CN104263827A - Novel probe design method for ligase reaction - Google Patents
Novel probe design method for ligase reaction Download PDFInfo
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Abstract
The invention relates to a probe design method and application in ligase-mediated nucleic acid amplification reaction. In the ligase-mediated nucleic acid amplification reaction, one target sequence can be amplified by using 1-2 pairs of probes, and each probe is characterized by comprising a complementary area which is strictly complementary to the target sequence, a filling area (optional) for adjusting the length of each probe, a primer area (optional) used as a PCR primer and an extension area, wherein the length of the extension area is 5-20bp, and the extension area is complementary to a nucleotide sequence of the front end or tail end of the complementary area, namely, the nucleotide sequence of the extension area is determined by the target sequence; when each probe is not combined with the target sequence, the probe is of a stem-and-loop structure; and under a certain condition, a pair of the probes are combined with the target sequence and are connected with the target sequence to form a complete oligonucleotide chain by virtue of the ligase. The probe design method disclosed by the invention can be used for reducing non-specific amplification and avoiding false positive, and is particularly suitable for multiple detection of nucleic acids.
Description
Technical field
The present invention relates to the probe design process of molecular biology and medical science amplifying nucleic acid amplified reaction, be specifically related to the probe design process in ligase enzyme mediation nucleic acid amplification reaction and application.
Technical background
Multiplex ligation-dependent probe amplification (multiplex ligation-dependent probe amplification, MLPA) is a kind of new technology of carrying out quantitative and semi-quantitative analysis for DNA sequence dna to be checked first reported by Schouten etc. for 2002.The ultimate principle of MLPA is: probe and target sequence DNA are hybridized, and afterwards by ligase enzyme connection, pcr amplification, product is by capillary electrophoresis separation, and analysis software is reached a conclusion to after the data analysis collected.In MLPA reaction, each target sequence that increases needs a pair probe, and each probe comprises one section of primer sequence and one section of specific sequence.After this pair probe and target sequence are hybridized, then be connected to become an oligonucleotide chain with ligase enzyme.Ligation high special, only have when two probes and target sequence are hybridized completely, i.e. target sequence and probe-specific sequence complete complementary, two sections of probes could be connected into a complete single nucleic acid strands by ligase enzyme; Otherwise, if target sequence and probe sequence are not exclusively complementary, even if only have the difference of a base, also can cause hybridization not exclusively, ligation cannot be carried out.After ligation completes, to increase the probe connected with a pair universal primer, the length of the amplified production of each probe is unique, and scope is between 130 ~ 480bp.Finally, by capillary electrophoresis separation amplified production, reach a conclusion after analyzing with Genemarker software.If the target sequence origination point sudden change detected or disappearance, amplification sudden change, so the amplified peak of correspondent probe just can lack, reduces or increase, therefore, just can judge whether target sequence has the exception of copy number or point mutation existence according to the change of amplified peak.Current MLPA technology has been applied to the research of multiple field, various diseases.
Ligase chain reaction (ligase chain reaction, LCR) by Landegree in first Application in 1988 in the molecular diagnosis of sickle-cell anemia.Be after PCR, emerging a kind of more perfect DNA amplification in vitro and detection technique, have unique advantage in check point sudden change etc.Synthesize a pair probe A, B, their complete and target sequence strictly complementaries.After annealing is combined with target sequence, between A, B, stay next breach (nick), after linked enzyme connection, become complete, and the nucleotide chain of target complement sequence.If have point mutation in target sequence, probe accurately can not be combined with target sequence, and near breach, the space structure of Nucleotide changes, and ligation can not be carried out, and after sex change, probe is still two.The people such as Wu introduce again the principle of PCR, and after ligation, sex change, annealing, to connect, a small amount of target sequence is just amplified out again.Barany reported the discovery of thermostable ligase-Taq ligase enzyme and the application in LCR thereof in 1991, for the practical of LCR is laid a good foundation.During actually operating, probe is 2 right, A, A ' and B, B ', respectively with the positive strand complementary of target sequence.The product of each ligation can work as template again in next round reaction, increases out with exponential form by target sequence.
MLPA technology and LCR technology all can be used for Multiple detection, by rational probe design, can detect multiple target sequence in primary first-order equation simultaneously.When adopt capillary electrophoresis amplified production is separated, analyze, the target sequence simultaneously detected can reach 45.Multiple detection method is conducive to improving detection efficiency, reduces the usage quantity of template, is particularly useful for the few and situation that target spot to be measured is more of template amount.But in traditional MLPA, LCR and other ligase enzymes mediation nucleic acid amplification reaction, due to the kind of probe and quantity more, usually there is non-specific amplification band, affect detected result.The present invention improves on traditional probe structure, changes linear probe into cycling probe, can reduce non-specific amplification, improves detection sensitivity.
Summary of the invention
The invention provides a kind of method of design at ligase enzyme mediation nucleic acid amplification reaction middle probe and application, effectively can reduce non-specific amplification, avoid false positive.
This probe design process is applicable to MLPA nucleic acid amplification reaction, and be particularly useful for MLPA multiple nucleic acid amplified reaction, probe structure and principle of design are shown in Fig. 1.Need a pair probe to detect target sequence in the method, contain with the complementary district of target sequence strictly complementary in the structure of probe, regulate the fill area of probe length, as the guiding region of PCR primer and the extension area (B, D district see in Fig. 1) complementary with the Nucleotide (A, C district see in Fig. 1) of front end, complementary district or end.The length of extension area is 5 ~ 20 Nucleotide, and with the Nucleotide strictly complementary of front end, complementary district or end, namely A, B district is complementary; C, D district is complementary.The sequence in B district is determined by A district; The sequence in D district is determined by C district.The primer sequence in PCR primer district can be universal primer or Auele Specific Primer.Can not comprise sudden change in probe or comprise sudden change, mutating alkali yl position within the probe can be positioned at last (when complementary district is positioned at 3 ' end of probe) or first (when complementary district is positioned at 5 ' end of probe).
This probe design process is applicable to LCR nucleic acid amplification reaction, and be particularly useful for LCR multiple nucleic acid amplified reaction, probe structure and principle of design are shown in Fig. 2.A pair probe or two pairs of probes can be used in the method to detect target sequence, when use two pairs of probes, the two pairs of probes respectively with the positive strand complementary of target sequence.Contain with the complementary district of target sequence strictly complementary in the structure of probe, regulate the fill area of probe length and the extension area (B, D district see in Fig. 2) complementary with the Nucleotide (A, C district see in Fig. 2) of front end, complementary district or end.The length of extension area is 5 ~ 20 Nucleotide, and with the Nucleotide strictly complementary of front end, complementary district or end, namely A, B district is complementary; C, D district is complementary.The sequence in B district is determined by A district; The sequence in D district is determined by C district.Can not comprise sudden change in probe or comprise sudden change, mutating alkali yl position within the probe can be positioned at last (when complementary district is positioned at 3 ' end of probe) or first (when complementary district is positioned at 5 ' end of probe).
This probe design process is equally applicable to the ligase enzyme mediation nucleic acid amplification reaction except MLPA, LCR.
The methods such as agarose electrophoresis, polyacrylamide gel electrophoresis and capillary electrophoresis can be adopted to detect the nucleic acid fragment of amplification.
Because the Nucleotide quantity of extension area is less than complementary district, the combination in complementary district and target sequence will do not affected when annealing; The probe be not combined with target sequence will revert to stem loop type, effectively prevent the non-specific binding with template, avoid non-specific amplification and false-positive result, improve the sensitivity of detection.
When carrying out Multiple detection, due to the kind of probe and quantity more, probably occur that different probes matches situation about combining mutually, causes dimeric appearance, interference detection results.And probe of the present invention when not with target sequence in conjunction with will revert to stem ring-type, effectively prevent dimeric generation.
Accompanying drawing explanation
Fig. 1 is structure and the principle of design of MLPA nucleic acid amplification reaction middle probe.
Fig. 2 is structure and the principle of design of LCR nucleic acid amplification reaction middle probe.
Fig. 3 is capillary electrophoresis collection of illustrative plates.
Embodiment
With specific embodiment, the present invention is described in further detail below.Should be understood that, specific embodiment is only make clearer explanation to the present invention, instead of limitation of the present invention.
Embodiment 1:
Take clinical diagnosis as the blood of non-Hodgkin lymphoma (non-Hodgkin ' s lymphoma, NHL) patient be sample, adopt
dNA Blood Mini Kit (Qiagen) test kit extracts poba gene group DNA, detects IL-10 promoter region pleomorphism site rs1800896 and rs1800871 by MLPA method.
According to probe design process of the present invention, design is applicable to this probe detected, and probe sequence is in table 1:
Table 1: the probe sequence detecting rs1800896 and rs1800871
Hybridization: probe is dissolved in TE, is mixed with the solution of 5nM.According to the hybridization system preparation reaction solution of table 2 in PCR pipe, hybridize in 60 DEG C of insulation 16 ~ 20h.
Connect: after hybridization terminates, in PCR pipe, add ligase enzyme damping fluid 3uL, ligase enzyme 0.5uL, ultrapure water 6.5uL, in 54 DEG C of insulation 20min, 98 DEG C of heating 5min, then be cooled to 20 DEG C.
PCR: connect after terminating, add universal primer 1uL, polysaccharase 0.5uL, water 3.5uL in PCR pipe, the PCR response procedures according to table 3 carries out PCR reaction.
Table 2: hybridization system
Table 3:PCR response procedures
After PCR reaction terminates, get after 10uL amplified production mixes with 6 × DNA Loading Buffer, add in the well of 4% sepharose, with 5V/cm electrophoresis after 45 minutes, under gel imaging instrument, observe electrophoresis result.If there is pcr amplification band, illustrate that detected result is for positive.When wherein rs1800896 is positive, pcr amplification band is about 90bp; When rs1800871 is positive, amplified band is about 110bp.
In this embodiment, with the blood of 27 parts of non-Hodgkin lymphoma patients for sample, wherein in 9 increment product, rs1800896 is positive; In 16 increment product, rs1800871 is positive.
Embodiment 2:
With the DNA extracted in bone-marrow-derived lymphocyte LY-01 for sample, LCR method is adopted whether extremely to detect the copy number of gene C 1qA.
According to probe design process of the present invention, design is applicable to this probe detected, and probe sequence is in table 1:
Table 1: the probe sequence detecting gene C 1qA
LCR reacts: be dissolved in by probe in TE, be mixed with the solution of 5nM.According to the LCR reaction system preparation reaction solution of table 2 in PCR pipe, PCR pipe is placed in PCR instrument, reacts by the response procedures of table 3.
Table 2:LCR reaction system
Table 3:LCR response procedures
After LCR reaction terminates, adopt capillary electrophoresis to analyze amplified production, C1qA gene amplification fragment length is 100bp, and Fig. 3 is shown in by capillary electrophoresis collection of illustrative plates.Compared with the copy number of house-keeping gene, in this embodiment, in bone-marrow-derived lymphocyte LY-01 there is abnormal increasing in the copy number of C1qA gene.
Claims (7)
1. the novel probe method of design for ligase enzyme reaction, it is characterized in that: the target sequence that increases can use 1 ~ 2 pair of probe, contain with the complementary district of target sequence strictly complementary in the structure of probe, regulate the fill area of probe length (optional), as the guiding region (optional) of PCR primer and extension area.The length of extension area is 5 ~ 20bp, with the nucleotide sequence complementary of front end, complementary district or end.
2. as claimed in claim 1 a kind of for ligase enzyme reaction novel probe method of design, it is characterized in that: the nucleotide sequence complementary of extension area and front end, complementary district or end, namely the sequence of extension area is determined by target sequence.
3. as claimed in claim 1 a kind of for ligase enzyme reaction novel probe method of design, it is characterized in that: the length of extension area is 5 ~ 20bp.
4. as claimed in claim 1 a kind of for ligase enzyme reaction novel probe method of design, it is characterized in that: the target sequence that increases can use 1 ~ 2 pair of probe, in a pair probe, wherein 3 ' the end of is target sequence cog region, and another 5 ' end is target sequence cog region.
5. a kind of novel probe method of design for ligase enzyme reaction as claimed in claim 1, is characterized in that: not with target sequence in conjunction with time, 5 ' end of probe hold combine with 3 ', formation loop-stem structure.
6. as claimed in claim 1 a kind of for ligase enzyme reaction novel probe method of design, it is characterized in that: can not comprise sudden change in probe or comprise sudden change, mutating alkali yl position within the probe can be positioned at last (when complementary district is positioned at 3 ' end of probe) or first (when complementary district is positioned at 5 ' end of probe).
7. a kind of novel probe method of design for ligase enzyme reaction as claimed in claim 1, is characterized in that: the method is applicable to the nucleic acid amplification reaction of the ligase enzyme such as MLPA, LCR mediation.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105525010A (en) * | 2016-01-28 | 2016-04-27 | 陕西师范大学 | Stem-loop structured combined probe and application thereof |
| CN105821138A (en) * | 2016-05-12 | 2016-08-03 | 陕西师范大学 | Method for constructing double-stem-loop structure DNA template to detect nucleic acid based on ligation reaction |
| CN112553310A (en) * | 2020-08-07 | 2021-03-26 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Method and kit for detecting cytogenetic abnormality by applying MLPA (MLPA) |
| CN114410754A (en) * | 2022-01-27 | 2022-04-29 | 国科宁波生命与健康产业研究院 | CDA probe primer group and kit for detecting miRNA and application of CDA probe primer group and kit |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101200769A (en) * | 2007-11-22 | 2008-06-18 | 上海交通大学 | Detection method discriminating medicinal lucid ganoderma by using special gene sequence |
| CN102559661B (en) * | 2012-01-18 | 2014-06-04 | 厦门基科生物科技有限公司 | Novel amplification method and application of ligase reaction mediate |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101200769A (en) * | 2007-11-22 | 2008-06-18 | 上海交通大学 | Detection method discriminating medicinal lucid ganoderma by using special gene sequence |
| CN101200769B (en) * | 2007-11-22 | 2010-06-30 | 上海交通大学 | Detection method for discriminating medicinal lucid ganoderma by using special gene sequence |
| CN102559661B (en) * | 2012-01-18 | 2014-06-04 | 厦门基科生物科技有限公司 | Novel amplification method and application of ligase reaction mediate |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105525010A (en) * | 2016-01-28 | 2016-04-27 | 陕西师范大学 | Stem-loop structured combined probe and application thereof |
| CN105821138A (en) * | 2016-05-12 | 2016-08-03 | 陕西师范大学 | Method for constructing double-stem-loop structure DNA template to detect nucleic acid based on ligation reaction |
| CN105821138B (en) * | 2016-05-12 | 2018-05-08 | 陕西师范大学 | A kind of method that double loop-stem structure DNA profiling detection nucleic acid are built based on coupled reaction |
| CN112553310A (en) * | 2020-08-07 | 2021-03-26 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Method and kit for detecting cytogenetic abnormality by applying MLPA (MLPA) |
| CN114410754A (en) * | 2022-01-27 | 2022-04-29 | 国科宁波生命与健康产业研究院 | CDA probe primer group and kit for detecting miRNA and application of CDA probe primer group and kit |
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Application publication date: 20150107 |