CN102102122B - Design method of oligonucleotide probe and application thereof - Google Patents
Design method of oligonucleotide probe and application thereof Download PDFInfo
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- CN102102122B CN102102122B CN 200910214074 CN200910214074A CN102102122B CN 102102122 B CN102102122 B CN 102102122B CN 200910214074 CN200910214074 CN 200910214074 CN 200910214074 A CN200910214074 A CN 200910214074A CN 102102122 B CN102102122 B CN 102102122B
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Abstract
The invention relates to a design method of an oligonucleotide probe, and the oligonucleotide probe designed by the method can effectively distinguish the mononucleotide polymorphism. The design method of an oligonucleotide probe of the invention is applicable to all probe design fields including the preparation of gene chips. The method does not use base analogues, but uses A/T/G/C and adopts a way of manual addition or reduction of bases. Based on the detection effects, the probe designed by the method of the invention is superior to traditional probes in specificity, and no significant difference exists between the two probes in sensitivity.
Description
Technical field
The present invention relates to a kind of method of design of oligonucleotide probe, utilize the oligonucleotide probe of this method design can effectively distinguish single nucleotide polymorphism.The method of design of oligonucleotide probe of the present invention can be applicable to comprise the field of all probe design of the preparation of gene chip.
Background technology
(single nucleotide polymorphism SNP), mainly refers on genomic level by the caused dna sequence polymorphism of the variation of single Nucleotide single nucleotide polymorphism.It is modal a kind of in human heritable variation, accounts for more than 90% of all known polymorphisms.SNP extensively exists in human genome, just has 1 in average per 500～1000 base pairs, estimates that its sum can reach 3,000,000 even more.
Estimate that SNP will play a significant role in following field from now on: (1) carries out genetic linkage analysis (linkage analysis) and the association analysis (association analysis) of simple and complex disease; (2) in " pharmacogenomics " (pharmacogenomics) studied, can be by Different Individual among the genetic polymorphism mark announcement crowd who detects SNP to the basic reason of the sensitivity differences of different pharmaceutical.(3) also can be used for the discriminating, paternity test etc. of criminal's identity of legal medical expert research, in the pairing selection in this external organ transplantation between donor and acceptor and the research of spore all with significant.
Can reach than higher sensitivity and specificity by designing specific probe in detecting SNP, but single nucleotide mutation requires high to probe design, how fast and effectively the probe of single nucleotide mutation can be correctly distinguished in design, is the problem that academia and company researcher put concern always.People (Loakes D such as Loakes D, et al.NucleicAcids Res.1994,22 (20): 4039-43) propose 5-Nitroindole as base analogue, people (David Loakes such as David Loakes subsequently, et al.Nucleic Acids Res.1995,23 (13): 2361-6) further propose to use 3-Nitropyrrole and 5-nitroindole as order-checking and PCR analogue, people such as Guo Z report (Guo Z on this basis, et al.NatBiotechnol.1997,15 (4): result of study 331-5) is a landmark achievement, main points have following several: (1) mononucleotide is the most indistinguishable, and two or more Nucleotide then can effectively be distinguished; (2) single nucleotide mutation detects the location dependency, and specifically, sudden change position separating capacity in the middle of probe is the strongest, and more past probe two ends separating capacity is more low; (3) base analogue is set and forms artificial mispairing in probe sequence, can effectively improve the mononucleotide separating capacity.Subsequently, many pieces of further research reports are arranged.By model (Pozhitkov AE, the et al.BMC Bioinform 2002 that makes up; 3:9; Held GA, et al.Proc Natl Acad Sci USA, 2003; 100:7575-80) select the reasonableness of designing probe to be paid close attention to widely, yet other investigator finds that but still there are big difference (Naef F in theoretical simulation and experimental result, et al.Phys Rev E Stat Nonlin Soft Matter Phys, 2003; 68:011906; Mei R et al.Proc Natl Acad SciUSA, 2003; 100:11237-42), that is to say that probe design is still mysterious, theoretical investigation does not also reach the degree of depth of practical application.Nonetheless, people (Held GA, et al.Proc Natl Acad Sci USA, 2003 such as Held GA; 100:7575-80) think that still probe design need follow certain principle, pattern remains effectively, is mandatory and the probe mutational site is in the probe mid-way.
3-Nitropyrrole and 5-nitroindole are not proved validity widely as base analogue in some experiments, therefore, people such as BurgnerD (Burgner D, et al.Nucleotides Nucleic Acids.2004,23 (5): 755-65) propose the approach of another solution, specifically, in probe, directly add base analogue in the hope of being able to usually in the method for all SNPs probe design.After this, still constantly appear at various research periodicals and patent database about probe design and Study on Theory, confirm that from a side people's such as Burgner D method does not obtain a general effect.DF doctor's Qualley in 2007 a report (http: // 66.102.1.104/scholar? q=cache:rGEh8ixkw60J:scholar***.com/+%22Synthesis+and+Survey+of+Non-natural+Bases+in+Nucleic+Acids+%22﹠amp; Hl=en) propose, the hydrogen bond of base is formed the position sealing, make this analogue not form combination with any base of target gene, cooperate with mutator gene in conjunction with empty thereby so just between probe and target gene, form one, be equivalent to double alkali yl and suddenly change.A weak point of this method is: the sealing base needs special reaction to increase synthetic cost, and can not finish corresponding service in commercial Synesis Company, therefore still can't effectively promote.Nearest another piece of writing report (Hwang GT, Nucleic Acids Res.2009Jun 10.[Epub ahead ofprint]) thinking of another solution then is provided: the aromatic nucleus to base carries out the modification of different sites and the modification of a plurality of parts, d34DMPy is mixed in final discovery can reach best effect as artificial mutation, yet this conclusion and DF doctor's Qualley method faces same problem, namely only can can't enter the commercial applications stage in the laboratory realization.
Gene chip is the forward position biotechnology that the nineties grows up.It has merged multi-disciplinary state-of-the-art technologies such as life science, chemistry, microelectronics, computer science, statistics and life-information.Gene chip claims oligonucleotide chip or DNA chip again, by a large amount of dna segments in addressable mode, be fixed to nylon membrane to high-density, on the carriers such as sheet glass and silicon chip, utilize the pairing between the nucleic acid base, be used for carrying out the instrument of sample DNA high-throughput, parallel analytical information.The sharpest edges of gene chip are high-throughput, parallel parsing.And this advantage just also becomes the weakness of gene chip maximum, and except distinguishing the single nucleotide mutation, a large amount of probes also will be in parallel detection under the identical conditions, and this just has higher requirement to probe design.People (S Choi such as S Choi for example, et al.US PatentApp.11/335,229) method of a kind of obstacle DNA and artificial mismatch probe (hurdle DNA and artificially mismatched probe) is disclosed, this method needs two probes to realize the detection in a site, because just a probe can detect a site usually, the quantity of probe must double like this, undoubtedly the point sample of probe and the preparation of superchip have been increased very big difficulty, realize that as far as possible a probe just effectively detects a site, this remains the Consideration of actual application.
So far, as if probe design enters into the artificial reconstructed of an eternal end and seeks a kind of wrong path of omnipotent method.The present invention just is being based on such technical bottleneck, proposes a kind of new probe design method, can design the probe of effective differentiation single nucleotide mutation faster.
Summary of the invention
The method of design that the purpose of this invention is to provide a kind of oligonucleotide probe utilizes the oligonucleotide probe of this method design can effectively distinguish single nucleotide polymorphism.
The differentiation effect of single nucleotide mutation depends on the separating capacity of probe in homology sequence, and this has also directly determined the accuracy of gene chip (microarray) experimental result.From initial Southern Blot probe to the Microarray probe, investigators a large amount of between decades have carried out a large amount of theoretical investigationes and experiment confirm to the method for design of probe, and it has been generally acknowledged that the probe of higher separating capacity possesses following feature: (1) probe self does not have hairpin structure; (2) design of probe mutational site is at the middle part of probe; (3) GC content is at 40-60%; (4) there is not serious complementary pairing between probe and the probe, as basic theories, derived multiple predictive model, wherein, what extensively admitted is ortho position hybridization predictive model (nearestneighbor hybridization potential), it has been generally acknowledged that, low 3-25 ℃ of the neighbour position hybridization of the hybridization temperature of probe predicted temperature, a plurality of probe design softwares as model are arranged, as: melting (Le Novere, 2001) or mfold (Zuke r, 2003), because the separating capacity of probe is low, theoretical prediction is inaccurate to be still main bottleneck.Therefore, the method for design of probe normally is lower than many probes of 5-20 ℃ of design of theoretical prediction value, screens by experiment then.Recently (Xu Q, et al.Proc Natl Acad Sci USA.2009,106 (7): thus 2289-94.) just reported result of study by gene chip designing probe matrix screening probe.
The method of design of probe provided by the present invention is as follows:
1) according to the specific nucleic acid sequence synthesising probing needle of target gene to be detected.Require probe to cover base to be detected, length is 14～30 bases, and probe sequence can be positive-sense strand or its reverse complementary sequence of target-gene sequence;
2) probe sequence should not have higher similarity in template, otherwise causes mispairing easily.Probe sequence avoids occurring the continuous base more than 3.
3) the GC content of probe sequence is generally 40～60%, the too high or too low specific detection that all is unfavorable for.
4) avoid forming hairpin structure more than 3 in the probe sequence.
5) probe of single base mutation, mutational site are in the middle of the probe as much as possible, are at least in the penult base of probe.
6) preferred positions is that four bases of penult to the of probe manually increase or reduce, and the base of sudden change is in the other end of probe, but is not in end, and the position is the 3rd in the middle of the base number usually.
7) probe design method provided by the present invention, preferred probes length is 14～30bp.
Have stronger separating capacity according to the designed probe of method provided by the invention, thereby significantly reduced the screening operation amount of probe.
Probe design method of the present invention, an embodiment preferred is that its middle probe manually increases base, may further comprise the steps:
(1) according to the both sides that increase base, if T (A) * T (A), then the base of Tian Jiaing (*) is G or C;
(2) according to the both sides that increase base, if G (C) * G (C), then the base of Tian Jiaing (*) is A or T;
(3) according to the both sides that increase base, if T (A) * G (C), then the base of Tian Jiaing (*) is T or A;
(4) according to the both sides that increase base, if G (C) * T (A), then the base of Tian Jiaing (*) is T or A.
Probe design method of the present invention, an embodiment preferred is that its middle probe manually reduces base, may further comprise the steps:
(1) according to the both sides of reducing base, if T (A) * T (A), then the base of Jian Shaoing (*) is G or C;
(2) according to the both sides of reducing base, if G (C) * G (C), then the base of Jian Shaoing (*) is A or T;
(3) according to the both sides of reducing base, if T (A) * G (C), then the base of Jian Shaoing (*) is T or A;
(4) according to the both sides of reducing base, if G (C) * T (A), then the base of Jian Shaoing (*) is T or A.
The Application Areas of probe design method of the present invention is very extensive, and every field of using probe design is all applicable, comprising based on the gene chip of nylon membrane with based on the gene chip of slide, but not only is confined to the application in gene chip field.
The innovation part of people's such as the present invention and Burgner D method maximum is not use base analogue, and is to use A/T/G/C, and base is optionally used to make and distinguished effect and reach best; Utilize artificial increasing or the minimizing base simultaneously, need not special reagent, program that can be identical with common probe is synthetic, thereby does not need to increase extra cost.Utilize present method designed probe to compare with traditional probe, no significant difference in the sensitivity, and specificity is better than conventional probe.Based on above reason, method of the present invention can be promoted in actual applications.The method of the disappearance base that the present invention proposes is not seen relevant report as yet in disclosed data.
Description of drawings
Fig. 1 probe of the present invention and conventional probe detect MDRI gene C-4T and A61G site result contrast.Wherein A is that C-4T is normal, and A61G is normal; B is the sudden change of C-4T homozygote, and A61G is normal; C is that C-4T is normal, the sudden change of A61G homozygote.
Fig. 2 probe of the present invention and conventional probe detect beta Thalassemia CD26 and CD43 site result contrast.Wherein 1 is that CD26 is normal, and CD43 is normal; 2 are the sudden change of CD26 homozygote, and CD43 is normal; 3 is that CD26 is normal, the sudden change of CD43 homozygote.
Embodiment
The description of embodiment of the present invention is intended to illustrate by way of example and understands essence of the present invention better, rather than restriction the present invention.
Embodiment 1 detects the probe design of the gene chip of MDRI gene SNP s
(multi-drug resistance gene MDRI) is positioned at No. 7 karyomit(e) q21.1 of people to multidrug resistance gene, is the method for design of example explanation probe with C-4T.Be the sequence (LOCUS:AY910577) in MDRIC-4T site below:
1 CTCTGGCTTC?GACGGGGGAC?TAGAGGTTAG?TCTCACCTCC?AGCGCGCCTG?AGGCTCATGC
61 ATTTGGCTAA?TGAGCTGCGG?TTTCTCTTCA?GGT
GGGATG?GATCTTGAAG?GGGACCGCAA
121?TGGAGGAGCA?AAGAAGAAGA?ACTTTTTTAA?ACTGAACAAT?AAAAGGTAAC?TAGCTTGTTT
181?CATTTTCATA?GTTTACATAG?TTGCGAGATT?TGAGTAATTT?ATTTCTAGCC?TCCAGCTCTG
241?AAATAAATGA?CATGTTGTTG?TTTTTAATTA?TTTTTAAGAA?ACGCAAGCTA?GCCTTTGGAA
301?TCAATATCCC?TGCTTAGAGC?AGAAGTTTGT?TGGCTGAGTG?GAGCACAGCA?TATGCATTTT
In above-mentioned sequence,
Be the site of undergoing mutation, namely wild-type (Normal) is C, and mutant (Mutation) is T.Mutational site and artificial insertion site are distributed in the two ends of probe during probe design, the rule of probe design is considered in best site simultaneously, comprise: the secondary structure of annealing temperature (Tm), GC% content, base quantity and probe, use method of the present invention, the wild-type probe sequence of design is 5 '-AGGTCGGGATGGATAC-3 ' (SEQ IDNO:2), 5 '-AGGTCGGGATGGATCT-3 ' (SEQ IDNO:3); The mutant probe sequence is 5 '-AGGTTGGGATGGAGTC-3 ' (SEQ ID NO:5), 5 '-AGGTTGGGATGGATCT3 ' (SEQ IDNO:6).The probe design method in A61G site can be with reference to C-4T.
The gene chip of embodiment 2 slide carriers detects MDRI gene C-4T and A61G site
The gene chip that utilizes embodiment 1 designed probe to prepare the slide carrier detects MDRI gene SNP s, and compares with the gene chip of the probe preparation of bibliographical information.
(multi-drug resistance gene MDRI) is positioned at No. 7 karyomit(e) q21.1 of people to multidrug resistance gene, and its protein product p-GP is important a member of ABC haulage system, belongs to ABCB1 family.The MDRI gene expression dose can be used as a reference index of prediction chemotherapy effect, predicts that by detecting the MDRI gene pleiomorphism chemotherapeutic efficacy of tumour patient becomes a kind of feasible clinical detection means.The PCR primer sequence that uses is respectively:
1F：5’TTGGCTAATGAGCTGCGGTTTCTC?3’(SEQ?ID?NO：25)；
1R：5’cy3-GATTCCAAAGGCAGCTTGCGTTTC?3’(SEQ?ID?NO：26)。
The amplified fragments of above-mentioned sequence is 240bp, detects two SNPs site C-4T, A61G of exon 2.
A difference of the present invention and bibliographical information probe is to use artificial mismatch probe, thereby further improves the differentiation mononucleotide ability of probe.The probe that uses is as shown in the table:
Annotate: SEQ ID NO:1～SEQ ID NO:12 be the connecting arm that 5 ' amino labeled connects 6 T (5 '-NH2-spacer-)
The probe array of gene chip is:
First row---1N1,1N1,1N2,1N2,1N3, the 1N3; Second row---1M1,1M1,1M2,1M2,1M3, the 1M3;
Second row---2N1,2N1,2N2,2N2,2N3, the 2N3; Fourth line---2M1,2M1,2M2,2M2,2M3,2M3
25ul PCR reaction system includes: 1 * PCR damping fluid (10mM Tris-HCl (pH8.30), 50mM KCl, 1.5mM MgCl
2), 200mM dNTPs, 10uM 1F, 200uM 1R, the 100ng genomic dna, 1.5U Taq enzyme, reaction system is at 94 ℃ of pre-sex change 5min, then with 94 ℃, 30s → 58 ℃, 30s → 72 ℃, 30s moves 40 circulations, and last 72 ℃ are extended 5min.42 ℃ of hybridization of PCR product 2 hours, other NM working method and reagent preparation are with reference to " biochip technology and application detailed annotation " ([U.S.] G. Ha Deman, Chen Zhongbin etc. translate, Chemical Industry Press in 2006).
Utilization confirms it is respectively C-4T wild-type, A61G wild-type through order-checking, C-4T suddenlys change, and homozygote, A61G wild-type and C-4T are normal, A61G sudden change homozygote three routine samples, use bibliographical information probe and the inventive method designing probe to detect, experimental result confirms: method designed probe of the present invention, specificity is better than the bibliographical information probe, sensitivity simultaneously and bibliographical information probe no significant difference.The result is referring to accompanying drawing 1.
Embodiment 3 detects the probe design of the gene chip of beta Thalassemia transgenation
(β-Globin) bunch is positioned at 11p15.5 to the human beta globin protein gene.The generation of beta Thalassemia (it is poor to be called for short β ground) mainly is that minority is genetically deficient owing to gene mutations.Be β-Globin CD26 (sequence (LOCUS:NC 000011) of G → A) below
1 TGACTCCTGA?GGAGAAGTCT?GCCGTTACTG?CCCTGTGGGG?CAAGGTGAAC?GTGGATGAAG
61 TTGGTGGT
A?GGCCCTGGGC?AGGTTGGTAT?CAAGGTTACA?AGACAGGTTT?AAGGAGACCA
121?ATAGAAACTG?GGCATGTGGA?GACAGAGAAG?ACTCTTGGGT?TTCTGATAGG?CACTGACTCT
181?CTCTGCCTAT?TGGTCTATTT?TCCCACCCTT?AGGCTGCTGG?TGGTCTACCC?TTGGACCCAG
241?AGGTTCTTTG?AGTCCTTTGG?GGATCTGTCC?ACTCCTGATG?CTGTTATGGG?CAACCCTAAG
301?GTGAAGGCTC?ATGGCAAGAA?AGTGCTCGGT?GCCTTTAGTG?ATGGCCTGGC?TCACCTGGAC
In above-mentioned sequence,
Be the site of undergoing mutation, namely wild-type (Normal) is G, and mutant (Mutation) is A.Mutational site and artificial insertion site are distributed in the two ends of probe during probe design, the rule of probe design is considered in best site simultaneously, comprise: the secondary structure of annealing temperature (Tm), GC% content, base quantity and probe, use method of the present invention, the wild-type probe sequence of design is 5 ' GACCCAGGGCCTCACC-3 ' (SEQ ID NO:14), 5 ' AGAAGTTGGTGGTGAGG-3 ' (SEQ ID NO:15); The mutant probe sequence is 5 '-GTAAGTTGGTGGTAAGG-3 ' (SEQ ID NO:17), 5 ' AGAAGTTGGTGGTAAGG-3 ' (SEQ IDNO:18).(the probe design method in the site of G → T) can be with reference to CD26 (G → A) for CD43.
Embodiment 4 nylon membranes are that the gene chip of carrier detects beta Thalassemia gene C D26 and CD43 site
The gene chip that utilizes embodiment 3 designed probe to prepare the slide carrier detects beta Thalassemia gene C D26, the CD43 site mutation, and compare with the gene chip of the probe preparation of bibliographical information.
β-thalassemia is a kind ofly to cause the peptide chain imbalance of expression unusually and the monogenic inheritance hemopathy that produces by beta globin genes, how by due to the beta-globin point mutation, is one of inherited disease that each province, China south is the most common, harm is maximum.Still do not have specificity therapeutic method at present, and carry out effective molecular diagnosis and prenatal gene diagnosis is the effective ways of present prenatal and postnatal care.The PCR primer sequence that uses is respectively:
2F：5’Biotin-GTACGGCTGTCATCACTTAGACCTCA-3’(SEQ?ID?NO：27)；
2R：5’-Biotin-TGCAGCTTGTCACAGTGCAGCTCACT-3’(SEQ?ID?NO：28)
The amplified fragments of above-mentioned sequence is 602bp, detects the CD26 of β-Globin, the CD43 site.A difference of the present invention and bibliographical information probe is to use artificial mismatch probe, thereby further improves the differentiation mononucleotide ability of probe.The probe that uses is as shown in the table:
Annotate: SEQ ID NO:13～SEQ ID NO:24 be 5 ' amino labeled (5 '-NH2-)
The probe array of gene chip is:
First row---3N1,3N2,3N3,3M1,3M2, the 3M3; Second row---4N1,4N2,4N3,4M1,4M2, the 4M3;
25ul PCR reaction system includes: 1 * PCRbuffer (10mM Tris-HCl (pH8.30), 50mM KCl, 1.5mM MgCl2), 200mMdNTPs, 200uM 1F, 200uM 1R, the 100ng genomic dna, 1.5U the Taq enzyme, reaction system is at 94 ℃ of pre-sex change 5min, then with 94 ℃, 30s → 55 ℃, 30s → 72 ℃, 45s moves 40 circulations, and last 72 ℃ are extended 5min.45 ℃ of hybridization of PCR product 2 hours, other NM working method and reagent preparation are with reference to people (Chan V, et al.Br J Haematol, 1999,104 (3): experimental technique 513-5.) such as Chan V.
Utilization confirms it is respectively the normal people through order-checking, beta Thalassemia CD26 sudden change homozygote and beta Thalassemia CD43 sudden change homozygote three routine samples, use bibliographical information probe and the inventive method designing probe to detect, experimental result confirms: method designed probe of the present invention, specificity is better than the probe of bibliographical information, the probe no significant difference of sensitivity simultaneously and bibliographical information.The result is referring to accompanying drawing 2.
Sequence table
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＜120〉a kind of method of design of oligonucleotide probe and application thereof
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<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>19
cagaggttctttgagtcctt
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<211>21
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＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>20
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<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>21
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<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>22
agaagttggtggtaagg
<210>23
<211>20
<212>DNA
＜213〉artificial sequence
<220>
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<400>23
ctccagaggttcttttagtc
<210>24
<211>20
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<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>24
gaccagaggttcttttagtc
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<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as the PCR primer.
<400>25
ttggctaatgagctgcggtttctc
<210>26
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as the PCR primer.
<400>26
gattccaaaggcagcttgcgtttc
<210>27
<211>26
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＜213〉artificial sequence
<220>
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<400>27
gtacggctgtcatcacttagacctca
<210>28
<211>26
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<220>
＜223〉according to the specific nucleotide sequence design, to be used as the PCR primer.
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tgcagcttgtcacagtgcagctcact
Claims (1)
1. the method for design of an oligonucleotide probe is characterized in that this method of design comprises:
(1) require probe to cover base to be detected, length is 14～30 bases, and probe sequence is positive-sense strand or its reverse complementary sequence of target-gene sequence;
(2) probe sequence does not have higher similarity in template, and probe sequence avoids occurring the continuous base more than 3;
(3) the GC content of probe sequence is 40～60%;
(4) avoid forming the hairpin structure that 3 and above base form in the probe sequence;
(5) probe of single base mutation, mutational site are in the middle of the probe as much as possible, are at least in the penult base of probe;
(6) four bases of penult to the at probe manually increase or reduce, and the base of sudden change is in the other end of probe, but is not in end, and the position is the 3rd in the middle of the base number usually;
Its middle probe manually increases base, may further comprise the steps:
1) according to the both sides that increase base, if T (A) * T (A), then the base of Tian Jiaing (*) is G or C;
2) according to the both sides that increase base, if G (C) * G (C), then the base of Tian Jiaing (*) is A or T;
3) according to the both sides that increase base, if T (A) * G (C), then the base of Tian Jiaing (*) is T or A;
4) according to the both sides that increase base, if G (C) * T (A), then the base of Tian Jiaing (*) is T or A;
Its middle probe manually reduces base, may further comprise the steps:
1) according to the both sides of reducing base, if T (A) * T (A), then the base of Jian Shaoing (*) is G or C;
2) according to the both sides of reducing base, if G (C) * G (C), then the base of Jian Shaoing (*) is A or T;
3) according to the both sides of reducing base, if T (A) * G (C), then the base of Jian Shaoing (*) is T or A;
4) according to the both sides of reducing base, if G (C) * T (A), then the base of Jian Shaoing (*) is T or A;
The target gene that is further characterized in that this method of design is MDRI gene and β-thalassemia gene, and wherein the MDRI gene with the probe of this method preparation is:
Primer 1F:5 '-TTGGCTAATGAGCTGCGGTTTCTC-3 ' (SEQ ID NO:25);
The probe of 1R:5 '-cy3-GATTCCAAAGGCAGCTTGCGTTTC-3 ' (SEQ ID NO:26) preparation is:
5 '-AGGTCGGGATGGATAC-3 ' (SEQ ID NO:2), called after 1N2;
5 '-AGGTCGGGATGGATCT-3 ' (SEQ ID NO:3), called after 1N3;
5 '-AGGTTGGGATGGAGTC-3 ' (SEQ ID NO:5), called after 1M2;
5 '-AGGTTGGGATGGATCT-3 ' (SEQ ID NO:6), called after 1M3;
5 '-GAACAATAAAAGGTAATC-3 ' (SEQ ID NO:8), called after 2N2;
5 '-GAACAATAAAAGGTAACA-3 ' (SEQ ID NO:9), called after 2N3;
5 '-GAACGATAAAAGGTAATC-3 ' (SEQID NO:11), called after 2M2;
5 '-GAACGATAAAAGGTAACA-3 ' (SEQ ID NO:12), called after 2M3;
Wherein β-thalassemia gene with the probe of this method preparation is:
Primer 2 F:5 '-Biotin-GTACGGCTGTCATCACTTAGACCTCA-3 ' (SEQ ID NO:27);
2R:5 '-Biotin-TGCAGCTTGTCACAGTGCAGCTCACT-3 ' (SEQ ID NO:28) preparation probe is:
5 '-GACCCAGGGCCTCACC-3 ' (SEQID NO:14), called after 3N2;
5 '-AGAAGTTGGTGGTGAGG-3 ' (SEQ ID NO:15), called after 3N3;
5 '-GTAAGTTGGTGGTAAGG-3 ' (SEQID NO:17), called after 3M2;
5 '-AGAAGTTGGTGGTAAGG-3 ' (SEQID NO:18), called after 3M3;
5 '-ACTCCAGAGGTTCTTTGAGTC-3 ' (SEQ ID NO:20), called after 4N2;
5 '-GACCAGAGGTTCTTTGAGTC-3 ' (SEQ ID NO:21), called after 4N3;
5 '-CTCCAGAGGTTCTTTTAGTC-3 ' (SEQ ID NO:23), called after 4M2;
5 '-GACCAGAGGTTCTTTTAGTC-3 ' (SEQID NO:24), called after 4M3.
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| CN1461811A (en) * | 2002-05-31 | 2003-12-17 | 中科开瑞生物芯片科技股份有限公司 | Designing method of oligonucleotide probe |
| CN101003836A (en) * | 2006-01-20 | 2007-07-25 | 株式会社东芝 | Primer design method, target nucleic acid detection method, single base mutation detection method and detection kit |
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| CN1461811A (en) * | 2002-05-31 | 2003-12-17 | 中科开瑞生物芯片科技股份有限公司 | Designing method of oligonucleotide probe |
| CN101003836A (en) * | 2006-01-20 | 2007-07-25 | 株式会社东芝 | Primer design method, target nucleic acid detection method, single base mutation detection method and detection kit |
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