CN101329250B - Method for detecting sequence of PCR-LCR gene polymorphism of fluorescence labeling probe - Google Patents
Method for detecting sequence of PCR-LCR gene polymorphism of fluorescence labeling probe Download PDFInfo
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Abstract
The invention provides a PCR-LDR gene polymorphism detection sequencing method for a fluorescent probe. The invention uses the fluorescent probe to carry out the PCR-related LDR and sequencing technique so as to carry out the gene polymorphism site detection. The method of the invention comprises the steps as follows: primer and probe designing, PCR detecting, LDR detecting, and sequencing instrument result detecting. Due to the directness of sequencing results, the method of the invention solves the false positive and the false positive of the results; owning to the sample high throughput, a sequencing platform can carry out the detection of hundreds of samples at the same time; the high throughput of the site can utilizes the 5-colour fluorescent detection system of the sequencing instrument and four fluorescence at the same time so as to lead the sequencing instrument system to carry out the detection of hundreds of sites at the same time, thus quickening the detection speed and reducing the experiment cost.
Description
Technical field
The present invention relates to biotechnology, the PCR-LDR gene pleiomorphism that is specifically related to a kind of fluorescence labeling probe detects sequence measurement.
Background technology
Along with the development of human medicine genomics, increasing achievement in research will apply in the clinical diagnosis, and this has just required large-scale gene polymorphism sites detection method.In recent years, coupled reaction is used to detect the attention that gene polymorphism sites more and more causes people, scholars have been a large amount of research (France Barany et al., Proc.Nat ' l Acad.Sci.USA, 88:189-93 (1991), The Ligase Chain Reaction (LCR) in a PCR World, PCR Methods and Applications, 1:5-16 (1991)).(ligasechain reaction LCR) is meant in reaction two pairs of probes of design to ligase chain reaction, makes the coupled reaction can the index amplification template.(ligase detection reaction is only to add a pair of probe in reaction LDR) to the ligase detection reaction, and template is by linear amplification.
At present, occurred the related use of LDR technology with round pcr, and LDR technology, round pcr and biochip technology, the sequencing technologies association is used for gene polymorphism sites and detects (Norman P.Gerryl et al., J.Mol.Biol. (1999) 292,251-262, U.S.Pat.Pub.No.2003/0032016A1), the appearance of these technology greatly facilitates the detection of gene loci, but in these technology, need a large amount of fluorescently-labeled probes, and the probe cost costliness of these marks, for development and application brings inconvenience, thereby, limited their application.
Summary of the invention
The technical problem to be solved in the present invention is to overcome above-mentioned weak point, the PCR-LDR gene pleiomorphism order-checking detection method that research and design is easy.
The invention provides a kind of PCR-LDR gene pleiomorphism order-checking detection method of fluorescence labeling probe.
Because gene pleiomorphism comprises multiple situations such as unit point gene mutation, gene delection, insertion and little satellite, it mainly is to suddenly change at the gene unit point that gene polymorphism sites of the present invention detects, be single base mutation, insertion and disappearance, and around the mutational site, do not have the other unit point mutation in about 20 bases.
Utilize a kind of fluorescence labeling probe provided by the invention, carry out gene polymorphism sites when detecting at the related sequenator that utilizes of PCR with LDR, at each mutational site, relate to two kinds of probes: first kind of probe is label probe, comprise three parts, wherein a part is and the template mating section, and another part is and is connected the template mating section, introduces the length that random probe is adjusted probe simultaneously in two parts sequence; Second kind of probe is detector probe, comprises two parts, and wherein a part is and the template mating section, and another part is then introduced the length that random series is adjusted the LDR product.Second kind of probe wherein, can be two also can be three or four, 3 ' end is detection site in the probe.Every group of detector probe that changes at nucleic acid list base by setting the length of detector probe, realizes the LDR product length that different nucleic acid bases is corresponding different.
The present invention transforms at first kind of label probe, at first introduce a general fluorescence labeling probe and a common template, 5 ' the sequence and the complementation of general fluorescence labeling probe sequence of common template part, and partial sequence is also arranged in above-mentioned first kind of label probe is sequence with the common template complementation, in the LDR process, by the complementary pairing of common template, realize being connected of general fluorescence labeling probe and label probe, realize mark to label probe.
LDR reaction is put into sequenator with the LDR product after finishing, and utilizes the genescan function of sequenator, confirms the length of Different L DR product by a series of confidential reference items of known molecular amount among the result, thus the polymorphism in interpretation site.
The unit point polymorphism order-checking of PCR-LDR association of the present invention detects, and through suitably adjusting, also can be used for the separate nucleic acid detection system of high resolving power such as HPLC and micro flow chip.
Specifically, the inventive method may further comprise the steps:
1. probe design
(1) at the site of detecting, design the molecular weight series of a series of LDR products, the difference of LDR product can be from a base to a hundreds of base;
(2) proprietary fluorescence labeling probe of design, this probe has high specificity, does not have homology with the sequence of known organism;
(3) proprietary template of design, this template has high specificity, does not have homology with the sequence of known organism;
(4) respectively design the detector probe and the label probe of 10-25 base length in the front and back in the mutational site that will detect, the detection site design of detector probe is at 3 ' end;
(5) introduce template matched sequence and random series at 3 ' end of label probe, and detector probe 5 ' end introduces random series (not having any sequence of homology with the sequence of PCR product), generally labeling probe and detector probe have close molecular weight.
2.PCR
Get more than the DNA1 μ l to be detected (5ng/ μ l), each 10-30pmol of PCR primer, pyro polymerase, the corresponding mixed liquor of pyro polymerase mixes mutually.The PCR reaction conditions is: 94 ℃ 30 seconds, 40-68 ℃ 30 seconds-2 minutes, 60-72 ℃ 45 seconds-2 minutes, circulation 25-40 time.
3.LDR
Get PCR product 1 μ l, each 40fmol-10pmol of probe, ligase, corresponding ligase damping fluid mixes mutually.The LDR reaction conditions is: 94 ℃ 30 seconds, 40-70 ℃ 1-10 minute (is best with 45-60 ℃), circulation 1-40 time.
4. the result detects
Get LDR product 1-5 μ l, mix the loading buffer of sequencing system, sample on the sequenator carries out LDR product isolation identification.
Step (one) (3) is introduced template matched sequence and random series and detector probe 5 ' end at 3 ' end of label probe and is introduced random series, and described random series is any sequence that does not have homology with the sequence of PCR product.
Step (one) (4) respectively designs the detector probe and the label probe of 10-25 base length in the front and back in the mutational site that will detect, the design of label probe detection site is at 3 ' end, and the detection site design of detector probe is at 5 ' end.Label probe and detector probe have close molecular weight.
(1) (4) described generally labeling probe comprises three parts, first is and purpose template mating section, second portion is and is connected the template mating section, the random series of third part in first and second partial sequences, introducing simultaneously, be used for adjusting the length of probe, length is 40-80bp; Described detector probe comprises two parts, and first is and purpose template mating section, and the random series of second portion for introducing is used for adjusting the length of LDR product, and length is 40-80bp; Detector probe can be two, three or four, and 3 ' end is detection site in the probe, and every group of detector probe that changes at nucleic acid list base by setting the length of detector probe, realizes the LDR product length that different nucleic acid bases is corresponding different.
The present invention has following effect:
1. owing to the substantivity of sequencing result, solved result's false positive and false negative problem.
2. the sample high flux property of order-checking platform can be carried out the detection of routine samples up to a hundred simultaneously, and chip technology then can't carry out the detection of multisample simultaneously.
3. the high flux in site utilizes 5 look fluorescence detecting systems of sequenator, utilizes 4 kinds of fluorescence simultaneously, and the sequenator system also can carry out the detection in more than 100 site simultaneously.The speed of acceleration detection greatly.
4. compare with existing gene polymorphism sites detection and sequencing technologies, significantly reduced application cost, and testing result is correct.
Description of drawings
Fig. 1-2 is HBV1896 sudden change testing result figure.
The result of Fig. 1 represents to have only the connection product of 82bp, promptly has only the wild type of HBV1896.
The result of Fig. 2 represents to have the product that is connected of 82bp and 87bp, and existing HBV1896 wild type also has the HBV1896 saltant.
The joint detection results of the YIDD/YVDD of Fig. 3-4 expression HBV1896 and HBV.
Fig. 3 has two of 82bp and 92bp to be connected product, and expression HBV1896 testing result is to have only the wild type product, and the YIDD/YVDD testing result of HBV is to have only the YIDD product.
Fig. 4 has the product that is connected of 82bp, 92bp and 97bp, and expression HBV1896 testing result has only the wild type product, and the testing result of the YIDD/YVDD of HBV is that existing YIDD product also has YVDD sudden change product.
The result of joint-detection is carried out in Fig. 5-7 expression to 5 SNP site rs4652678, rs199930, rs7043268, rs6424820 and rs4133072.
The result of Fig. 5 represents that rs4652678 has two of 82bp and 87bp to be connected product, is heterozygote C/T; Rs199930 has the connection product of 92bp, is homozygote C; Rs7043268 has two of 102bp and 107bp to be connected product, is heterozygote G/A; Rs6424820 has two of 112bp and 117bp to be connected product, is heterozygote C/G; Rs4133072 has the connection product of 122bp, is homozygote A.
The result of Fig. 6 represents that rs4652678 has 82bp to connect product, is homozygote C; Rs199930 has the connection product of 92bp, is homozygote C; Rs7043268 has 107bp to connect product, is homozygote A; Rs6424820 has 117bp to connect product, is homozygote G; Rs4133072 has the connection product of 122bp, is homozygote A.
The result of Fig. 7 represents that rs4652678 has two of 82bp and 87bp to be connected product, is heterozygote C/T; Rs199930 has the connection product of 92bp, is homozygote C; Rs7043268 has 102bp to connect product, is homozygote G; Rs6424820 has 112bp to connect product, is homozygote C; Rs4133072 has the connection product of 122bp, is homozygote A.
The result of Fig. 8-10 expression 6 SNP site rs348475, rs499776, rs2161811, rs561712, rs4646580 and rs4646642 joint-detection.
The result of Fig. 8 represents that rs348475 has two of 82bp and 85bp to be connected product, is heterozygote A/G; Rs499776 has the connection product of 91bp, is homozygote G; Rs2161811 has 94bp to connect product, is homozygote A; Rs561712 has 100bp to connect product, is homozygote A; Rs4646580 has two of 106bp and 109bp to be connected product, is heterozygote C/T; Rs4646642 has two of 112bp and 115bp to be connected product, is heterozygote C/T.
The result of Fig. 9 represents that rs348475 has two of 82bp and 85bp to be connected product, is heterozygote A/G; Rs499776 has the product that is connected of 88bp and 91bp, and is heterozygote A/G; Rs2161811 has 94bp to connect product, is homozygote A; Rs561712 has two of 100bp and 103bp to be connected product, is heterozygote A/G; Rs4646580 has two of 106bp and 109bp to be connected product, is heterozygote C/T; Rs4646642 has two of 112bp and 115bp to be connected product, is heterozygote C/T.
The result of Figure 10 represents that rs348475 has 85bp to connect product, is homozygote A/G; Rs499776 has 91bp to connect product, is homozygote G; Rs2161811 has 94bp to connect product, is homozygote A; Rs561712 has 100bp to connect product, is homozygote A; Rs4646580 has 109bp to connect product, is homozygote T; Rs4646642 has the connection product of 115bp, is homozygote T.
Embodiment
The present invention is described further below in conjunction with this patent embodiment and accompanying drawing thereof.
Embodiment one: the dna polymorphism to the unit point sudden change detects
With hepatitis type B virus (HBV) DNA1896 site mutation for example, the mutational site is 5 bases at interval.
Sequence is near the HBV1896 site:
5’GCTGTGCCTTGGGTGGCTTTG/AGGGCATGGACATTGACCCG3’
1. primer and probe design
(1) regular-PCR design of primers
| Primer sequence (5 ' → 3 ') | Length (nt) | ||
| | CACCTCTGCCTAATCATCTCATGTTCATGT | 30 | |
| | ACACAGAATAGCTTGCCTGAGTGCTGTATG | 30 |
(2) fluorescence labeling probe: GCATCACTCAGAGGG
Template: CCCTCTGAGTGATGCGAGTACAGGTTTGCG
Fluorescence labeling probe will carry out 5 ' phosphorylation and 3 ' probe mark (this example FAM).
(3) label probe
| The probe title | Probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | AAAGCCACCCAAGGCACA | 18 | |
| With the common template mating section | CGCAAACCTGTACTC | 15 | |
| Random series | ACCTACCT | 8 | |
| The total sequence of probe ＊ | AAAGCCACCCAAGGCACA ACCTACCTCGCAAACCTGTACTC | 41 |
Annotate: * represents that probe will carry out 5 ' phosphorylation
(4) detector probe one
| The probe title | Probe sequence (5 ' → 3 ') | Length (nt) |
| With the template mating section | GGTCAATGTCCATGCCCC | 19 |
| Random series | ACCTACCTACCTACCTACCTA | 22 |
| The total sequence of probe | GGTCAATGTCCATGCCCCACCTACCTACCTACCTACCTAC | 41 |
(5) detector probe two
| The probe title | Probe sequence (5 ' → 3 ') | Length (nt) |
| With the template mating section | GGTCAATGTCCATGCCCT | 19 |
| Random series | ACCTACCTACCTACCTACCTACCTAC | 27 |
| The total sequence of probe | GGTCAATGTCCATGCCCTACCTACCTACCTACCTACCTACCTAC | 46 |
2.PCR reaction
Reaction mixture is: 200 μ M dATP, 200 μ M dTTP, 200 μ M dCTP, 200 μ MdGTP, 2mM MgCl
2, 2U Taq archaeal dna polymerase, the upstream and downstream primer of each 1 μ M, 1 μ l genomic templates (concentration is greater than 5ng/ μ l).Reaction conditions is: 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 45 seconds, circulate 30 times.
3.LDR reaction
Reaction mixture is: 20mM Tris-HCl, pH8.3 (at25 ℃), 50mM KCl, 10mM MgCl
2, 1mM EDTA, 1mM NAD+, 10mM DTT, 0.1% (v/v) Triton X-100, each 0.01 μ M of probe, Taq ligase 15U, 1 μ l pcr template DNA.Reaction conditions is: 94 ℃ 30 seconds, 60 ℃ 2 minutes, circulate 30 times.
4. going up the order-checking instrument detects
The results are shown in Figure, Fig. 1 shows to have only the 1896G wild-type strain, and Fig. 2 shows not only the 1896G wild-type strain, also has the strain of 1896A mutant virus.
Embodiment two: the dna polymorphism to two point mutation detects
Sport example with HBV1896 site and YIDD/YMDD the unit point dna polymorphism is detected, each mutational site is 5 bases at interval.
The HBV1896 sudden change:
The YIDD/YMDD sudden change of 5 ' GCTGTGCCTTGGGTGGCTTTG/AGGGCATGGACATTGACCCG3 ' HBV:
5’CTGTCTGGCTTTCAGTTATATG/TGATGATGTGGTATTGGGGG3’
1. primer and probe design
(1) regular-PCR design of primers
| HBV1896 primer sequence (5 ' → 3 ') | Length (nt) | ||
| | CACCTCTGCCTAATCATCTCATGTTCATGT | 30 |
| | ACACAGAATAGCTTGCCTGAGTGCTGTATG | 30 | |
| HBV YIDD/YMDD primer sequence (5 ' → 3 ') | Length (nt) | ||
| | CAAGGTATGTTGCCCGTTTGTCC | 30 | |
| Downstream primer | GG(CT)A(AT)AAAGGGACTCA(AC) |
30 |
(2) fluorescence labeling probe: GCATCACTCAGAGGG
Template: CCCTCTGAGTGATGCGAGTACAGGTTTGCG
Fluorescence labeling probe will carry out 5 ' phosphorylation and 3 ' probe mark (this example FAM).
(3) label probe
| The probe title | HBV1896 (G/A) label probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | AAAGCCACCCAAGGCACA | 18 | |
| With the common template mating section | CGCAAACCTGTACTC | 15 | |
| Random series | ACCTACCT | 8 | |
| The total sequence of probe ＊ | AAAGCCACCCAAGGCACA ACCTACCTCGCAAACCTGTACTC | 41 | |
| The probe title | HBV YIDD/YMDD (G/T) label probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template mating section | ATATAACTGAAAGCCAAACAG | 22 | |
| With the common template mating section | CGCAAACCTGTACTC | 15 | |
| Random series | ACCTACCTA | 9 | |
| The total sequence of probe * | ATATAACTGAAAGCCAAACAGACCTACCTACGCAAACCTGTACTC | 46 |
Annotate: * represents that probe will carry out 5 ' phosphorylation
(4) detector probe one
| The probe title | HBV1896_G probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template mating section | GGTCAATGTCCATGCCCC | 19 | |
| Random series | ACCTACCTACCTACCTACCTA | 22 | |
| The total sequence of probe | GGTCAATGTCCATGCCCCACCTACCTACCTACCTACCTAC | 41 | |
| The probe title | HBV YIDD/YMDD_G probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template mating section | GCCCCCAATACCACATCATCC | 22 | |
| | ACCTACCTACCTACCTACCTACC | 24 | |
| The total sequence of probe | ACCTACCTACCTACCTACCTACCGCCCCCAATACCACATCATCC | 46 |
(5) detector probe two
| The probe title | HBV1896_A probe sequence (5 ' → 3 ') | Length (nt) |
| With the template mating section | GGTCAATGTCCATGCCCT | 19 |
| Random series | ACCTACCTACCTACCTACCTACCTAC | 27 |
| The total sequence of probe | GGTCAATGTCCATGCCCTACCTACCTACCTACCTACCTACCTAC | 46 |
| The probe title | HBV YIDD/YMDD_T probe sequence (5 ' → 3 ') | Length (nt) |
| With the template mating section | GCCCCCAATACCACATCATCA | 22 |
| Random series | ACCTACCTACCTACCTACCTACCTACCTAC | 29 |
| The total sequence of probe | ACCTACCTACCTACCTACCTACCTACCTACGCCCCCAATACCACAT CATCA | 51 |
2.PCR reaction
Reaction mixture is: 200 μ M dATP, 200 μ M dTTP, 200 μ M dCTP, 200 μ MdGTP, 2mM MgCl
2, 2U Taq archaeal dna polymerase, the upstream and downstream primer of each 1 μ M, 1 μ l genomic templates (concentration is greater than 5ng/ μ l).Reaction conditions is: 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 45 seconds, circulate 30 times.
3.LDR reaction
Reaction mixture is: 20mM Tris-HCl, pH8.3 (at25 ℃), 50mM KCl, 10mM MgCl
2, 1mM EDTA, 1mM NAD+, 10mM DTT, 0.1% (v/v) Triton X-100, each 0.01 μ M of probe, Taq ligase 15U, 1 μ l pcr template DNA.Reaction conditions is: 94 ℃ 30 seconds, 60 ℃ 2 minutes, circulate 30 times.
4. going up the order-checking instrument detects
The results are shown in Figure 3-4.
Embodiment three: polymorphism is carried out in a plurality of sites of people's gene group (SNP) simultaneously detect
Simultaneously 5 SNP sites (rs4652678, rs199930, rs7043268, rs6424820 and rs4133072) carried out the SNP somatotype, each SNP mutational site is 5 bases at interval.
1. primer and probe design
(1) regular-PCR design of primers
| Rs4652678 primer sequence (5 ' → 3 ') | Length (nt) | ||
| | CTTCTGTAGAATGAGGTATAGTGAGGAGGC | 30 | |
| | GTCAGCAGAGCCCAGATAATGTTGTCGTAG | 30 | |
| Rs199930 primer sequence (5 ' → 3 ') | Length (nt) | ||
| | GAGGGCTGGCAGGAGATTATGCTGTCATGC | 30 | |
| | GGTAAAAGCACAATGTTGCTCACCAAGAAG | 30 | |
| Rs7043268 primer sequence (5 ' → 3 ') | Length (nt) | ||
| | CTCTGGATTTGACATTGCTTTTCAGGAGTG | 30 | |
| | TTAGTGAGAGGGATCAGGAAACCAAGGAAG | 30 | |
| Rs6424820 primer sequence (5 ' → 3 ') | Length (nt) | ||
| | AGGACGGGACCTGATGTAGTGTGAACAGTC | 30 | |
| | ACCTCCTCCCACACTGGCACCAACACACCA | 30 | |
| Rs4133072 primer sequence (5 ' → 3 ') | Length (nt) | ||
| | AAGAACTGTAACAACAAAGCAGCAATATGA | 30 | |
| | CGATGGAAAATGTTTTACAAAATGGAAGAC | 30 |
(2) fluorescence labeling probe: GCATCACTCAGAGGG
Template: CCCTCTGAGTGATGCGAGTACAGGTTTGCG
Fluorescence labeling probe will carry out 5 ' phosphorylation and 3 ' probe mark (this example FAM).
(3) label probe
| The probe title | Rs4652678 (C/T) label probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template mating section | CTACATAGTTGCCTCTAAAAC | 20 | |
| With the common template mating section | CGCAAACCTGTACTC | 15 | |
| Random series | GTACGT | 6 | |
| The total sequence of probe * | CTACATAGTTGCCTCTAAAACGTACGTCGCAAACCTGTACTC | 41 | |
| The probe title | Rs199930 (C/T) label probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | TTCTCGTTCTGCCCTGATGGTGCGG | 25 | |
| With the common template mating section | CGCAAACCTGTACTC | 15 | |
| Random series | GTACGT | 6 | |
| The total sequence of probe * | TTCTCGTTCTGCCCTGATGGTGCGGGTACGTCGCAAACCTGTAC | 46 |
| TC | |||
| The probe title | Rs7043268 (G/A) label probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | GGGGTCCATACAAGCCAACTGGAA | 25 | |
| With the common template mating section | CGCAAACCTGTACTC | 15 | |
| | GTACGTACGTA | 11 | |
| The total sequence of probe * | GGGGTCCATACAAGCCAACTGGAAGTACGTACGTACGCAAACC TGTACTC | 51 | |
| The probe title | Rs6424820 (C/G) label probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | CTCCTATATTTGGATTTCATGATG | 25 | |
| With the common template mating section | CGCAAACCTGTACTC | 15 | |
| | GTACGTACGTACGTAC | 16 | |
| The total sequence of probe * | CTCCTATATTTGGATTTCATGATGGTACGTACGTACGTACCGCAA ACCTGTACTC | 56 | |
| The probe title | Rs4133072 (A/G) label probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template mating section | CATAACAAAAGCAAGCAATGCTA | 23 | |
| With the common template mating section | CGCAAACCTGTACTC | 15 | |
| Random series | GTACGTACGTACGTACGTACGTA | 23 | |
| The total sequence of probe * | CATAACAAAAGCAAGCAATGCTAGTACGTACGTACGTACGTACG TACGCAAACCTGTACTC | 61 |
Annotate: * represents that probe will carry out 5 ' phosphorylation
(4) detector probe one
| The probe title | Rs4652678_C probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | CTTCACCTCCATCTTGTTTTTCTCG | 25 | |
| | GTACGTACGTACGTAC | 16 | |
| The total sequence of probe | GTACGTACGTACGTAC CTTCACCTCCATCTTGTTTTTCTCG | 41 | |
| The probe title | Rs199930_C probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | GGCCAGATCGGTGCCGCAGCGTTCG | 25 | |
| Random series | GTACGTACGTACGTACGTACG | 21 | |
| The total sequence of probe | GTACGTACGTACGTACGTACGGGCCAGATCGGTGCCGCAGCGTT CG | 46 | |
| The probe title | Rs7043268_G probe sequence (5 ' → 3 ') | Length (nt) |
| With the template | GTTCTTGGAACTGTCCGTCATCGGC | 25 | |
| Random series | GTACGTACGTACGTACGTACGTACGT | 26 | |
| The total sequence of probe | GTACGTACGTACGTACGTACGTACGTGTTCTTGGAACTGTCCGT CATCGGC | 51 | |
| The probe title | Rs6424820_C probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | ACATTGTGCATCAATATGTACTTG | 25 | |
| | GTACGTACGTACGTACGTACGTACGTACGTA | 31 | |
| The total sequence of probe | GTACGTACGTACGTACGTACGTACGTACGTAACATTGTGCATCAA TATGTACTTG | 56 | |
| The probe title | Rs4133072_A probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | CAACTACAGTATCTAGAACAGTGAT | 25 | |
| | GTACGTACGTACGTACGTACGTACGTACGTACGTAC | 36 | |
| The total sequence of | GTACGTACGTACGTACGTACGTACGTACGTACGTACCAACTACA GTATCTAGAACAGTGAT | 61 |
(5) detector probe two
| The probe title | Rs4652678_T probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | CTTCACCTCCATCTTGTTTTTCTCA | 25 | |
| Random series | GTACGTACGTACGTACGTACG | 21 | |
| The total sequence of probe | GTACGTACGTACGTACGTACGCACCTCCATCTTGTTTTTCTCA | 46 | |
| The probe title | Rs199930_T probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | GGCCAGATCGGTGCCGCAGCGTTCA | 25 | |
| Random series | GTACGTACGTACGTACGTACGTACGT | 26 | |
| The total sequence of probe | GTACGTACGTACGTACGTACGTACGTGGCCAGATCGGTGCCGCAG CGTTCA | 51 | |
| The probe title | Rs7043268_A probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | GTTCTTGGAACTGTCCGTCATCGGTGGC | 25 | |
| | GTACGTACGTACGTACGTACGTACGTACGTA | 31 | |
| The total sequence of probe | GTACGTACGTACGTACGTACGTACGTACGTAGTTCTTGGAACTGTC CGTCATCGGTGGC | 56 | |
| The probe title | Rs6424820_G probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | ACATTGTGCATCAATATGTACTTC | 25 | |
| | GTACGTACGTACGTACGTACGTACGTACGTACGTAC | 36 | |
| The total sequence of | GTACGTACGTACGTACGTACGTACGTACGTACGTACACATTGTGCA | 61 |
| TCAATATGTACTTC | |||
| The probe title | Rs4133072_G probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | TCAACTACAGTATCTAGAACAGTGAC | 25 | |
| Random series | GTACGTACGTACGTACGTACGTACGTACGTACGTACGTACG | 41 | |
| The total sequence of probe | GTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTCAAC TACAGTATCTAGAACAGTGAC | 66 |
2.PCR reaction
Reaction mixture is: 200 μ MdATP, 200 μ MdTTP, 200 μ M dCTP, 200 μ MdGTP, 2mM MgCl
2, 2U Taq archaeal dna polymerase, the upstream and downstream primer of each 1 μ M, 1 μ l genomic templates (concentration is greater than 5ng/ μ l).Reaction conditions is: 94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 45 seconds, circulate 30 times.
3.LDR reaction
Reaction mixture is: 20mM Tris-HCl, pH8.3 (at25 ℃), 50mM KCl, 10mM MgCl
2, 1mM EDTA, 1mM NAD+, 10mM DTT, 0.1% (v/v) Triton X-100, each 0.01 μ M of probe, Taq ligase 15U, 1 μ l pcr template DNA.Reaction conditions is: 94 ℃ 30 seconds, 60 ℃ 2 minutes, circulate 30 times.
4. going up the order-checking instrument detects
Result such as following table:
| rs4652678(C/T) | rs199930(C/T) | rs7043268(G/A) | rs6424820(C/G) | rs4133072(A/G) | |
| Fig. 5 | C/T | C | G/A | C/G | A |
| Fig. 6 | C | C | A | G | A |
| Fig. 7 | C/T | C | G | C | A |
Embodiment four: polymorphism is carried out in a plurality of sites of people's gene group (SNP) simultaneously detect
Simultaneously 6 SNP sites (rs348475, rs499776, rs4646642, rs2161811, rs561712 and rs4646580) carried out the SNP somatotype, each SNP mutational site is adjusted into 3 bases at interval.
1. primer and probe design
(1) regular-PCR design of primers
| Rs348475 primer sequence (5 ' → 3 ') | Length (nt) | ||
| | ACATAAGTTTAATTGAACACAGCACATAGC | 30 | |
| | ATACCAGATAAGACGTGGAAAAAAATCTTC | 30 | |
| Rs499776 primer sequence (5 ' → 3 ') | Length (nt) | ||
| | GGTTCAGTGGCACAAAAGCACAAAAGTGGC | 30 | |
| | GAGGAATCTGAGGCTAATAGGCAGCAGAGG | 30 | |
| Rs4646642 primer sequence (5 ' → 3 ') | Length (nt) | ||
| | TCTCGTGTCTTCATTTTCCTCCTTCCAGGC | 30 | |
| | GCTCGTGGCATTTTCATTCTGGTCTGACTC | 30 | |
| Rs2161811 primer sequence (5 ' → 3 ') | Length (nt) | ||
| | GTGGCGGGTACAAACCAACTTAATGTTTCA | 30 | |
| | TTTACAGATGGCTCCTGTCCAGTGGGGGGA | 30 | |
| Rs561712 primer sequence (5 ' → 3 ') | Length (nt) | ||
| | TAACCTCCTCCCTTCTCTGCTGAATCCTCA | 30 | |
| | TCGCCCACATCAAAACCACTTCTGCTTTTC | 30 | |
| Rs4646580 primer sequence (5 ' → 3 ') | Length (nt) | ||
| | ATACTACTTACTTTGAGGACAGTGCTGTGG | 30 | |
| | CATCTTAGAATAACACTTTGGGGAAATAAG | 30 |
(2) fluorescence labeling probe: GCATCACTCAGAGGG
Template: CCCTCTGAGTGATGCGAGTACAGGTTTGCG
Fluorescence labeling probe will carry out 5 ' phosphorylation and 3 ' probe mark (this example FAM).
(3) label probe
| The probe title | Rs348475 (A/G) label probe sequence (5 ' → 3 ') | Length (nt) |
| With the template mating section | GAACAGCTAGCTTGCTACTTC | 20 |
| With the common template mating section | CGCAAACCTGTACTC | 15 |
| Random series | TTCGTT | 6 |
| The total sequence of probe * | GTAGAACAGCTAGCTTGCTACTTCGTTCGCAAACCTGTACTC | 41 |
| The probe title | Rs499776 (A/G) label probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template mating section | ATTTACAGCAATGGGGCAGGTATG | 23 | |
| With the common template mating section | CGCAAACCTGTACTC | 15 | |
| Random series | TTCGTT | 6 | |
| The total sequence of probe * | ATTTACAGCAATGGGGCAGGTATGTTCGTTCGCAAACCTGTACT C | 44 | |
| The probe title | Rs2161811 (A/G) label probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | TGGTGTCAAACAGTCAACTTGGGA | 25 | |
| With the common template mating section | CGCAAACCTGTACTC | 15 | |
| Random series | TTCGTTC | 7 | |
| The total sequence of probe * | TGGTGTCAAACAGTCAACTTGGGA TTCGTTCCGCAAACCTGTACTC | 47 | |
| The probe title | Rs561712 (A/G) label probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | GGCTACCTACATGATCGTAAACAGA | 25 | |
| With the common template mating section | CGCAAACCTGTACTC | 15 | |
| Random series | TTCGTTCGTT | 10 | |
| The total sequence of probe * | GGCTACCTACATGATCGTAAACAGATTCGTTCGTTCGCAAACCT GTACTC | 50 | |
| The probe title | Rs4646580 (C/T) label probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | TTTAAGAAGTGTAACAAAGAAGAAA | 25 | |
| With the common template mating section | CGCAAACCTGTACTC | 15 | |
| | TTCGTTCGTTCGT | 13 | |
| The total sequence of probe * | TTTAAGAAGTGTAACAAAGAAGAAATTCGTTCGTTCGTCGCAA ACCTGTACTC | 53 | |
| The probe title | Rs4646642 (C/T) label probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | AAAATTGTTCCCGGCCCAAACATC | 25 | |
| With the common template mating section | CGCAAACCTGTACTC | 15 | |
| | TTCGTTCGTTCGTTCG | 16 | |
| The total sequence of probe * | AAAATTGTTCCCGGCCCAAACATCTTCGTTCGTTCGTTCGCGCA AACCTGTACTC | 56 |
Annotate: * represents that probe will carry out 5 ' phosphorylation.
(4) detector probe one
| The probe title | Rs348475_A probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | GTGTTCCTATAATATATTAATATAT | 25 | |
| | TTCGTTCGTTCGTTCG | 16 | |
| The total sequence of probe | TTCGTTCGTTCGTTCGGTGTTCCTATAATATATTAATATAT | 41 | |
| The probe title | Rs499776_A probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | CATGCCTTTGACCCCCACACAGT | 25 | |
| Random series | TTCGTTCGTTCGTTCGTTC | 19 | |
| The total sequence of probe | TTCGTTCGTTCGTTCGTTCCATGCCTTTGACCCCCACACAGT | 44 | |
| The probe title | Rs216181_A probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | CAGACAGGAAGCCAAACACAAAGGT | 25 | |
| Random series | TTCGTTCGTTCGTTCGTTCGTT | 22 | |
| The total sequence of probe | TTCGTTCGTTCGTTCGTTCGTTCAGACAGGAAGCCAAACACAAAG GT | 47 | |
| The probe title | Rs561712_A probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | GATTACAGGCATTTCCCAAGTCAAT | 25 | |
| | TTCGTTCGTTCGTTCGTTCGTTCGT | 25 | |
| The total sequence of probe | TTCGTTCGTTCGTTCGTTCGTTCGTGATTACAGGCATTTCCCAAGT CAAT | 50 | |
| The probe title | Rs4646580_C probe sequence (5 ' → 3 ') | ||
| With the template | CTCAGAAGATTATCTGATACCCCGG | 25 | |
| | TTCGTTCGTTCGTTCGTTCGTTCGTTCG | 28 | |
| The total sequence of probe | TTCGTTCGTTCGTTCGTTCGTTCGTTCGCTCAGAAGATTATCTGATA CCCCGG | 53 | |
| The probe title | Rs4646642_C probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | GATAATTTGGCTTTACAACCTTGG | 25 | |
| | TTCGTTCGTTCGTTCGTTCGTTCGTTCGTTC | 31 | |
| The total sequence of probe | TTCGTTCGTTCGTTCGTTCGTTCGTTCGTTCGATAATTTGGCTTTAC AACCTTGG | 56 |
(5) detector probe two
| The probe title | Rs348475_G probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | GTGTTCCTATAATATATTAATATAC | 25 | |
| Random series | TTCGTTCGTTCGTTCGTTC | 19 | |
| The total sequence of probe | TTCGTTCGTTCGTTCGTTC GTGTTCCTATAATATATTAATATAC | 44 | |
| The probe title | Rs499776_G probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | CATGCCTTTGACCCCCACACAGC | 25 | |
| Random series | TTCGTTCGTTCGTTCGTTCGTT | 22 | |
| The total sequence of probe | TTCGTTCGTTCGTTCGTTCGTT CATGCCTTTGACCCCCACACAGC | 47 | |
| The probe title | Rs2161811_G probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | CAGACAGGAAGCCAAACACAAAGGC | 25 | |
| | TTCGTTCGTTCGTTCGTTCGTTCGT | 25 | |
| The total sequence of probe | TTCGTTCGTTCGTTCGTTCGTTCGTCAGACAGGAAGCCAAACACAA AGGC | 50 | |
| The probe title | Rs561712_G probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | GATTACAGGCATTTCCCAAGTCAAC | 25 | |
| | TTCGTTCGTTCGTTCGTTCGTTCGTTCG | 28 | |
| The total sequence of probe | TTCGTTCGTTCGTTCGTTCGTTCGTTCGGATTACAGGCATTTCCCAA GTCAAC | 53 | |
| The probe title | Rs4646580_T probe sequence (5 ' → 3 ') | ||
| With the template | CTCAGAAGATTATCTGATACCCCGA | 25 | |
| | TTCGTTCGTTCGTTCGTTCGTTCGTTCGTTC | 31 | |
| The total sequence of probe | TTCGTTCGTTCGTTCGTTCGTTCGTTCGTTCCTCAGAAGATTATCTGA TACCCCGA | 56 | |
| The probe title | Rs4646642_T probe sequence (5 ' → 3 ') | Length (nt) | |
| With the template | GATAATTTGGCTTTACAACCTTGA | 25 | |
| Random series | TTCGTTCGTTCGTTCGTTCGTTCGTTCGTTCGTT | 34 | |
| The total sequence of probe | TTCGTTCGTTCGTTCGTTCGTTCGTTCGTTCGTTGATAATTTGGCTTT ACAACCTTGA | 59 |
2.PCR reaction
Reaction mixture is: 200 μ M dATP, 200 μ M dTTP, 200 μ M dCTP, 200 μ MdGTP, 2mM MgCl
2, 2U Taq archaeal dna polymerase, the upstream and downstream primer of each 1 μ M, 1 μ l genomic templates (concentration is greater than 5ng/ μ l).Reaction conditions is: 94 ℃ 30 seconds, 62 ℃ 30 seconds, 72 ℃ 45 seconds, circulate 30 times.
3.LDR reaction
Reaction mixture is: 20mM Tris-HCl, pH8.3 (at25 ℃), 50mM KCl, 10mM MgCl
2, 1mM EDTA, 1mM NAD+, 10mM DTT, 0.1% (v/v) Triton X-100, each 0.01 μ M of probe, Taq ligase 15U, 1 μ l pcr template DNA.Reaction conditions is: 94 ℃ 30 seconds, 60 ℃ 2 minutes, circulate 30 times.
4. going up the order-checking instrument detects
Result such as following table:
| rs348475(A/G) | rs499776(A/G) | rs2161811(A/G) | rs561712(A/G) | rs4646580(C/T) | rs4646642(C/T) | |
| Fig. 8 | A/G | G | A | A | C/T | C/T |
| Fig. 9 | A/G | A/G | A | A/G | C/T | C/T |
| Figure 10 | G | G | A | A | T | T |
Claims (7)
1. the PCR-LDR gene pleiomorphism of a fluorescence labeling probe detects sequence measurement, it is characterized in that this method may further comprise the steps:
(1) probe design
(1), designs the molecular weight series of a series of LDR products at the site of detecting;
(2) proprietary fluorescence labeling probe of design, this probe has high specificity, does not have homology with the sequence of known organism;
(3) proprietary template of design, this template has high specificity, does not have homology with the sequence of known organism;
(4) adjust label probe and detector probe length at 3 ' end of label probe and 5 ' end introducing random series of detector probe;
(2) PCR detects
Get DNA 1 μ l to be detected, each 10-30pmol of PCR primer, pyro polymerase, the pyro polymerase mixed liquor mutually mixes, the PCR reaction conditions is: 94 ℃ 30 seconds, 40-68 ℃ 30 seconds-2 minutes, 60-72 ℃ 45 seconds-2 minutes, circulation 25-40 time;
(3) LDR detects
Get PCR product 1 μ l, each 40fmol-10pmol of probe, ligase, the ligase damping fluid mutually mixes, the LDR reaction conditions is: 94 ℃ 30 seconds, 40-70 ℃, 1-10 minute, circulation 1-40 time;
(4) result detects
Get LDR product 1-5 μ l, mix the loading buffer of sequencing system, sample on the sequenator carries out LDR product isolation identification.
2. the PCR-LDR gene pleiomorphism of the described a kind of fluorescence labeling probe of root a tree name claim 1 detects sequence measurement, it is characterized in that wherein step () (4) is introduced template matched sequence and random series at 3 ' end of label probe, and 5 ' end introducing random series of detector probe, described random series is any sequence that does not have homology with the sequence of PCR product.
3. the PCR-LDR gene pleiomorphism of the described a kind of fluorescence labeling probe of root a tree name claim 1 detects sequence measurement, it is characterized in that step () (4) wherein respectively designs the detector probe and the label probe of 10-25 base length in the front and back in the mutational site that will detect, the design of label probe detection site is at 3 ' end, and the detection site design of detector probe is at 5 ' end.
4. the PCR-LDR gene pleiomorphism of the described a kind of fluorescence labeling probe of root a tree name claim 1 detects sequence measurement, it is characterized in that wherein step () (4) label probe and detector probe have close molecular weight.
5. the PCR-LDR gene pleiomorphism of the described a kind of fluorescence labeling probe of root a tree name claim 1 detects sequence measurement, it is characterized in that wherein the described label probe of step () (4) comprises three parts, first is and purpose template mating section, second portion is and is connected the template mating section, the random series of third part in first and second partial sequences, introducing simultaneously, be used for adjusting the length of label probe, length is 40-80bp; Described detector probe comprises two parts, and first is and purpose template mating section, and the random series of second portion for introducing is used for adjusting the length of LDR product, and length is 40-80bp; Detector probe can be two, three or four, and 5 ' end is detection site in the probe, and every group of detector probe that changes at nucleic acid list base by setting the length of detector probe, realizes the LDR product length that different nucleic acid bases is corresponding different.
6. the PCR-LDR gene pleiomorphism of the described a kind of fluorescence labeling probe of root a tree name claim 1 detects sequence measurement, it is characterized in that wherein step (three) LDR reaction conditions is: 94 ℃ 30 seconds, 45-60 ℃, 1-10 minute, circulation 1-40 time.
7. a PCR-LDR gene pleiomorphism as claimed in claim 1 detects the application of sequence measurement in HPLC or the high-resolution separate nucleic acid detection system of micro flow chip.
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| CN102586457B (en) * | 2012-03-14 | 2014-04-23 | 东华大学 | Single nucleotide polymorphism (SNP) classification method for identifying inbred line mice |
| CN103436603B (en) * | 2013-07-15 | 2015-01-21 | 东华大学 | PCR method for identification of genotypes of offspring mice |
| CN103352086A (en) * | 2013-07-29 | 2013-10-16 | 山东省农业科学院高新技术研究中心 | PCR-LDR kit for detecting 11 deaf susceptibility genes |
| JP6871160B2 (en) | 2014-10-08 | 2021-05-12 | コーネル・ユニバーシティーＣｏｒｎｅｌｌ Ｕｎｉｖｅｒｓｉｔｙ | Methods for Identifying and Quantifying Nucleic Acid Expression, Splice Variants, Translocations, Copy Counts, or Methylation Changes |
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| CN1330153A (en) * | 2000-06-19 | 2002-01-09 | 苏荣仁 | Pylorospirobacillus antigen in blood |
| US6368801B1 (en) * | 2000-04-12 | 2002-04-09 | Molecular Staging, Inc. | Detection and amplification of RNA using target-mediated ligation of DNA by RNA ligase |
| CN1632132A (en) * | 2003-12-22 | 2005-06-29 | 华东理工大学 | Probe and its use in associated detection of gene polymorphous site |
| CN1908627A (en) * | 2006-08-16 | 2007-02-07 | 南通大学附属医院 | Method for HCV RNA real-time fluorescent detection with two probes |
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| US6368801B1 (en) * | 2000-04-12 | 2002-04-09 | Molecular Staging, Inc. | Detection and amplification of RNA using target-mediated ligation of DNA by RNA ligase |
| CN1330153A (en) * | 2000-06-19 | 2002-01-09 | 苏荣仁 | Pylorospirobacillus antigen in blood |
| CN1632132A (en) * | 2003-12-22 | 2005-06-29 | 华东理工大学 | Probe and its use in associated detection of gene polymorphous site |
| CN1908627A (en) * | 2006-08-16 | 2007-02-07 | 南通大学附属医院 | Method for HCV RNA real-time fluorescent detection with two probes |
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