is methylated, marked with -mC. After the above-mentioned double-stranded DNA fragments are denatured and unwound into a single-stranded form, after bisulfite conversion treatment, the unmethylated (-mC) modified C in the original upper strand and the original lower strand is converted into uracil (U ), while the methylated C remains C. In the subsequent PCR amplification process, due to the complementary pairing of uracil (U) and adenine (A), the base paired with adenine (A) introduced in the PCR amplification of DNA is thymine (T). In PCR amplification, the target upper strand complementary strand (CTOT) complementary to the original upper strand with uracil (U) after bisulfite treatment is first formed, and the target upper strand complementary strand (CTOT) with the original upper strand after bisulfite treatment is formed. The target lower strand complementary strand (CTOB) to which the original lower strand of uracil (U) is complementary. In the subsequent PCR amplification process, the target upper strand (OT) transformed from the original upper strand and complementary to CTOT, and the target lower strand (OB) transformed from the original lower strand complementary to CTOB were formed. The comparison shows that the unmethylated C in the original upper strand and the original lower strand is replaced by T in the target upper strand and the target lower strand, while the methylated C (underlined) remains unchanged . According to this feature, the number and position of methylated C can be identified by measuring the C after bisulfite conversion treatment, so as to achieve the purpose of DNA methylation detection. For the sake of brief description, the above-mentioned process is expressed in the description herein as C being converted to T, C being replaced by T, C being replaced by T, and the like.

An ideal situation as shown in Figure 5, that is, the methylation status of the original strand is known. However, in actual operation, whether C in the target chain (OT, OB) is replaced by T is unknown, and it needs to be determined by detection, and in unknown cases, it is necessary to hybridize and capture the designed probes for OT and OB chains, so This application makes the following two assumptions:

1) All Cs in the target strand (OT, OB) are not modified by methylation, so after bisulfite conversion and PCR treatment, all Cs are replaced by T, and the corresponding complementary strands are designed accordingly (CTOT, CTOB).

2) All Cs in the target strands (OT, OB) are modified by methylation, so after bisulfite conversion and PCR treatment, all Cs remain unchanged, and the corresponding complementary strands ( CTOT, CTOB).

First, the capture probes of the present application can be designed based on target regions that are assumed to be unmethylated. Taking Figure 5 as an example, convert all C in the original upper strand to T as the first strand, and the sequence corresponding to Figure 5 is: T T GGTATGTTTAAA T GTT, and design a first probe complementary to the first strand; All C in the original lower strand is converted to T as the second strand, corresponding to the sequence in Figure 5: AGTGTTTAAATATGTTGG, design a second probe complementary to the second strand; at the same time, use the complementary strand of the first strand as the third strand , corresponding to the sequence in Figure 5 is: AACATTTAAACATACCAA, design the third probe complementary to the third strand; at the same time, according to the complementary strand of the second strand as the fourth strand, corresponding to the sequence in Figure 5 is: CC A ACATATTTAAAC A CT to design a fourth probe complementary to the fourth strand. In addition to the two target strands derived from the original strands, the probes of the present application are also designed for the complementary strands of the two target strands, achieving good coverage. Proven to improve capture performance, such as probe accuracy and reproducibility. It should be noted that the above-mentioned FIG. 5 is only an example for convenience of explanation, and the number of target chains to be selected is actually very large, and is not limited to the sequence in FIG. 5 .

Preferably, the capture probe of the present application can also be further designed according to the target region assumed to be fully methylated. In the case of CpG islands as the main body of methylation determination, only base sequencing is considered (for example, in Figure 5 The direction of the arrow) means that the base C in "CG" will be methylated, and in other cases, it is considered that methylation will not occur. Also take Figure 5 as an example, convert only non-CpG C in the original upper chain to T as the fifth chain, and the sequence corresponding to Figure 5 is: T C GGTATGTTTAAA C GTT, design the fifth chain complementary to the fifth chain Probe; only non-CpG C in the target lower strand (OB) is converted into T as the sixth strand, corresponding to the sequence in Figure 5: AGCGTTTAAATATGTCGG, the sixth probe is designed to be complementary to the sixth strand; simultaneously according to The complementary strand of the fifth strand is used as the seventh strand, corresponding to the sequence in Figure 5: AACGTTTAAACATACCGA, the seventh probe complementary to the seventh strand is designed; at the same time, the complementary strand of the sixth strand is used as the eighth strand, corresponding to The sequence in Fig. 5 is: CCGACATATTTAAACGCT, the eighth probe complementary to the eighth strand is designed.

In designing capture probes, the target regions of the present application are preferably about 10,000 or more.

Example 3

Performance testing of capture probes

In this application, the performance of the capture probe combination is detected through specific methylation standards, and the final probes used in the probe set are determined.

For 20% and/or 50% methylation standards (known in specific regions, the methylation level is 20% and/or 50% standard test samples), to detect the accuracy and repeatability of the capture probes of the present application .

accuracy

The detection deviation is calculated as the difference between the methylation level detected by the candidate capture probe combination and the actual (or theoretical) methylation level/the actual (or theoretical) methylation level. Suitable capture probe combinations have a detection fluctuation, i.e., the difference between the maximum and minimum values, of about 25% or less. More specifically, the difference=maximum value−minimum value.

When using the above formula to evaluate the methylation level of the sample to be tested, the collection of all probe combinations should cover more than 90% of the target region. Preferably, the collection of all probe combinations should cover more than 95% of the target area. Further preferably, the collection of all probe combinations should cover more than 99% of the target area. Even more preferably, the collection of all probe combinations should cover 100% of the target area.

Refer to the exemplary illustration in Figure 6. As the sequencing depth increases, multiple reads will cover a single CpG site, and different reads may have different methylation detection results for the same CpG site. . For example, for the CpG-2 site, reads 1-4 showed results that were positive for methylation (indicated by black dots ●), but reads 5-6 showed results that were negative for methylation (indicated by white dots ○ ), calculate the methylation status of all sites in all reads, instead of forcing a single site to qualitatively select a positive or negative status, which can avoid errors caused by artificial intervention in the methylation signal in the sequencing results.

repeatability

The repeatability RMSE is calculated as the mean squared error of the methylation levels detected by the candidate capture probe combination for two or more replicate measurements for a standard test sample with a specific methylation level of 20% and/or 50% . The reproducibility, ie, the median squared error of the methylation level between duplicate wells, for a suitable capture probe combination is about 9E-05 or less.

Optionally, the uniformity and bias of the combination of capture probes is tested.

Uniformity

The uniformity CV is calculated as,

where, for k capture probes in a capture probe combination, d _i represents the sequencing depth of the i-th probe,

Indicates the mean of the sequencing depth of all probes. The coverage uniformity CV of a suitable capture probe combination should be less than 1; preferably, the CV should be less than 0.5; more preferably, the CV should be less than 0.3; further preferably, the CV should be less than 0.2.

preference

The calculation method of preference R is,

Among them, for the m capture probes in the capture probe combination, x _i represents the sequencing depth of the i-th probe for the target strand (OT+OB),

Indicates the mean value of the sequencing depth of all probes for the target strand, y _i represents the sequencing depth of the i-th probe for the complementary strand (CTOT+CTOB), and y represents the average value of the sequencing depth of all probes for the complementary strand.

Among them, OT represents the target upper strand of the target region, CTOT represents the complementary strand of the target upper strand; OB represents the target lower strand of the target region, and CTOB represents the complementary strand of the target lower strand.

Optionally, the capture probes in the capture probe set are about 80 to about 120 bases in length. Optionally, the region of overlap between any two capture probes in the capture probe set comprises about 10 to about 110 bases. Optionally, the region of the capture probe set to which the capture probes are complementary does not contain 10 or more contiguous bases that overlap the repeat region. Repeated regions are described in what is known in the art, for example repeats described in repeatmasker.org.

Example 4

Construction of methylated standards

The current methylation standards come from samples obtained by whole genome amplification. However, in the process of obtaining "zero methylation standards" in the amplification process, it may appear that all cytosines in the standards are actually unmethylated, making the above samples After bisulfite conversion, there is no cytosine, and it is prone to large capture deviation, so it is not suitable as a standard for evaluating the performance of capture methods.

The present application provides a method for the construction of a methylation standard for a capture probe pair. For nucleic acid samples derived from human cell lines, treated with a methyltransferase (such as M.sssI) to obtain a "perfectly methylated standard (PC)", for the corresponding Nucleic acid samples, as "zero methylation standard (NC)". Methylation sequencing was carried out for "zero methylated standard" and "full methylated standard"; A specific region with a methylation level of 100% in the “Standards for Methylation” was used as the standard region. When the "zero methylation standard" and "full methylation standard" are mixed in any ratio, the methylation level in the standard area is the actual methylation level of the methylation standard in this application level. For example: after mixing 20% full methylation standard with 80% zero methylation standard, in the selected specific region, it can be regarded as the actual methylation level (also called theoretical methylation level ) is 20%.

The reaction conditions for the methyltransferase (eg M.SssI) enzyme are: react at 37° C. for 15 minutes, and react at 65° C. for 20 minutes. The left and right sides of Figure 1 and Figure 3 show the methylation measurement results of the "zero methylation standard" and "full methylation standard" of the present application. Through multiple measurements, the methylation level of NC is 0-0.002; the methylation level of PC is 0.97-1.00, and the methylation standard of this application is suitable for the evaluation of capture probes.

Among them, the complementary strand group means that the capture probe is only designed for the complementary strand (CTOT+CTOB), the target strand group means that the capture probe is designed for the target strand (OT+OB), and the double-strand group means the double-strand for the target strand and the complementary strand Design capture probes. Among them, OT means the upper chain of the target, CTOT means the complementary chain of the upper chain of the target; OB means the lower chain of the target, and CTOB means the complementary chain of the lower chain of the target.

Example 5

Performance results of this application capture probe combination

Take the 20% and 50% standard in the 20% and 50% ratio as an example, this standard can be used for the accuracy evaluation of different probe batches. Figure 1 shows the methylation measurement results of "20% standard" and "50% standard" of the present application. The result is as follows:

Table 1: 20% and 50% probe accuracy evaluation results

(1) Evaluation of the deviation between the theoretical methylation level and the actual test methylation level. The horizontal axis in Figure 3 represents the theoretical methylation level, and the vertical axis represents the actual test methylation level. The results are as follows: double-stranded probe design The average value of the measured methylation signal is closer to the theoretical methylation level; when the methylation level is 20%, the deviation between the average value of the methylation detection of the double strand, the target strand, and the complementary strand and the theoretical value (deviation=(detection value -theoretical value)/theoretical value) are respectively: 0.28, 0.32, 0.28; when the methylation level is 50%, the deviations between the average value of the methylation detection of the double strand, the target strand and the complementary strand and the theoretical value are respectively: 0.14, 0.15 , 0.13.

(2) Fluctuation evaluation between the theoretical methylation level and the actual test methylation level, the double-strand probe design has the smallest fluctuation of the measured methylation signal, and the maximum value of the three probes designed for the double-strand, target strand, and complementary strand The difference of minimum value, the fluctuation of 20% methylation level is 0.22, 0.24, 0.25, and the fluctuation of 50% methylation level is 0.22, 0.25, 0.27.

The uniformity of the capture probe combination is used to evaluate the uniformity of probe coverage on different target regions, and the CV range of the coefficient of variation. The horizontal axis of the figure below represents different methylation levels, and the vertical axis represents the sequencing depth. Figures 2A-2C show the uniformity measurement results of the three probes designed by the present application for the double strand, the target strand, and the complementary strand. The results showed that the uniformity of the double-strand probe design was better than that of the complementary-strand probe design alone, and was close to that of the traditional target-strand probe design.

The evaluation of repeatability, using the mean square error of detection of different methylation levels to evaluate the repeatability of different probe designs, the horizontal axis represents different methylation levels, the vertical axis represents the deviation between repeated samples, the smaller the value represents the detection The method is more stable.

Table 2: 20% and 50% probe repeatability evaluation results

边界boundary	理论甲基化水平theoretical methylation level	双链重复性duplex repeatability	目标链重复性target chain repeatability	互补链重复性Complementary strand repeatability
最小值minimum value	0.50.5	1.34E-051.34E-05	2.49E-052.49E-05	1.67E-051.67E-05
中值median value	0.50.5	7.03E-057.03E-05	1.23E-041.23E-04	9.16E-059.16E-05
最大值maximum value	0.50.5	2.19E-042.19E-04	3.54E-043.54E-04	3.04E-043.04E-04
最小值minimum value	0.20.2	1.55E-051.55E-05	2.60E-052.60E-05	2.51E-052.51E-05
中值median value	0.20.2	8.05E-058.05E-05	1.22E-041.22E-04	1.12E-041.12E-04
最大值maximum value	0.20.2	2.67E-042.67E-04	3.58E-043.58E-04	3.40E-043.40E-04

Figure 3 shows the repeatability measurement results of the three probes designed by the present application for the double strand, the target strand, and the complementary strand. The results showed that the reproducibility of the double-strand probe design was better than that of the complementary-strand probe design alone, and was close to that of the target-strand probe design.

For the target strand, the median estimated repeatability was 1.22E-04 for the 20% methylated standard and 1.23E-04 for the 50% methylated standard; for the complementary strand, 20% methylated Median repeatability of assessments for methylated standards was 1.12E-04, median repeatability for assessments of 50% methylated standards was 9.16E-05; for double strands preferred for this application, 20% methylated standards assessed The median repeatability value of 8.05E-05 was 8.05E-05, and the repeatability median value of 50% methylation standard evaluation was 7.03E-05.

The capture strand preference evaluates the depth of capture of the target strand (OT+OB) and complementary strand (CTOT+CTOB) by different probes. The horizontal axis of the figure indicates the coverage depth of the target strand, and the vertical axis indicates the sequencing depth of the complementary strand. The results showed a lower strand preference R^2 for capture using double-stranded probes.

Figures 4A-4C show the bias measurement results of the three probes designed by the present application for the double strand, the target strand, and the complementary strand. The results showed that the design bias of the double-strand probe was better than that of the complementary-strand probe design alone, and was close to that of the traditional target-strand probe design.

The foregoing detailed description has been offered by way of explanation and example, not to limit the scope of the appended claims. Variations on the presently recited embodiments of this application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims

A nucleic acid molecule combination, the nucleic acid molecule combination comprising at least one nucleic acid probe set covering a target region of a nucleic acid to be detected, characterized in that the nucleic acid probe set at least comprises: (1) complementary to the first strand The first probe of the first strand is the sequence of the target region after base substitution; (2) the second probe complementary to the second strand, the second strand is the complementary sequence of the target region The sequence of the region after base substitution; and contains any one or both of the following two probes: (3) a third probe that is complementary to the third strand, and the third strand is complementary to the first strand complementary; (4) a fourth probe complementary to a fourth strand which is the complementary sequence of the second strand.
The combination of nucleic acid molecules according to claim 1, wherein the free energy of binding of the nucleic acid molecules in the combination of nucleic acid molecules to the nucleic acid sequence derived from the target region and the free energy of binding to the nucleic acid sequence derived from the non-target region The difference is greater than a certain threshold value, said certain threshold value being about 12 to 50 kcal/mol; preferably, said certain threshold value being preferably about 20-30 kcal/mol.
The combination of nucleic acid molecules according to any one of claims 1-2, wherein the base substitution comprises a nucleic acid sequence in which cytosine is replaced by thymine or uracil through chemical and/or biological processes.
The nucleic acid molecule combination according to any one of claims 1-3, wherein the nucleic acid probe set further comprises: (1) a fifth probe complementary to the fifth strand, the fifth strand being The sequence of the target region without base substitution; (2) the sixth probe complementary to the sixth strand, the sixth strand being the sequence of the complementary region of the target region without base substitution; (3) A seventh probe complementary to the seventh strand, the seventh strand being complementary to the fifth strand; (4) an eighth probe complementary to the eighth strand, the eighth strand being the complementary sequence of the sixth strand .
The nucleic acid molecule combination according to any one of claims 1-4, wherein the detection result of the standard substance of the 20% methylation level in the nucleic acid molecule combination meets the following index: the fluctuation is 25% or more Low, and/or repeatability is 9E-05 or lower; preferably, the fluctuation is the difference between the maximum value and the minimum value of the detection result, and the repeatability is the median value of the mean square error of the methylation level between multiple wells .
The nucleic acid molecule combination according to any one of claims 1-5, the length of the nucleic acid molecule in the nucleic acid molecule combination is about 80 to about 120 bases, any two nucleic acid molecules in the nucleic acid molecule combination The region of overlap comprises about 10 to about 110 bases and/or the region to which the nucleic acid molecules in the combination of nucleic acid molecules are complementary does not comprise 10 or more contiguous bases overlapping the repeat region.
Use of the nucleic acid molecule combination according to any one of claims 1-6 in the preparation of human tumor gene detection preparations.
The use according to claim 7, wherein the detection preparation is used to detect the base modification level of the target region; preferably, the base modification comprises methylation modification.
The application according to any one of claims 7-8, wherein the human tumors are from homogenous tumors, heterogeneous tumors, blood cancers and/or solid tumors; preferably, the Human tumor One or more cancers from the following group: brain cancer, lung cancer, skin cancer, nasopharyngeal cancer, throat cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, bowel cancer, rectal cancer , thyroid cancer, bladder cancer, kidney cancer, oral cancer, stomach cancer, solid tumors, ovarian cancer, esophagus cancer, gallbladder cancer, biliary tract cancer, breast cancer, cervical cancer, uterine cancer, prostate cancer, head and neck cancer, sarcoma, thoracic malignancies (except lung), melanoma, and testicular cancer.
A standard substance nucleic acid molecule used as the accuracy of the degree of base modification detected in the application of any one of claims 7-9, characterized in that, the standard substance nucleic acid molecule comprises a base modification degree of about 0% candidate Region, the total length of the candidate region is about 1bp-about 10000bp.
The standard nucleic acid molecule according to claim 10, wherein the standard nucleic acid molecule is selected from one or more of the following cell lines: GM24385, GM12878, GM12877, and GM24631.
The standard nucleic acid molecule according to any one of claims 10-11, wherein the degree of base modification comprises the degree of methylation of cytosine in the candidate region.
The standard nucleic acid molecule according to any one of claims 10-12, characterized in that, the standard substance without base modification and all nucleic acid molecules or standards that have been processed through base modification are mixed in a predetermined ratio , to obtain methylated standards with a predetermined degree of base modification.
The nucleic acid molecule according to claim 13, wherein the proportion of all the nucleic acid molecules or standards subjected to base modification is 20% or 50%.
The nucleic acid molecule according to any one of claims 10-14, wherein the base modification treatment comprises contacting the nucleic acid molecule with a methyltransferase.