CN101130823A - A reverse hybridization coupling extension DNA sequencing method - Google Patents

Abstract

The invention discloses a reverse hybridization coupling extension DNA sequencing method, in which an unknown DNA template is hybridized in parallel with multiple probes whose 3' ends are 3 or more known bases, and the labeled bases are extended, Detect whether there is marker extension, determine whether the unknown DNA template has corresponding four (or more) consecutive base information according to the principle of base complementarity, and splice out the DNA sequence information to be tested; including repeated hybridization, extension and detection steps . The method of the invention adds an extension step to the traditional hybridization sequencing method, which can solve the problems of false positives and high cost, and realize high throughput, low cost, high sensitivity and rapid determination of short-segment DNA sequences.

Description

A reverse hybridization coupling extension DNA sequencing method

technical field

The present invention relates to a DNA sequencing method, in particular to a DNA hybrid sequencing method.

Background technique

With the deepening of genome research, it will become possible to understand the differences in life, the rules of disease occurrence and development, and the interaction between drugs and living organisms at the genetic level. Although numerous factors contribute to the occurrence of disease, genetic mutations (single nucleotide polymorphisms, methylation, etc.) are widely recognized as an important intrinsic factor. In terms of basic research, gene mutation sites are helpful for the precise positioning of genomes, the study of genetic laws of disease genes, and the cloning of disease-causing genes; in terms of applications, it is of great value to directly find disease-susceptible gene mutation sites. Most complex diseases, such as cancer, diabetes, cardiovascular disease, depression, asthma, etc., are affected by many genes and environmental factors. Through large-scale identification and detection of mutant genotypes in a large number of genomic samples of a specific disease, Information on genotypes associated with the disease is available. More importantly, by screening drug-sensitive gene mutation sites, it will provide a basis for realizing individualized drug use in the future. By discovering the gene mutation sites of disease susceptibility, people can prevent diseases early and achieve the purpose of avoiding the occurrence of diseases.

Therefore, just after the draft of the human genome was drawn, finding the gene mutation sites in the human genome immediately became one of the main tasks of the Human Genome Project. There are currently many methods for mutation and SNP detection, such as DNA sequencing, restriction length polymorphism, single-strand conformation polymorphism, pyrosequencing, and allele-specific oligonucleotides acid hybridization etc. Although these technologies can complete the detection of mutations or nucleotide polymorphisms to a certain extent, they cannot simultaneously determine the sequence of the target fragment in an all-round way. A method capable of comprehensive sequence detection, that is, a whole genome sequence determination method. However, the cost of sequencing the whole genome is too high. The cost of sequencing the first human genome was about 1 billion US dollars, which has been reduced to about 20 million US dollars at present. Therefore, the progress of functional genome research is still limited by DNA sequencing technology, and a substantial reduction in the cost of DNA sequencing will greatly promote the research of life sciences and medicine, and even bring about revolutionary changes.

In addition to improving the existing electrophoresis-based DNA sequencing technology, new sequencing technologies currently under development mainly focus on non-electrophoretic means. Generally speaking, such technologies can be divided into four categories: the first type is sequencing by synthesis, which detects during the process of adding bases to the extended DNA chain; the second type is hybrid sequencing, by preparing a set of high-density The hybridization signal of the oligonucleotide microarray chip is used to identify the sequence of the target gene; the third type is molecular imaging technology, a series of technologies that can be sequenced at the single-molecule level; the last type of technology is to induce DNA molecule winding Through very small holes, the bases are read out by means of electronic or optical methods in this process, also known as nanopore sequencing.

The traditional hybridization sequencing method is to immobilize a series of ^4k sequences with a length of K bases on the chip, then hybridize the labeled DNA template to be tested, and determine the hybridization sequence spectrum of the sequence by detecting the hybridization signal. If a long DNA sequence is to be determined, a large amount of synthesized probes is required, and the cost is multiplied. More importantly, this method has the disadvantage of high false positives.

Contents of the invention

Technical problem: The purpose of the present invention is to provide a reverse hybridization coupling extension DNA sequencing method, which can overcome the defects of high false positives in the traditional hybridization sequencing method, and solve the problem of large amount of probe synthesis and high cost in the existing method, Achieve high-throughput, low-cost, high-sensitivity and rapid determination of short-segment DNA sequences.

Technical solution: The present invention adopts a reverse hybridization coupling extension DNA sequencing method to determine the base information of the unknown DNA template by hybridizing the DNA template with multiple probes in parallel and extending the labeled bases, and by detecting whether there is a marker Extension, according to the principle of base complementarity, determine whether the unknown DNA template has corresponding four (or more) continuous base information. That is, it is achieved by repeating the steps of "hybridization-extension-detection" on the DNA template, that is, adding an extension step to the traditional hybridization sequencing method, which can solve the problems of false positives and high cost.

The present invention adopts following technical scheme:

A reverse hybridization coupling extension DNA sequencing method, characterized in that the DNA sequence is determined by hybridization primers with known bases at the 3' end in parallel with the DNA sequence to be tested through multiple hybridization, extension, and detection, including Follow the steps below:

Step 1) The hybridization primer with three or more known bases at the 3' end is heated in the hybridization solution with the immobilized unknown DNA template, and then cooled and annealed for hybridization;

Step 2) After washing the hybridization solution with washing liquid, the labeled monomer is catalyzed by the polymerase, and one or more bases are extended at the 3' end of the hybridization primer; the information of the extended base is read by a chip scanner to determine A sequence containing several bases on an unknown DNA template;

Step 3) Elute the hybridization primers with extended bases from the unknown DNA template with a denaturing solution, and then hybridize the hybridization primers with different known base sequences at the 3' end to the unknown DNA template according to step 1) and perform step 2), Determine the different sequences containing several bases on the unknown DNA template; repeat the above process with different hybridization primers until the DNA sequence determination is completed.

The hybridization primer refers to an oligonucleotide fragment that can and can only hybridize with a general specific sequence in all DNA templates. In the process of DNA template preparation, a general sequence complementary to a specific hybridization primer is introduced by ligation or in the amplification process, wherein the general sequence introduced should be different from any sequence fragment in the template, that is, only the A general sequence can hybridize with the specific hybridization primer.

In the hybrid primer of the present invention, 3 or more bases at its 3' end are known bases, preferably 3 to 5 bases are known bases, and most preferably 3' ends are 3 known bases. Base; 5' end with 7 or more bases, preferably 7 to 13 bases. In the most preferred case, that is, the 3' end is three known bases, and the sequence characteristics of the hybridization primer can be expressed as 5'(N)nXYZ3', where N is four bases T, A, G, and C. Any of the defined ones, or can be replaced by universal base I, n is an integer of 7 to 13; X, Y, and Z are all selected from T, A, G, and C. A definite base. In this way, the sequence XYZ has 4 ³ =64 different combinations, that is, the method of the present invention can be realized under the conditions of synthesizing at least 64 different hybridization primers; and the length of the hybridization primers can only contain 10-16 bases.

The unknown DNA template refers to the DNA sequence to be tested or its amplified method for increasing the amount of the target sequence of interest in the genome by conventional PCR, rolling circle amplification, solid phase amplification, and bridge amplification. sequence fragment. Amplification can be single-plexed, that is, one target segment is amplified at a time, or multiplexed, that is, multiple target segments are amplified at a time. Unknown DNA templates can be immobilized on flat substrates by chemical or physical methods, and can also be immobilized on carriers such as "96, 384-well plates" or various modified beads. Multiple DNA templates can also be immobilized at the same time, including the immobilization of a single DNA template.

Step 1) is the hybridization step. According to the method of the prior art, the hybridization primers can be dissolved in high temperature conditions (about 60° C.) and heated for a short time, and then annealed at low temperature (about 4° C.) to complete the hybridization process.

Step 2) is the extension step, when the hybridization primer is hybridized with the immobilized unknown DNA template, by extending one or more labeled monomers (four kinds of A, G, C, and T) on the hybridization primer, and passing through the chip scanner By detecting the extended bases, the base information of the unknown DNA template in the extension can be determined at different positions to be detected. At this time, the sequence containing several bases in a short DNA sequence on a certain point to be tested (monoclonal template DNA fixed on the substrate) in the unknown DNA template has been determined, that is, it is consistent with one or more bases contained in the labeled monomer. A base and a sequence composed of 3 or more known bases at the 3' end of the hybridization primer form a base-complementary DNA sequence.

The extension of the labeled monomer adopts a known general method, that is, in the buffer, four different labeled monomers, such as dATP (ddATP), dCTP (ddCTP), dGTP (ddGTP) and dUTP (ddUTP), are added. Under the action of enzyme, it is extended at 4-10°C for 5-15 minutes, so that the 3' end of the hybridization primer is extended with the labeled base.

The labeling monomer refers to the base monomer (A, G, C, T) containing groups or particles that can be detected directly or indirectly, and the 3' is dideoxy or blocked by a cleavable group. Detectable groups or particles, ie labels, such as modified fluorophores, biotin and quantum dots, or unmodified monomers that generate light signals during the reaction. The marking monomers can be monochromatic, that is, the four monomers are all labeled with the same marker, or polychromatic, that is, the four monomers are labeled with different or not completely identical markers. For example, the labeled monomer can be four different fluorescently labeled dATP (ddATP), dCTP (ddCTP), dGTP (ddGTP) and dUTP (ddUTP).

The extension of the labeled monomer is the extension of the four labeled monomers (A, G, C, T). If the labeled monomers are monochromatic, for any hybridization primer, the different four monomers (A, G, C, T) of the same label should be extended separately and read the base extension information, and the different monomers should be read After extraction, the marker should be removed, and then another marker monomer should be extended, so that the chip scanner can read the information of different bases. It is easy to understand that if the labels of the four monomers are not the same, the four monomers can be simultaneously extended and the information of the extended base can be read.

The extension of the monomer can be extended by one base, or multiple bases can be extended by multiple steps. In multi-base extension, one base is extended at a time, and the extension information of the sub-base occurs at the point to be tested on the unknown DNA template by detection; then the label removal method is used, such as the method of cutting off the fluorescence , and then extend the next base, or use marker superposition, such as fluorescence superposition, to determine that multiple base extensions have occurred at the points to be tested on the unknown DNA template.

After completing the determination of several base sequences in the DNA sequence at a certain point to be tested, the hybrid primers with extended bases can be eluted from the unknown DNA template with a denaturing solution. The denaturation solution contains components capable of lowering the DNA denaturation temperature, such as urea, NaOH, ethylene glycol, glycerol and formamide.

After the hybridization primer for extending the labeled monomer is separated from the DNA template, the method from step 1) to step 2) can be repeated, and another hybridization primer with a different known base sequence at the 3' end is hybridized with the unknown DNA template to extend the monomer. , and read out the base sequence information of the unknown DNA sequence on the point to be tested. With different hybridization primers, cycle the above-mentioned "hybridization-extension-detection" process until the sequence spectrum of the unknown DNA template is measured, or ^4K species (K is the number of known bases at the end of the hybridization primer) hybridization primers have passed through the above steps , to complete the DNA sequence determination.

Beneficial effect: compared with the prior art, the present invention has the following advantages:

1. The greatest advantage of the present invention is correctness and reliability. In the traditional hybridization sequencing method, if there is one or more base mismatches at the end, it is also possible to read the signal, but the read signal is wrong. And the method of the present invention, only reads the last several bases of 3 ' terminal, and adds extension step (polymerase is very sensitive to 3 ' terminal mismatched base) has increased correctness greatly.

2. The sequencing method of the present invention is low in cost, and at least only needs to synthesize 64 hybridization primers (about 10-15 bases in length each), common polymerase, fluorescently labeled dNTP and a chip scanner to perform large-scale sequencing. DNA sequence determination.

3. The present invention can realize high-throughput DNA sequencing. High-density DNA chips prepared by methods such as polony or emulsion PCR can easily read the sequences on these chips by using reverse hybridization coupling extension DNA sequencing method.

The method proposed by the present invention can solve the problems of false positive and high cost due to the addition of an extension step. Moreover, the application of hybridization extension sequencing on the chip can reach a high-throughput level. In addition, the present invention does not need to remove hybrid primers by destroying or cutting markers, and can establish a fast, accurate and cheap genome sequence determination method.

The present invention will be described in further detail below in combination with specific embodiments.

Description of drawings

Fig. 1 is the schematic diagram of reverse hybridization coupled extension DNA sequencing method of the present invention

Among them: 1 is an unknown DNA template; 2 is a hybridization primer; 3 is a fluorescent group; 4 is a substrate; i, j, k, l, m are points to be measured on the chip respectively.

Figure 2 is a schematic diagram of DNA sequence splicing

Among them: 4 is the substrate; 1 is the unknown DNA template fixed on the substrate, 2 is the hybridization primer; 3 is the extended labeled base; e is the sequence obtained through splicing.

Detailed ways

Embodiment one

A DNA sequence to be tested is amplified by conventional PCR and fixed on an acrylamide substrate to make an unknown DNA template. Prepare 64 hybrid primers, with three known bases at the 3' end and 8 bases at the 5' end. The sequence characteristics of the hybrid primers can be expressed as 5, NNNNNNNNNXYZ 3', where N is four bases T, A , any one of G, C not limited, or can be replaced by universal base I, X, Y, Z are all selected from T, A, G, a certain base in C, such as NNNNNNNNACG, NNNNNNNNGAG etc., XYZ constituted 64 different sequences in the hybridization primers.

1) Hybridization: hybridize the hybridization primer NNNNNNNNACG with the immobilized unknown DNA template. Dissolve the hybridization primers at 100uM concentration in 1X hybridization solution, take an appropriate amount (submerge the template to be tested completely) and place the hybridization primers in the template to be tested, denature at 60°C for 2 minutes, then anneal at 4°C for 3 minutes , washed with 1 times 4°C stored polymerase buffer (10mM Tris-HCl (pH 7.5), 5mM MgCl2, 7.5mM DTT) for 30 seconds to complete the hybridization process.

2) Extension: in 1X EcoPol buffer (10mM Tris-HCl (pH 7.5), 5mM MgCl2, 7.5mM DTT), add four kinds of labeled monomers dATP (ddATP), dCTP (ddCTP), dGTP ( ddGTP) and dUTP (ddUTP), under the action of polymerase (Klenow fragment (3'-->5'exo-)), an extension reaction occurs at 4°C for 10 minutes to extend the 3' end of the hybridization primer by one labeled bases.

3) Detection: read the information of the extended bases by a chip scanner, and determine the sequence information containing 4 bases at a point to be tested in the unknown DNA sequence. That is, it can be known whether the sequence contains a sequence of four bases in total that is complementary to the known three bases GCA and one base extended on the hybridization primer.

As shown in Figure 1, the unknown DNA template 1 is immobilized on the solid-phase carrier acrylamide substrate 4, the hybridization primer NNNNNNNNACG 2 hybridizes with the DNA template 1, and after adding the labeled monomer, the hybridization primer 2 undergoes a labeled monomer extension reaction, which can be known through detection In this extension, among the points i, j, k, l, and m to be tested at different positions, the unknown DNA templates at i, j, l, and m respectively contain bases complementary to the sequences ACGT, ACGC, ACGG, and ACGA. sequence of bases.

4) After the detection of the hybrid primer NNNNNNNNACG, repeat steps 1) to 3) for the rest of the hybrid primers in turn, and detect the remaining 63 hybrid primers at most until the sequence profile of each point to be tested is obtained. By analyzing these sequence profiles After data sorting and splicing, these sequences can be read out.

The splicing of DNA sequences is shown in Figure 2. A DNA template 1 is immobilized on the substrate 4, and a base 3 with a label is extended after the hybridization primer 2 is hybridized. There are 19 hybridization primers in a sequence in the figure, and the sequence e can be obtained after splicing. Then there is a DNA sequence complementary to the sequence e base in the DNA sequence to be detected.

Embodiment two

Whole-genome sequencing of a single human by reverse hybridization-coupled extension DNA sequencing

DNA templates can be prepared as follows:

1. Extract the human genome.

2. DNA fragmentation. Use ultrasonic treatment to make the average size of DNA around 50bp.

3. Add linker primer a. Firstly, the fragmented genomic DNA is blunted with polymerase, and its 3-terminus is treated with a protruding A. Then, under the action of ligase, the linker primer with Mme I endonuclease recognition site is connected to the small fragment of DNA.

4. Enzyme digestion treatment. Under the action of MmeI endonuclease, the whole genome was cut into a genome library with a fragment size of 19 bp.

5. Same as step 3, add adapter primer b to the other end.

6. Microemulsion PCR amplifies a single-clonal genome library.

7. Whole genome microarray fabrication. Fix the amplified product from the previous step on an acrylamide substrate.

The prepared immobilized DNA template was subjected to hybridization-extension sequencing using the method of the present invention, and the sequencing method was the same as in Example 1. After the hybrid sequences are assembled and spliced, the whole genome information is obtained.

Embodiment three

RNA Expression Profiling by Reverse Hybridization Coupled with Extended DNA Sequencing

DNA templates can be prepared as follows:

1. Extract mRNA from normal cells and diseased cells respectively, and use magnetic beads to fix 30 T nucleotide chains to purify mRNA so that the magnetic beads can absorb mRNA.

2. Reverse transcribe into cDNA, and use RNaseH, DNA polymerase, etc. to synthesize the second strand.

3. Digest the product of step 2 with an endonuclease (Sau 3AI) that recognizes four bases. Wash the magnetic beads with binding buffer.

4. Add adapter primer A (with endonuclease Acu I recognition site on the primer) under the effect of ligase.

5. Digest the product of step 4 with Acu I. A cDNA library of adapter primer A followed by 16 unknown sequences to be tested was obtained.

6. Add another linker primer B. Amplification was then performed using emulsion PCR.

7. The amplified product is immobilized on the chip.

The prepared fixed DNA template is subjected to hybridization-extension sequencing using the method of the present invention, and the sequencing method is the same as

Embodiment one. After data analysis, the DNA sequence to be determined is obtained by splicing.

Embodiment four

Reverse Hybridization Coupled Extension DNA Sequencing Method to Detect Human Genome Containing

The SNP site number is rs 11053646 a DNA fragment

Design a pair of PCR primers, one of which is modified with an acrylamide group. Use PCR primers to amplify a DNA sequence fragment containing SNP site number rs 11053646 in human samples (such as blood, saliva, etc.). The PCR product is fixed on a glass slide by polymerization method, and the unfixed PCR chain and other impurities are removed by denaturation and electrophoresis methods to obtain a pure DNA chain.

Using the method described in Example 1, hybridize the hybridization primers with the above-mentioned chip, insert fluorescently labeled A, G, C, T under the extension reaction conditions, and wash the hybridized primers after the data is read. And repeat hybridization, extension and detection for other hybridization primers until all hybridization primers complete the hybridization-extension process, and sequence spectrum analysis is performed on the sequences corresponding to different samples, and the base situation of the SNP site numbered rs 11053646 can be known .

Claims (10)

1. sequencing method for reverse hybridized coupling extended DNA is characterized in that dna sequence dna determines terminally to realize through repeatedly hybridizing, extend, detecting with dna sequence dna to be measured abreast for the hybridized primer of known base by 3 ', comprises the steps:

Step 1) 3 ' end is that the hybridized primer and the unknown dna profiling of fixed of known base more than three or three heats in hybridization solution, and cooling annealing is hybridized then;

Step 2) with behind the clean hybridization solution of washing lotion, labeled monomer is under the catalysis of polysaccharase, in the one or more bases of 3 ' terminal extension of hybridized primer; Read the information of extending base by chip scanner, determine the sequence that comprises several bases on the unknown dna profiling;

Step 3) will be extended hybridized primer wash-out from unknown dna profiling of base with sex change liquid, again that 3 ' terminal known base sequence is different hybridized primer set by step 1) with unknown dna profiling hybridization and carry out step 2), determine the different sequences that comprise several bases on the unknown dna profiling; Repeat said process with different hybridized primers, up to finishing determined dna sequence.

2. dna sequencing method according to claim 1 is characterized in that described hybridized primer 5 ' end has 7 or 7 above bases.

3. dna sequencing method according to claim 2 is characterized in that described hybridized primer 3 ' end is 3 known bases, and 5 ' end has 7～13 bases, and the hybridized primer sequence signature is 5 ' (N) _nXYZ3 ', wherein for N is four kinds of base T, A, G, any one that does not limit among the C, or universal base I, X, Y, Z be for being selected from T, A, G, the arbitrary definite base among the C, n is 7～13 integer.

4. dna sequencing method according to claim 1, it is characterized in that described unknown dna profiling, be PCR, rolling circle amplification, solid-phase amplification or bridge-type amplification method, increase dna sequence dna to be measured or sequence fragment after the aim sequence amount in the genome by routine.

5. dna sequencing method according to claim 1 is characterized in that in the step 1), and described unknown dna profiling is fixed on the planar substrates by chemistry or physical method, or on " 96,384 orifice plate ", perhaps on the pearl carrier of various modifications; Fixedly the single DNA template is perhaps fixed a plurality of dna profilings.

6. dna sequencing method according to claim 1 is characterized in that described labeled monomer, is meant that comprise on the base monomer (A, G, C or T) can be directly or the group of indirect detection or particle, and 3 ' by two deoxidations or can be cut group and be sealed.

7. dna sequencing method according to claim 6 is characterized in that described detectable group or particle, for modify fluorophor, vitamin H or quantum dot, the optical signal that the monomer of perhaps not modifying produces in reaction.

8. dna sequencing method according to claim 6 is characterized in that described labeled monomer is four kinds of fluorescently-labeled dATP of difference (ddATP), dCTP (ddCTP), dGTP (ddGTP) and dUTP (ddUTP).

9. dna sequencing method according to claim 1 is characterized in that containing in the described sex change liquid urea, NaOH, ethylene glycol, glycerol or the methane amide component that can reduce the DNA denaturation temperature.

10. dna sequencing method according to claim 1 is characterized in that determining the dna sequence dna of unknown dna profiling by the base complementrity principle according to known base of hybridized primer and the base of being extended.

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