CN106591425A - Method of multiple-target detection of nucleic acid indicator based on ligation reaction - Google Patents
Method of multiple-target detection of nucleic acid indicator based on ligation reaction Download PDFInfo
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Abstract
The invention provides a method for simultaneously conducting multiple detection of specific nucleic acid indicator in a biological sample, wherein an RNA indicator comprises an RNA expression quantity and sequence information, DNA features comprise copy numbers and base variations. The method comprises the steps of conducting hybridization and connection between nucleic acid molecules in the biological sample and multiple probes, adding a forward primer compatible to a sequenator to conduct PCR database construction and sequencing and read sequence information, and finally using biological information to analyze abundance of all the probes representing specific RNA sequences so as to determine characteristics of the nucleic acid. The method of multiple-target detection of nucleic acid indicator based on ligation reaction is characterized in that parallel detection of multiple kinds of RNA transcript features and DNA variation can be conducted with low cost.
Description
Technical field
The present invention relates to molecular biology, it is qualitative with molecular method quantification to be related to nucleic acid sequencing, quantitative to RNA and DNA molecular especially with high-flux sequence method.
Background technology
Used as the index of the drug reaction for assessing normal living process, pathological development and to treating, biomarker can be used as the effective surrogate end point for distinguishing morbid state, progression of disease, the prognosis being estimated to clinical intervention and prediction.Biomarker is in extensive range, not only including the index of cellular level, protein level and metaboilic level, further comprises the transcriptional level difference and somatic mutation of specific gene.
Present analysis genetic transcription rna expression amount mainly has two detection directions of rna transcription group level and single-gene rna expression.The detection of rna transcription group generally uses high flux RNAseq either expressing gene chip or numeral expression spectral technology；The detection of single-gene transcriptional level relies primarily on reverse transcription-fluorescent quantitative PCR technique（RT-qPCR）1.Current study show that rna expression level often needs a gene incessantly as effective biomarker, but need the expression of related multiple genes as composite index, such as breast cancer relapse index 21gene of ER+2Or 70gene3.The gene expression detection of transcript profile level is generally used for the screening of the biomarker without assumed condition, but because its higher RNA mass and quantitative requirement are not appropriate for as Clinical detection.RT-qPCR is often used for the rna expression detection of small throughput as the goldstandard of detection rna expression level, is not suitable for the horizontal joint-detection of multiple gene transcription.
The amplification technique of multiple join dependency（muitiplex ligation-dependent probe amplication,MLPA）Moderate fluxes gene tester, up to 45 kinds different nucleotide sequences can be detected and quantitative analyses in same reaction tube4.MLPA technologies have been applied to many fields, such as single nucleotide polymorphism and detection in Gene Mutation, genetic fragment disappearance and repetition, numerical abnormalities of chromosomes, gene methylation detection and mRNA analyses5-9.But due to fragmentation assay of its detection technique based on capillary electrophoresis, so its Multiple detection power limitations is in 45 weights（Clip size at least 4bp difference）, and can only the change of semi-quantitative analyses copy number or rna expression amount, its quantitative sensitivity is high.
The present invention in the technical foundation of MLPA probe principles using high-flux sequence replace capillary electrophoresis fragmentation assay, eliminate PCR primer clip size restriction and can be with accurate quantitative analysis rna expression amount and DNA copy number and nucleotide variation.From detection rna expression amount aspect analysis, the conservative based on nucleotide sequence this invention can detect in theory 20,000 all gene expression amounts of human body and different spliced bodies in same tube reaction.Relative to qRT-PCR, the advantage of the invention is also resided in and has used hybridization probe and unified PCR primer, the efficiency difference between PCR primer can be effectively eliminated, so as to reach more accurately relative expression quantity.The left end probe of the invention and the hybridization sequences of right-hand member probe only need 25-35bp, as long as so the cDNA length of reverse transcription is not less than 70bp it is ensured that detection rna expression amount, the serious RNA that degrades in FFPE paraffin embedding samples can be detected in theory, so detecting that the rna expression of pathology sample is especially suitable when target gene number is more.In addition the RNA initial amounts of the present invention can reach 1ng, greatly reduce requirement of many target gene detection of expression to RNA sample sizes and quality.
The content of the invention
The invention provides a kind of probe combinations, at least include two fragments of left probe and right probe, as shown in Figure 1 for the probe of specific nucleic acid template.Far Left probe includes 5 ' universal sequences and 3 ' specific hybridization sequences, and rightmost probe includes 5 ' the specific hybridization sequences and 3 ' universal sequences of phosphorylated modification；It is closely adjacent without base breach between probe, can hybridize simultaneously in same single nucleic acid strands template, adjacent probe can be connected as same nucleic acid molecules by nucleic acid ligase catalysis；5 ' universal sequences and 3 ' universal sequences can be the joint sequence compatible with sequenator or biochip, including but not limited to illumina HiSeq, MiSeq, NextSeq microarray datasets, Life Ion microarray datasets, Illumina or Affymetrix chip platforms；Universal sequence can select all or part of joint sequence；Left and right probe can add 8-15 randomized bases between universal sequence and hybridization sequences（dN）8-15, shown in Fig. 1（dN）8-15It is added in left probe.
The invention also discloses the method that a kind of combination probe combinations as described above detect multi-target rna expression amount, ultimate principle is as shown in Fig. 2 main implementation method is as follows：
（1）Preparation can embody the total nucleic acid of biological specimen RNA characteristic informations；（2）The multiple probe group matched with target RNA transcript is added in total nucleic acid；（3）Hybridize at a certain temperature and excite coupled reaction using nucleic acid ligase；（4）With（3）The connection product of middle gained is template, the universal primer performing PCR reaction matched using 5 ' universal sequences described in Fig. 2 and 3 ' universal sequences；（5）Will（4）Middle PCR primer purification, using the sequenator compatible with universal linker sequence or chip hybridization scanner the sequence abundances information of all probe connection products is obtained；（6）Informatics Method determines the expression and sequence information of different rna transcription sheets.
The total nucleic acid that biological specimen RNA information is embodied in the method can be the RNA of extracting directly, or extract the cDNA obtained by reverse transcription after RNA.If the total nucleic acid for preparing is RNA, then specific base sequence should be with the base sequence reverse complemental of corresponding rna transcription sheet in probe groups, and nucleic acid ligase used should have single stranded DNA nicking coupled reaction ability, including but not limited to T4 DNA Ligase and SplintR Ligase in catalysis RNA-DNA hybridization chains；If the total nucleic acid for preparing is cDNA, then specific base sequence should be consistent with the base sequence of correspondence rna transcription sheet in probe groups, and nucleic acid ligase used should have single stranded DNA nicking coupled reaction ability in catalytic dna-DNA hybridization chain, including but not limited to T4 DNA Ligase, T3 DNA Ligase, Taq DNA Ligase, T7 DNA ligase, Taq DNA liagase, 9 ° of N DNA Ligase and Ligase-65.In addition, the purification of probe connection product can may be based on other affine mode purification by clip size principle purification, way of purification is included but is not limited to：（1）If the total nucleic acid for preparing is RNA, can be by being marked with Oligo（dT）N（15≤N≤35）Magnetic bead or affinity column purifying RNA-probe double-strand connection product complex, can effectively remove the adverse effect of free probe；（2）If the total nucleic acid for preparing is cDNA, select with biotinylated oligo（dN）6Or oligo（dT）N（15≤N≤35）Total nucleic acid cDNA, and the magnetic bead using Avidin labelling or affine column purification cDNA- probes double-strand connection product complex are prepared as reverse transcriptase primer.
The absolute expression information of rna transcription sheet can be by comprehensive claim 5（5）In obtain the abundance messages and claim of probe connection product（4）In random tags remove PCR repeat after obtain；The relative expression quantity information of specific rna transcription sheet can be by comprehensive claim 5（5）In obtain probe connection product abundance messages and the abundance messages of the particular probe connection product that represents reference gene obtain, the detection accuracy of relative expression quantity also can be further optimized further combined with random tags；Reference gene is not quite similar according to purpose different choice, including but not limited to ACTB, GAPDH, TFRC, RPLPO, GUSB.
The invention also discloses a kind of can be in the method for DNA copy number and nucleotide variation in Multiple detection biological specimen, ultimate principle is as shown in Fig. 2 main implementation method includes：（1）Preparation can embody the total nucleic acid of biological specimen DNA characteristic informations；（2）The multiple probe group matched with specific DNA sequence described in Fig. 2 is added in total nucleic acid；（3）Hybridize at a certain temperature and excite coupled reaction using nucleic acid ligase；（4）With（3）The connection product of middle gained is template, and the universal primer performing PCR matched using the 5 ' universal sequences and 3 ' universal sequences described in Fig. 2 is reacted；（5）Will（4）Middle PCR primer uses after purification the sequenator compatible with universal linker sequence or chip hybridization scanner to obtain the sequence abundances information of all probe connection products；（6）Informatics Method determines the copy number and nucleotide variation and respective frequencies of specific DNA.
Wherein, the corresponding probe sequence design of detection DNA base variation should be with the DNA of variation as complementary template, becoming isobase should be located within ± the 4bp of left and right probe nicking junction, be to reduce false positive coupled reaction to optimize change change isobase 1-8bp probe sequences nearby.The nucleic acid ligase of selection should have DNA nicking coupled reaction abilities in catalytic dna-DNA hybridization chain, including but not limited to T4 DNA Ligase, T3 DNA Ligase, Taq DNA Ligase, T7 DNA ligase, Taq DNA liagase, 9 ° of N DNA Ligase and Ligase-65.The absolute copy number information of specific DNA fragments can be removed by the abundance messages of comprehensive probe connection product and random tags and obtained after duplicate message；The abundance messages of abundance messages and the particular probe connection product for representing reference gene that the Relative copy number variation information of specific DNA fragments can pass through comprehensive probe connection product are obtained, also can be further combined with random tags optimization copy number variation testing result；Reference gene is not quite similar according to purpose different choice, can include any genome stability region target sequence, it is also possible to select total abundance of a plurality of specific probe as internal reference.
In addition, disclosed by the invention can simultaneously arrange multigroup probe for same specific nucleic acid template, using the independent detection ability between each probe groups the accuracy of the nucleic acid-templated characteristic is improved.
Finally, in order to realize detecting the accuracy and uniformity of nucleic acid characteristic, the invention also discloses being used to manufacture the preparation and the purposes of equipment of examination and diagnosis nucleic acid characteristic change relevant disease using probe combinations, relevant disease includes tumor, microorganism infection, infantile autism or genetic diseasess.There are many Testing index to be rna expression, DNA copy number and nucleotide variation in tumor diagnosis and treatment typing, the including but not limited to detection of breast carcinoma 21-gene expression, 70-gene expressions detects the HER2 amplification related to Herceptin medication curative effect, tumor staining body copy number variation, targeting medication effect and mutant target gene detection etc.；Microbial nucleic acids specific fragment can also rapidly understand infection state to instruct clinical decision in by detecting body fluid；The invention simultaneously can provide powerful for the gene diagnosises of heredopathia, infantile autism, Jessica Lynch's syndrome of MLH1/MSH2 Exon deletions initiation and congenital heart disease that including but not limited to microdeletion causes.
Description of the drawings：
Fig. 1. left and right probe combinations structural representation.
Fig. 2. for the probe combinations detection principle diagram of specific nucleic acid template.
Fig. 3. fluorescent quantitative PCR experiment verifies the expression of GAPDH, ERBB2 and ESR1 gene.
Embodiments of the present invention are illustrated
Unless stated otherwise, the implication of term used herein understands according to broader sense known to association area.
Specific embodiment one.
Detected using probe combinations targeting
FFPE
Sample is multiple
RNA
Transcript is expressed
In order to verify that whether the probe combinations of design are capable of the expression of the accurately intact RNA of multiple targeting detection quality and serious degradation of rna, this experiment selects respectively A549 cell lines and breast cancer tissue's FFPE sample extraction RNA, and assesses final data reliability according to following experiment flow：
（1）RNA takes Million cell of normal culture A549 cell lines 4, adds Trizol to blow even to clarifying, and extracts total serum IgE according to Invitrogen Trizol kit standards method and measures concentration using Nanodrop；8 μ m-thick roll film of breast cancer tissue FFPE samples two is selected, using Tiangeng paraffin embedding total RNA extraction reagent box total serum IgE is extracted, Nanodrop measurements concentration -80 is frozen standby.
（2）Reverse transcription using MMLV reverse transcriptases in the case where there is system in Rnase inhibitor by step（1）The A549 cells of middle extraction and breast cancer tissue RNA difference reverse transcriptions are cDNA, the Oligo (dN) of reverse transcriptase primer position biotin modification6。
（3）Respectively take A549 cells and breast cancer tissue's 5ul cDNA solution（Equivalent to 200ng RNA before transcription）Add containing in targeting ACTB, GAPDH, HER2 and ESR1 the hybridization buffer of totally 6 pairs of probe groups, a pair of each left and right probes of wherein ACTB and GAPDH, HER2 and ESR1 or so probe is each 2 pairs, and all probe particular sequences are as shown in table 1.60 DEG C hybridize 3 hours after 98 DEG C of degeneration 5min.
Table
1
| SEQ ID NO | Probe title | Sequence information |
| 1 | ACTBL2 | TACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGCACCCAGCACAATGAAGATCAAGA |
| 2 | ACTBR2 | TCATTGCTCCTCCTGAGCGCAAGTACTCAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC |
| 3 | GAPDHL1 | TACACTCTTTCCCTACACGACGCTCTTCCGATCTCTCAACGACCACTTTGTCAAGCTCATTTCCT |
| 4 | GAPDHR1 | GGTATGACAACGAATTTGGCTACAGCAACAGGAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC |
| 5 | ESR1L1 | TACACTCTTTCCCTACACGACGCTCTTCCGATCTACATGAGTAACAAAGGCATGGAGCATCTGTA |
| 6 | ESR1R1 | CAGCATGAAGTGCAAGAACGTGGTGCCCCTAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC |
| 7 | ESR1L2 | TACACTCTTTCCCTACACGACGCTCTTCCGATCTGACATGCTGCTGGCTACATCATCTCGGTTC |
| 8 | ESR1R2 | CGCATGATGAATCTGCAGGGAGAGGAGTTTAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC |
| 9 | ERBB2L1 | TACACTCTTTCCCTACACGACGCTCTTCCGATCTGAAGTGCAGCAAGCCCTGTGCCCGAGTGT |
| 10 | ERBB2R1 | GCTATGGTCTGGGCATGGAGCACTTGCGAGAAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC |
| 11 | ERBB2L2 | TACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCTCCAAGTGTTTGAGACTCTGGAAGAGA |
| 12 | ERBB2R2 | TCACAGGTTACCTATACATCTCAGCATGGCAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC |
（4）Add Taq ligase
4 DEG C of terminating reactions and using Avidin magnetic beads for purifying connection product after 45 DEG C of incubation 15min.（5）It is template to take connection product 10ul in step 4, last storehouse of building is carried out in 50ul PCR reaction systems using universal PC R primer pairs 5UAP/3UAP and is processed.Universal PC R primer sequences are as shown in table 2.
Table
2
| SEQ ID NO | Universal primer | Sequence information |
| 13 | 5UAP | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA |
| 14 | 3UAP | CAAGCAGAAGACGGCATACGAGATCGTACGTAGTGACTGGAGTTCAGACGTG |
（6）Library Quality Control is simultaneously sequenced using the SE50 of HiSeq 2500, and data volume is 0.5M reads.
（7）Data analysiss：6 target sequence of the clean reads for obtaining and target ACTB, GAPDH, ERBB2 and ESR1 mRNA are compared and count read quantity on each target respectively by sequencing data after Quality Control removes low quality reads, as shown in table 3.
Table
3
| Target site | A549 cells | Breast cancer tissue |
| ACTB_2 | 396671 | 389936 |
| GAPDH_1 | 115063 | 100800 |
| ERBB2_1 | 2134 | 2800 |
| ERBB2_2 | 3093 | 3928 |
| ESR1_2 | 3 | 1446 |
| ESR1_2 | 4 | 1907 |
All very high according to the visible ACTB and GAPDH expressions as house-keeping gene of result, the higher ESR1 of mammary gland tissue specificity is not expressed in A549 cell lines, meets rule.ERBB2_1 and ERBB2_2 in breast cancer tissue's sample is contrasted in addition（2800：3928）, and ESR_1 and ESR_2（1446:1907）, same gene transcript different targeting regions gained expression it is similar, equally illustrate technically reliable.In order to further verify the reliability of this invention, we verify the relative expression quantity of GAPDH, ERBB2 and ESR1 relative to ACTB in A549 samples using fluorescence quantifying PCR method.Comparative result shows that the technology height of two clocks detection rna expression is consistent, as shown in Figure 3.
Specific embodimentTwo.
Genome is detected using probe combinations targeting DNA Copy number
In order to prove can effectively to detect DNA copy number for genomic DNA design probe groups, we select the peripheral blood genomic DNA of normal person as original nucleic acid template and therefrom select 5 specific regions design probes and test as follows：
（1）DNA extraction takes normal human blood and uses Tiangeng poba gene group DNA extraction kit (DP348) to extract gDNA and use Nanodrop to measure concentration；
（2）Two parts of 50ng gDNA are taken as repeating to be separately added into containing targeting BIRC5, GAPDH and ESR1 the hybridization buffer of totally 5 pairs of probe groups, wherein a pair of GAPDH or so probes, BIRC5 and ESR1 or so probe is each 2 pairs, and ESR1 probes still use the middle probe of table 1（SEQ ID NO 5/6/7/8）, BIRC5 and GAPDH probe particular sequences are as shown in table 4.60 DEG C hybridize 3 hours after 98 DEG C of degeneration 5min.
Table
4
| SEQ ID NO | Probe title | Sequence information |
| 15 | BIRC5L1 | TACACTCTTTCCCTACACGACGCTCTTCCGATCTGAGGCTGGCTTCATCCACTGCCCCACTGAGA |
| 16 | BIRC5R1 | ACGAGCCAGACTTGGCCCAGTGTTTCTTCTAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC |
| 17 | BIRC5L2 | TACACTCTTTCCCTACACGACGCTCTTCCGATCTTAAGTCATTGGGGAAACGGGGTGAACTTCAG |
| 18 | BIRC5R2 | GTGGATGAGGAGACAGAATAGAGTGATAGGAAGCGAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC |
| 19 | GAPDHL2 | TACACTCTTTCCCTACACGACGCTCTTCCGATCTCGATTTCTCCTCCGGGTGATGCTTTTCCTA |
| 20 | GAPDHR2 | GATTATTCTCTGATTTGGTCGTATTGGGCGAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC |
（3）94 DEG C of degeneration 5min terminating reactions after 54 DEG C of incubation 15min of Ligase-65 are added, using magnetic beads for purifying connection product buffer is changed.
（4）It is template to take connection product 10ul in step 4, carries out last storehouse of building using universal PC R primer pair 50ul PCR reaction systems and processes.General PCR primer 5UAP/3UAP sequence is as shown in table 2.
（5）Library Quality Control is simultaneously sequenced using the SE50 of HiSeq 2500, and final data amount is 0.2M reads.
（6）Data analysiss：Sequencing data, by the clean reads for obtaining, is then compared with 5 target sequence of target GAPDH, BIRC5 and ESR1 genomic DNA and counts read quantity on each target respectively, as shown in table 5 after Quality Control removes low quality reads.
Table
5
| Target site | gDNA-1 | gDNA-2 |
| GAPDH_2 | 27063 | 28603 |
| BIRC5_1 | 33855 | 32678 |
| BIRC5_2 | 28089 | 29284 |
| ESR1_2 | 25986 | 25301 |
| ESR1_2 | 29978 | 31684 |
In theory genome is complete 2 times body, and the sequencing abundance ratio obtained for the probe groups of all of specific sequence stencil design all should be near 1.From the gDNA-1 interpretations of result of table 5, the minimum ESR1_2 probes ratio of abundance highest BIRC5_1 and abundance is 1.3（33855/25986）, two targeting regions ratios of BIRC5 are 1.2（33855/28089）, the ratio of two targeting regions of ESR1 is 1.15.All be in two technology repeated samples gDNA-1 and gDNA-2 the abundance of BIRC5_1 probe groups with respect to highest, the abundance of ESR1_2 probe groups is minimum, and the ratio between different target is consistent.These as shown by data present invention can be used to detect the copy number characteristic of specific genome area, and technology repeatability is very well.
Reference material
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Paik, S., et al., A multigene assay to predict
recurrence of tamoxifen-treated, node-negative breast
cancer. N Engl J Med, 2004. 351(27): p. 2817-26.
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Knauer, M., et al., The predictive value of the
70-gene signature for adjuvant chemotherapy in early breast cancer. Breast
Cancer Res Treat, 2010. 120(3): p. 655-61.
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Schouten, J.P., et al., Relative quantification of 40 nucleic acid sequences by
multiplex ligation-dependent probe amplification. Nucleic
Acids Res, 2002. 30(12): p. e57.
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Eldering, E., et al., Expression profiling via novel
multiplex assay allows rapid assessment of gene regulation in defined signalling pathways. Nucleic Acids Res,
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SEQUENCE LISTING
<110>
Beijing is sought because of bio tech ltd
<120>
The method that multiple targeting based on coupled reaction detects indicator nucleic acid
<130> 2015
<160> 20
<170> PatentIn version 3.5
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Claims (14)
1. one group of probe that can be used for making nucleic acid molecular hybridization, it is characterised in that：
（1）At least include two probes in left and right, Far Left probe includes 5 ' universal sequences and 3 ' distinguished sequences, and rightmost probe includes the 5 ' distinguished sequences and 3 ' universal sequences of phosphorylated modification, positioned at middle probe the distinguished sequence of 5 ' phosphorylation modifications is；
（2）Tight adjacent non-notch, can hybridize in same single nucleic acid strands template simultaneously between probe, and adjacent probe can be connected as same nucleic acid molecules by nucleic acid ligase catalysis.
2. in probe sequence as described in claim 1,5 ' universal sequences and 3 ' universal sequences can be the joint sequence compatible with sequenator or biological chip instrument, including but not limited to illumina HiSeq, MiSeq, NextSeq microarray datasets, Life
Ion microarray datasets, Illumina or Affymetrix chip platforms；Universal sequence can select all or part of joint sequence.
3. distinguished sequence can be combined with template nucleic acid molecule reverse complemental in probe as described in claim 1, and template nucleic acid includes RNA, cDNA and genome double-stranded DNA.
4. probe as stated in claim 2, may be inserted into 8-15 randomized bases label between universal sequence and distinguished sequence（N）8-15, this label can remove in information analysiss PCR amplification efficiencies different band come deviation.
5. it is a kind of can in the method for rna expression amount and sequence in Multiple detection biological specimen, including：
（1）Preparation can embody the total nucleic acid of biological specimen RNA characteristic informations；
（2）The multiple probe group from this matching of different rna transcriptions described in claim 1 is added in total nucleic acid；
（3）Hybridize at a certain temperature and excite coupled reaction using nucleic acid ligase；
（4）With（3）The connection product of middle gained is template, using the universal primer performing PCR reaction matched with 5 ' universal sequences and 3 ' universal sequences described in claim 2；
（5）Will（4）After purification usage right requires that instrument described in 2 obtains the sequence abundances information of all probe connection products to middle PCR primer；
（6）Informatics Method determines the expression and sequence information of different rna transcription sheets.
6. such as claim 5（1）Described in, the total nucleic acid for embodying biological specimen RNA information can be the RNA of extracting directly, or extract the cDNA obtained by reverse transcription after RNA；
（1）If the total nucleic acid for preparing is RNA, specific base sequence should be with the base sequence reverse complemental of corresponding rna transcription sheet in probe groups, and claim 5（3）In nucleic acid ligase used should have single stranded DNA nicking coupled reaction ability, including but not limited to T4 DNA Ligase and SplintR Ligase in catalysis RNA-DNA hybridization chains；
（2）If the total nucleic acid for preparing is cDNA, specific base sequence should be consistent with the base sequence of correspondence rna transcription sheet in probe groups, and claim 5（3）In nucleic acid ligase used should have single stranded DNA nicking coupled reaction ability in catalytic dna-DNA hybridization chain, including but not limited to T4 DNA Ligase, T3 DNA Ligase, Taq DNA Ligase, T7 DNA ligase, Taq DNA liagase, 9 ° of N DNA Ligase and Ligase-65.
7. such as claim 5（4）Described in connection product purification, other affine mode purification can be may be based on by clip size principle purification, way of purification is included but is not limited to：
（1）If the total nucleic acid for preparing is RNA, can be by being marked with Oligo（dT）NMagnetic bead or affinity column purifying RNA-probe double-strand connection product complex, can effectively remove the adverse effect of free probe（15≤N≤35）；
（2）If the total nucleic acid for preparing is cDNA, select with biotinylated oligo（dN）6Or oligo（dT）NClaim 5 is prepared as reverse transcriptase primer（1）In total nucleic acid cDNA, and the magnetic bead using Avidin labelling or affine column purification cDNA- probes double-strand connection product complex（15≤N≤35）.
8. such as claim 5（6）Described in, the absolute expression information of rna transcription sheet can be by comprehensive claim 5（5）In obtain the abundance messages and claim of probe connection product（4）In random tags remove PCR repeat after obtain；The relative expression quantity information of specific rna transcription sheet can be by comprehensive claim 5（5）In obtain probe connection product abundance messages and the abundance messages of the particular probe connection product that represents reference gene obtain, the detection accuracy of relative expression quantity also can be further optimized further combined with random tags；Reference gene is not quite similar according to purpose different choice, including but not limited to ACTB, GAPDH, TFRC, RPLPO, GUSB.
9. it is a kind of can in the method for DNA copy number and nucleotide variation in Multiple detection biological specimen, including：
（1）Preparation can embody the total nucleic acid of biological specimen DNA characteristic informations；
（2）The multiple probe group matched with specific DNA sequence described in claim 1 is added in total nucleic acid；
（3）Hybridize at a certain temperature and excite coupled reaction using nucleic acid ligase；
（4）With（3）The connection product of middle gained is template, using the universal primer performing PCR reaction matched with 5 ' universal sequences and 3 ' universal sequences described in claim 2；
（5）Will（4）After purification usage right requires that instrument described in 2 obtains the sequence abundances information of all probe connection products to middle PCR primer；
（6）Informatics Method determines the copy number and nucleotide variation of specific DNA.
10. such as claim 9（2）Described in, the corresponding probe sequence design of detection DNA base variation should be with the DNA of variation as complementary template, becoming isobase should be located within ± the 4bp of left and right probe nicking junction, be to reduce false positive coupled reaction to optimize change change isobase 1-8bp probe sequences nearby.
11. such as claim 9（3）Described in, the nucleic acid ligase of selection should have single stranded DNA nicking coupled reaction ability in catalytic dna-DNA hybridization chain, including but not limited to T4 DNA Ligase, T3
DNA Ligase, Taq DNA Ligase, T7 DNA ligase, Taq DNA liagase, 9 ° of N DNA Ligase and Ligase-65.
12. such as claim 9（6）Described in, the absolute copy number information of specific DNA fragments can be by comprehensive claim 9（5）The abundance messages and claim of middle probe connection product（4）In random tags remove PCR repeat after obtain；The Relative copy number variation information of specific DNA fragments can be by comprehensive claim 5（5）In obtain probe connection product abundance messages and the abundance messages of the particular probe connection product that represents reference gene obtain, also can be further combined with random tags optimization copy number variation testing result；Reference gene is not quite similar according to purpose different choice, can include any genome stability region sequence, it is also possible to select total abundance of a plurality of specific probe as internal reference.
13. such as claim 5（2）With claim 9（2）Described in for same specific nucleic acid template, multigroup probe can be set simultaneously, improve the accuracy of the nucleic acid-templated characteristic using the independent detection ability between each probe groups.
Either the method for detection DNA characteristics described in the method or claim 9-12 of detection RNA characteristics described in claim 5-8 is used to manufacture the preparation and the purposes of equipment of examination and diagnosis nucleic acid characteristic change relevant disease probe combinations described in 14. claim 1-4, relevant disease includes tumor, microorganism infection, infantile autism or genetic diseasess.
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