CN109337984A - Nucleic acid molecule combination for fluorouracil metabolism related gene SNP detection - Google Patents
Nucleic acid molecule combination for fluorouracil metabolism related gene SNP detection Download PDFInfo
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Abstract
The invention discloses one group of nucleic acid molecule combinations for fluorouracil metabolism related gene SNP detection.The present invention also provides use the SNP fluorescence in situ hybridization of the nucleic acid molecule combine detection fluorouracil and its derivative drug metabolism related gene GSTP1 and DPYD that detection method is sequenced, the method includes extracting the DNA of sample to be tested, and single stranded is carried out as template using DNA and is derived, then the first sequencing probe and the second sequencing probe that different fluorescent markers are added simultaneously are hybridized with single stranded derivative, finally carry out interpretation to results of hybridization.This method accuracy is high, and stability is good, quickly, safety, easy automatic operation.The method can be completed to reach the prevention and the clinically guidance of personalized medicine for causing risk factor disease to understand the genotype of subject to the accurate parting of single nucleotide polymorphism in and hybridization reaction circulation derivative in a single stranded.
Description
Technical field
The invention discloses a kind of detection methods of single nucleotide polymorphism, belong to molecular genetics field.
Background technique
With being constantly progressive for medical science development, the whole world has many drugs to occur every year, because adverse reaction is too big
And it is eliminated in drug research.Although the effect of drug and adverse reaction are related with ethnic group and individual difference, person to person it
Between genome sequence hereditary difference be curative effect of medication and adverse reaction major reason.Single nucleotide polymorphism (Single
Nucleotide Polymorphisms；It SNP) is exactly difference between interpersonal 1 base, genome sequence difference has
Several kinds, SNP is only most common most common one kind, clinically significant.
SNP is the polymorphism of the DNA sequence dna as caused by single sequence change.Conversion including single base, overturn and
The insertion of single base or missing etc..Occurrence frequency of the SNP in human genome is relatively high, in about average every 1000 bases
Just there is a polymorphic site, be the third generation genetic marker after microsatellite, some SNP sites also will affect the function of gene
Can, change so as to cause biological character and even causes a disease.SNP is human heritable mutation kind one of the most common type, account for it is all
Know 90% or more of polymorphism, dbSNP has included about 5,000,000 mankind's SNP data having verified that at present.
In genetic analysis, SNP is widely applied as a kind of genetic marker, is mostly derived from these features:
(1) the high SNP of density is estimated as 1/1000bp in the averag density of human genome, reaches in the distribution of whole gene group
3×106A, genetic distance is 2~3cM, and density ratio microsatellite marker is higher, can be in the inside of any one gene to be studied
Or it is provided about a series of labels；
(2) typical certain SNP positioned at gene internal are possible to directly affect protein structure or expression,
Therefore, they may represent certain influencing factors in disease genetic mechanism；
(3) for genetic stability compared with the polymorphic repetitive sequences such as microsatellite label, SNP has higher inheritance stability
Property；
(4) easily realizing the automation SNP marker analyzed, only there are two types of allelotype (allele) in crowd.It is detecting in this way
When only need the mode of one "+- " or " complete without ", and need not be as detection limit fragment length polymorphism, microsatellite is right like that
The length of segment makes measurement, this makes the determination method based on SNP easily realize automation.
SNP also can be not only for the treatment of terminal to provide important target information on the source of medicament research and development
Body dosage regimen provides instruction, secondary anti-to provide the poison generated in curative effect of medication and reduction medicinal application to greatest extent
It answers, significance is embodied in:
(1) accuracy of new medicament screen being improved: recent research indicate that, by the SNP between measurement different people group, can have
Find the target molecule of new drug in effect ground.Due to the SNP difference between patient, the result of study of SNP can be used for reducing known drug
With the adverse reaction of clinical test drug, and its curative effect is improved.The excellent medicine being eliminated by erious adverse reaction can also be saved
Object is used for clinic as long as finding out SNP associated there and can serve as active drug.Because influencing each other or changing between drug
Become respective metabolic pathway, to generate synergistic effect and improve drug effect, patient can also be selected to carry out clinical test according to SNP,
Clinical Research Quality is improved, new drug success rate is enhanced, and from the SNP of illness gene, researching and developing drug target is very
Preferably.
(2) responsiveness and SNP of drug: when taking identical drug to the patient with same disease, different trouble
Person has different reactions, is divided into significant responder, responder, nonresponder.In clinical treatment, usual doctor can not be according to trouble
The symptom of person is most effective to what drug to determine patient, can only rely on the average statistics result and individual's warp of clinical data
It tests to select.But the patient of this symptom or disease, different treatment results can be generated using identical drug, this
Species diversity can be predicted by detection SNP.If SNP is placed exactly on the gene of expression, expression product-albumen for generating
Amino acid sequence is possible to different, thus may be that pharmaceutically-active target position changes, affinity decline to drug or
Person disappears.So carrying out snp analysis to patient can reach correctly diagnosis and effective therapeutic purposes.
(3) adverse reaction of SNP and drug: not only invalid after certain patient on medication in medical procedure, some is also
Serious adverse reaction can be generated, death can also be caused sometimes by such as dealing with improperly.According to statistics, annual 50000000 inpatient of China
In at least adverse reaction of 2,500,000 people being hospitalized for treatment with drug it is related, wherein 500,000 Genus Homos are in serious adverse reaction, because with greetings
Dead number is about 19.2 ten thousand people every year, and the number more lethal than infectious disease is higher by several times.In addition there is investigation, Chinese 1,800,000 is deaf
In mute children, 60% or more is caused by Irrational Use of Drugs.The SNP for understanding patient before the treatment, establishes Healthy People
With the snp database of patient, and it is particularly significant to carry out correlation analysis.
Fluorouracil (fluorouracil, FU) is tumor-inhibitory drug, is first changed into 5 fluoro- 2- deoxidations in vivo
Uridylate inhibits the biosynthesis of cancer cell DNA, RNA, is cell cycle specific medicine, main to inhibit S phase cell.
Fluorouracil is mainly used for treating tumor in digestive tract or larger dose fluorouracil in treatment chorioepithelioma, is also commonly used for controlling
Treat breast cancer, oophoroma, lung cancer, cervical carcinoma, bladder cancer and cutaneum carcinoma.Capecitabine is that a kind of Fluorouracil derivative class is anti-swollen
Tumor medicine is first converted into fluorouracil in vivo, then plays drug efficiency again, be mainly used for treat the carcinoma of the rectum, colon cancer,
The treatment of advanced stage or metastatic breast cancer and gastric cancer.Tegafur is the derivative of another fluorouracil, living through liver in vivo
Change is gradually converted into fluorouracil and plays antitumor action, is mainly used for treating tumor in digestive tract, to gastric cancer, colon cancer, straight
Intestinal cancer has certain curative effect.It can also be used for treatment breast cancer, lung bronchogenic carcinoma and liver cancer etc..Can also be used in bladder cancer, prostate cancer,
Kidney etc..It is one of most commonly used anti-tumor drug of clinical application in view of fluorouracil and its derivative, and existing research
It is 80 as many as several to show that gene relevant to fluorouracil drug effect has, therefore, understands SNP pairs of these related genes of patient
Have great importance in data for clinical drug use.Again in numerous related genes, study the most sufficient gene have GSTP1 and
DPYD。
Glutathione sulfydryl transferase (Glutathione S-transferase, GST) is that vivo biodistribution conversion is most important
One of II phase metabolic enzyme, be the main detoxification system that cell antibody Monoclonal, anticancer become.GSTs mainly includes the hypotypes such as α, μ, π, θ,
The gene GSTP1 for wherein encoding GST π is positioned at chromosome 1lq13, contains 7 exons and 6 intrones, and exon 5,6 is deposited
In gene pleiomorphism, A → G base substitution occurs for the 81st site of Exon 5, can lead to the 105th bit amino of protein peptide chain
Acid becomes GTC valine (Va1) from ATC isoleucine (Ile), is denoted as GSTP1-I105V, GSTP1-I105V polymorphism can be right
Tri- kinds of genotype of 105Ile/Ile, 105Ile/Val and 105Val/Val should be generated.GSTP1A114V (rs1138272) is then position
In the SNP of exon 6, researches show that these SNP can then influence the activity in vivo of fluorinated pyrimidine class drugs against tumor drug, from
And reduce the effect of oncotherapy.Therefore to being metabolized related SNP in the tumor therapeutic agents body such as GSTP1 and fluorinated pyrimidine class
The correct application for guaranteeing clinical cancer therapy drug is detected, prevention toxicity is of great significance.
Dihydropyrimidine dehydrogenase (DPYD) is the rate-limiting enzyme of fluorouracil catabolism, and active height will affect fluorine
The speed of uracil metabolism, and it is related to the catabolism rate-limiting enzyme of other fluorinated pyrimidine class drug toxicities.DPD is phonetic by dihydro
Pyridine dehydrogenase gene (DPYD) coding generates, and the mutation of base may cause DPYD activity shortage in DPYD sequence.Detect DPYD
Activity and DPYD gene pleiomorphism are advantageously possible for prediction fluorinated pyrimidine class drug toxicity and curative effect.
The high speed development of pharmacogenomics provides the foundation for individual administration, and really realizes individual administration, base
Because polymorphic detection becomes key.There are many methods can be used for SNP detection at present, and traditional method is using some existing
Mature technology, such as DNA double deoxidation sequencing, DNA pyrosequencing, genome sequencing, single-strand conformation polymorphism (SSCP), limitation
Property endonuclease bamhi length polymorphism (RFLP), denaturing gradient gel electrophoresis (DGGE) etc..The method of new high throughput assay SNP has
The special hybridization of chip technology, denaturing high-performance chromatography (DHPLC), Dynamic allele, Matrix Assisted Laser Desorption/electricity
From-flight time simple (MALDI-TOFMS) technology etc..
(1) DNA double deoxidation sequencing, pyrosequencing, genome sequencing: by Different Individual same gene or gene
Whether segment carries out sequencing and sequence compares, made a variation with the base for determining studied, recall rate is up to 100%.Although can be with
Obtain important parameter required for the SNP partings such as type and its accurate location of SNP.But the method is cumbersome, cost compared with
Height is not suitable for generally operating.
(2) SSCP: single stranded DNA will form secondary structure in neutral conditions, and different secondary structures can go out in electrophoresis
Existing different mobility.This secondary structure depends on the composition of base, and the change of single base also will affect its conformation, finally
It will lead to the change of the migration velocity on gel.On non-denaturing polyacrylamide gel, short single stranded DNA and RNA molecule according to
The difference of its single base sequence and form different conformations, migration rate in this way on gel is different, there is different bands,
So as to detect whether that there are SNP.The drawbacks of this method, is not can determine that mutation type and specific location.
(3) RFLP: using the specificity of the restriction enzyme site of restriction enzyme, in two or more restricted
Enzyme cutting acts on same DNA segment, and if there is SNP site, the length and quantity of digestion segment then will appear difference, according to electricity
The result of swimming is it may determine that whether there is SNP site.But the site that the premise of the technical application is SNP must contain the limit
The recognition site of restriction endonuclease processed.
(4) DGGE: it is the principle different using the identical double chain DNA fragment melting temperature of length, passes through denaturing gradient glue
The electrophoretic techniques that DNA fragmentation is separated.When electrophoresis starts, migration rate of the DNA in glue is only related with molecular size, and once
When DNA swimming is to certain point, that is, when reaching the denaturant concentration position DNA, so that DNA double chain starts to separate, to substantially reduce
Migration rate.When migration resistance and electric field dynamic balance, DNA fragmentation stops migration substantially in gel.Due to different DNA
The base composition of segment is variant so that its Denaturing generates difference, to form different bands on gel.But this
Method operation is excessively cumbersome.
(5) conventional hybridization sequencing-chip sequencing technologies: conventional hybridization sequencing technologies are in situ based on blot hybridization or nucleic acid
Hybridization approach is hybridized using oligonucleotide probe with target dna, entrained by fluorophor DNA can be marked, identify
Distinguished sequence, it is characterized in that sequencing reading length is shorter, it is will have particular bases that it is chip sequencing technologies which develops later
The probe of sequence is fixed on special carrier, and testing gene is extracted, after fluorescent marker, is carried out with the probe fixed miscellaneous
It hands over, the base classification of sequence to be measured is finally measured according to the intensity of fluorescence and type.There are some problems for such technology: only carrying out
Primary hybridization is difficult to screen nonspecific signals, it is thus possible to and cause sequence to be misread due to some non-specific hybridizations, in addition,
Chip cost is high, and required equipment is valuable, is unfavorable for popularization and application.
(6) DHPLC: target nucleic acid fragment PCR amplification, after the heat denatured of part, the DNA sequence dna containing mutating alkali yl due to
Base mismatch and normal base cannot match and form heteroduplex.Because the hybrid heterologous double stranded region comprising base mismatch is than complete
The homogenetic association area of pairing and the affinity of stationary phase are weak, are easier to be eluted from splitter, to reach the mesh of separation
's.The presence or absence of SNPs is eventually exhibited as the peak shape or number difference of chromatographic peak, can be easy to sentence from chromatogram according to this phenomenon
The base of disconnected mutation out.But DHPLC detection is higher to agents useful for same and environmental requirement, is easy to produce error, cannot detect
Homozygous mutation.
(7) AS-PCR technology: designing special primer according to SNP site, wherein 3 ' ends of chain (special chain) and SNP
The base complementrity (or identical) in site, another chain (common chain) are designed according to a conventional method, and therefore, AS-PCR technology is one
PCR label of the kind based on SNP.Because special primer has amplified production in a kind of genotype, do not have in another genotype
Amplified production can easily tell the presence or absence of amplified production with gel electrophoresis, so that it is determined that the SNP of genotype.But
It is that the method is also required to gel electrophoresis.
(8) MALDI-TOFMS: be the single stranded PCR products that will be denaturalized by with after the compound covalent bond on silicon chip,
The annealing of primer is carried out on the silicon die, and extension, the base of mutable site pairing and the base normally matched be not identical.Root
According to primer in extension in conjunction with the different qualities of different bases show different peaks on mass spectrograph and detect SNP.
(9) Real-Time Fluorescent Quantitative PCR Technique, the technology are released by Applied Biosystems company of the U.S., so-called reality
When fluorescent quantitative PCR technique, refer to and fluorophor be added in PCR reaction system, using fluorescence signal accumulation real-time monitoring it is whole
A PCR process, the method that quantitative analysis is carried out to unknown template finally by standard curve.The neck of quantitative fluorescent PCR application at present
Domain is extensive, is generally used for target template quantitatively or a certain specific nucleotide sequence detects.It is main in the other context of detection of genotype
It will be by single pass fluorescence signal value and CT (amplification cycles number) value to determine whether for effectively amplification.But PCR amplification walks
Suddenly not only excessively high to laboratory equipment requirement, great pollution risk also is brought for technology implementation, to seriously affect detection
As a result accuracy.Moreover, when target site is there are when multiple genotype, especially in the detection of single nucleotide polymorphism,
Single channel detection is just no longer practical, and the gene for needing to be detected target site using dual channel system is constituted.For binary channels
System as a result, often analyze two channels fluorescence curve CT value to determine whether for effectively amplification so that it is determined why
Genotype.This method for homozygous genotype and heterozygous genotypes fluorescence curve relatively when be easy error, can not
It provides accurately as a result, be easy to causeing false positive or erroneous judgement.
In view of the prior art carry out fluorouracil metabolism related gene single nucleotide polymorphism detection present in it is all
Such as False Rate is high, accuracy is poor, stability is low, the low technical problem of detection efficiency, this invention is intended to provide to the prior art into
Row improves, and derives boot sequence and probe sequence by design single stranded, and the optimization to each component provides one group and urinates for fluorine
The nucleic acid molecule combination of pyrimidine metabolic related gene SNP detection, and then provide and use the nucleic acid molecule combine detection fluorine
Detection method is sequenced in the SNP fluorescence in situ hybridization of uracil and its derivative drug metabolism related gene.
Summary of the invention
Based on above-mentioned purpose, the present invention discloses one group of core for fluorouracil metabolism related gene SNP detection first
Thuja acid molecular combinations, the combination is for fluorescence in situ hybridization sequencing detection fluorouracil and its derivative drug metabolism dependency basis
Because of the SNP of GSTP1 and DPYD, the combination includes that the first boot sequence and the second guidance are defined as used in single stranded is derivative
The oligonucleotide molecules of sequence and the sequencing by hybridization probe for being defined as the first sequencing probe and the second sequencing probe, described the
One boot sequence, the second boot sequence, the first sequencing probe and the second sequencing probe are following combination:
SEQ ID NO:1,2,9 and 10；
SEQ ID NO:3,4,11 and 12；
SEQ ID NO:5,6,13 and 14；With
SEQ ID NO:7,8,15 and 16.
Wherein, SEQ ID NO:1,2,9 and 10 are for detecting GSTP1 I 105/V (rs1695), SEQ ID NO:3,4,
11 and 12 for detecting GSTP1 A114V (rs1138272), and SEQ ID NO:5,6,13 and 14 are for detecting DPYD*2A
(rs3918290), SEQ ID NO:7,8,15 and 16 are for detecting DPYD*13 (rs55886062).
Secondly, the present invention also provides a kind of fluorouracil and its derivative drug metabolism related gene GSTP1 and DPYD
SNP fluorescence in situ hybridization be sequenced detection method, include the following steps:
(1) DNA of sample to be tested is extracted；
(2) DNA obtained using step (1) is template, in the presence of dNTP, reaction buffer, DNA cutting agent,
It carries out single stranded under the guidance of oligonucleotide molecules to derive, the oligonucleotide molecules are divided into the first boot sequence and second and draw
Sequence is led, while the different sequencing probe of two sequences is added, is respectively defined as the first sequencing probe and the second sequencing probe,
It is marked with fluorescent molecule different from each other respectively at 5 ' ends of two probes, is marked with respectively at its 3 ' end and base is quenched
Group, first boot sequence, the second boot sequence, the first sequencing probe and the second sequencing probe are selected from following combination:
SEQ ID NO:1,2,9 and 10；
SEQ ID NO:3,4,11 and 12；
SEQ ID NO:5,6,13 and 14；Or
SEQ ID NO:7,8,15 and 16；
(3) results of hybridization of probe the single stranded derivative of step (2) is sequenced respectively with the first sequencing probe and second
Carry out interpretation.
In a preferred embodiment, the fluorescent molecule of the first sequencing probe label is 5-carboxyfluorescein
(FAM), the fluorescent molecule of the second sequencing probe label is chlordene fluorescein (HEX), and the quenching group is BHQ.
The design of derivative guidance oligonucleotide sequence and probe sequence provided by the invention is different from general probe primer,
Probe is high to the identity of template.Fluorophor (FAX and HEX) is at 5 ' ends when designing probe, and quencher is at 3 ' ends.Due to
The mutating alkali yl of SNP site only one, which will targetedly carry out each SNP site only in the design of probe
Vertical design, and Accurate Analysis is carried out in the interpretation software in later period, make entirely to invent accurate, quick, economy to DNA sequence dna
Interpretation is carried out with SNP site.
In a preferred embodiment, sample to be tested described in step (1) is blood, body fluid, secretion, metabolism
Object, cast or tissue samples.
In a highly preferred embodiment, the sample to be tested DNA template concentration is 103A/uL or more.
In a preferred embodiment, the first boot sequence and the second boot sequence in step (2) reaction system
Ratio is 1:2-1:6, and the second boot sequence: the first sequencing probe: the second sequencing probe ratio is 1:1:1.
It is further preferable that the ratio of the first boot sequence and the second boot sequence is 1:4 in step (2) reaction system.
In the derivative system of the single stranded, the DNA cutting agent can for chemical small molecule cutting agent, endonuclease,
Exonuclease, archaeal dna polymerase etc., in a preferred embodiment, the cutting agent are the Exonucleolytic of 2.5U/ μ l
10 × buffer needed for enzyme, dNTP 5mM and cutting agent work.
In another preferred embodiment, the reaction condition of step (3) are as follows:
(1) 95 DEG C of initial denaturation, 10min, 1 circulation；
(2) it is denaturalized 95 DEG C, 30s, 60 DEG C~64 DEG C annealing to be hybridized, 75s, 55 circulations.
In still another preferred embodiment, the method for step (3) described interpretation be read derivative respectively with two kinds
(Sequencing Times, is sequenced number or sequencing depth to the St value of fluorescence intensity after probe hybridization, and 50 hybridization is 50
Secondary sequencing by hybridization) difference.
For the interpretation method of testing result, the present invention is divided on the basis of sequencing by hybridization depth by parsing fluorescence curve
The changing rule for analysing fluorescence signal, by reading the St value of binary channels fluorescence curve, so that directly interpretation goes out to be tested the DNA of template
Sequence and SNP genotype.
Compared with prior art, sequencing by hybridization technology provided by the invention needs more template nucleic acids (usually less than
3000 copies) sequencing could be completed, but need not move through PCR link.This feature has unique advantage: (1) being not easy dirt
Dye: the signal of a small amount of allogeneic dna sequence DNA pollution is very low, will not influence sequencing accuracy, such as the allogeneic dna sequence DNA dirt of 100-300 copy
Dye is catastrophic for PCR amplification, but in sequencing by hybridization, the intensity of Heterologous signal is less than total signal strength
10%, which can be used as background signal and is removed；(2) easy: sequencing by hybridization does not need PCR amplification, therefore common
Laboratory can carry out, and not need the forcible authentication in PCR amplification laboratory；(3) simple and direct: sequencing by hybridization does not have cumbersome sample
DNA is extracted and pcr amplification product extracts link, clinical samples simple process can be carried out sequencing by hybridization, therefore speed is sequenced
It is very fast, it is counted from sample is taken, can report result within 2-3 hours.
In short, being combined provided by the present invention for the nucleic acid molecule detected for fluorouracil metabolism related gene SNP
And detection is sequenced in the fluorescence in situ hybridization of the SNP of fluorouracil and its derivative drug metabolism related gene GSTP1 and DPYD
Method, combine sequencing by hybridization, fluorescence signal acquisition, using definition St value carry out analytic operation and accurately provide it is tested
The DNA sequence dna and genotype of sample.Succinct understandable, the easy to use, result of this method is accurately shown, is very beneficial for clinic and is answered
With.
Detailed description of the invention
Figure 1A fluorescence in situ hybridization sequencing detection single nucleotide polymorphism Method And Principle schematic diagram；
Figure 1B fluorescence in situ hybridization sequencing detection SNP result interpretation schematic diagram；
Snp analysis working-flow figure is sequenced in Fig. 1 C. fluorescence in situ hybridization；
The detection homozygosis GG genotype results figure of Fig. 2 .GSTP1 I 105/V gene rs1695；
The detection homozygosis AA genotype results figure of Fig. 3 .GSTP1 I 105/V gene rs1695；
The detection heterozygosis GA genotype results figure of Fig. 4 .GSTP1 I 105/V gene rs1695；
The detection homozygosis CC genotype results figure of Fig. 5 .GSTP1 A114V gene rs1138272；
The detection homozygosis TT genotype results figure of Fig. 6 .GSTP1 A114V gene rs1138272；
The detection heterozygosis CT genotype results figure of Fig. 7 .GSTP1 A114V gene rs1138272；
The detection homozygosis GG genotype results figure of Fig. 8 .DPYD*2A gene rs3918290；
The detection homozygosis AA genotype results figure of Fig. 9 .DPYD*2A gene rs3918290；
The detection heterozygosis GA genotype results figure of Figure 10 .DPYD*2A gene rs3918290；
The detection homozygosis CC genotype results figure of Figure 11 .DPYD*13 gene rs55886062；
The detection homozygosis AA genotype results figure of Figure 12 .DPYD*13 gene rs55886062；
The detection heterozygosis CA genotype results figure of Figure 13 .DPYD*13 gene rs55886062.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.
Reaction principle
Unlike common Taqman technology, the present invention does not use round pcr in the preparation of hybridization template,
But use it is single-stranded it is derivative guide oligonucleotide sequence, so that DNA double chain template is converted into single chain derivatives, and directly with label
Probe carries out sequencing by hybridization.
The derivative oligonucleotides guidance sequence for being required to combination complementary with template strand of the single stranded is caused, and is guided
Sequence can be held in the 3 ' of template strand and/or 5 ' ends are specifically bound with the template strand after annealing, and caused single stranded and derived, institute
The design principle for stating boot sequence and probe sequence is: being directed to specific SNP site, design 2 with special at 3 ' ends and 5 ' ends
The template strand that the opposite sex identifies the ability of simultaneously combining target SNP, and specific probe is blocked to combine is quick with its natural complementary strand
Annealing, so maintain the derivative boot sequence of the oligonucleotides of the single-stranded derivative state of template, 2 hold SNP mutation sites 5 ' respectively
Carry out the probe of fluorescent decoration.SNP mutation nucleotide is arranged within its preceding 3 base in 5 ' end in two kinds of probes, respectively using not
Same fluorescent marker, and quenching group is marked in the probe other end.Before probe is in conjunction with template strand, probe keeps returning because of sequence
The hairpin structure rolled over and formed, fluorophor cannot discharge fluorescence because spatially adjoining with quenching group at this time.Probe 5 '
The template sequence for holding 4~15bp of upstream is the zone of intersection.The single-stranded derivative end of boot sequence 5 ' has the sequence of zone of intersection complementation, this sequence
First base of column and 5 ' first, end bases of probe mismatch, and unmatched base forms " base tilting ", leads to probe
The zone of intersection and single-stranded derivative boot sequence form " hairpin structure ", the Tm temperature of this structure is 4~8 DEG C higher than probe, energy
Generally -8~-12kj.There are the interval region of 6~12bp in single-stranded derivative boot sequence and the sequencing probe zone of intersection, for keeping
" ring " region sequence in " hairpin structure "." hairpin structure " can be improved the accuracy of cutting agent identification.It is combined when based on probe
The exonuclease activity of efficiency and archaeal dna polymerase realizes the cutting with the probe of template specific bond, so that release is corresponding
Fluorescence signal.Figure 1A is fluorescence in situ hybridization sequencing detection single nucleotide polymorphism Method And Principle schematic diagram, by that can see in figure
Out, (black circle) is marked with fluorescein at 3 ' ends of oligonucleotide probe, quenching group (stain) is terminated with 5 ', non-hybridized
When, oligonucleotide probe be it is cricoid, do not issue fluorescence, when oligonucleotide probe with specific nucleotide SNP site (A)
When template combines, the ring-type of probe is opened, and for quenching group far from fluorescein base group, fluorescein base group issues fluorescence, glimmering at this time
Light is detected by machine.
In any sequencing by hybridization, two sequencing probes and single-stranded special or Non-specific hybridization to be sequenced all can
Generate fluorescence signal.When accumulating the fluorescence signal of sequencing of certain number, fluorescence signal intensity is likely to be breached a certain numerical value.This
The result interpretation of invention is the St value by calculating the fluorescence signal released, and the St value between difference SNP sequencing by hybridization
Come what is carried out, so-called St value is the abbreviation of Sequencing Times, i.e., sequencing number or sequencing depth, 50 hybridization are 50
Secondary sequencing by hybridization, sequencing by hybridization of every completion, calculates a St value.At this point, the sequencing time of setting wild type sequencing probe A1
Number is STA1, sets the sequencing number of mutability sequencing probe A2 as STA2.
The difference of St value: Δ ST=STA1-STA2
It in the detection of practical sequencing by hybridization, needs to set the threshold value being sequenced each time with standard form sequence, by as follows
Mode calculates:
(1) wild type standard sequence template, the Δ ST=STA1-STA2 obtained at this time are as follows: Δ ST1 are put into
(2) wild type and mutability standard sequence template are put into simultaneously, the Δ ST=STA1-STA2 obtained at this time are as follows: Δ
ST2
(3) wild type standard sequence template, the Δ ST=STA1-STA2 obtained at this time are as follows: Δ ST3 are put into
(4) setting of ST threshold value:
Threshold value 1: the median (Δ ST1+ Δ ST2)/2 between Δ ST1 and Δ ST2 is taken
Threshold value 2: the median (Δ ST1+ Δ ST2)/2 between Δ ST2 and Δ ST3 is taken
(5) determine: for any unknown nucleotide sequence to be measured, calculating its Δ ST:
If A, except threshold value 1 being wild pure and mild sequence；
If being B, the pure and mild sequence of mutation except threshold value 2；
If being heterozygous sequence C, between threshold value 1 and 2.
In specific measurement, as shown in Figure 1B, in the upper figure of Figure 1B, when wild type B1 template and wild type sequencing probe A1 are complete
It is complete complementary, it is not fully complementary with probe A2 is sequenced with saltant type.Reach the sequencing depth S T value of signal specific intensity, STA1 is less than
STA2.In this case, B1 template is wild type genotype.
In Figure 1B in figure, when template and sequencing probe A1 and A2 all complete complementaries, reach the ST value of signal specific intensity,
STA1 and STA2 very close to.In this case, template is heterozygous sequence.
It is not fully complementary with sequencing probe A12 when B2 template and sequencing probe A2 complete complementary in Figure 1B following figure.Reach
The sequencing depth S T value of signal specific intensity, STA1 are greater than STA2.In this case, B2 template is mutant-type genotype.
The present invention also to being distributed rationally in the configuration of experimental system component, passes through the derivative guidance sequence of various concentration
The Mg2 of combo is prepared, optimized to column+With dNTPs etc., available good testing result.This method is efficiently quick, as a result quasi-
It really, can one-time detection great amount of samples.Due to not having amplification link, high degree avoids bring pollution due to sample contamination and puts
Big effect and then the false positive for affecting detection, therefore, with high specificity.
Detect embodiment
First against SNP site sequence, single-stranded derivative boot sequence and probe sequence are designed using Beacon Designer 8
Column.Single-stranded derivative boot sequence and probe sequence are synthesized by epoch Gene science Development Co., Ltd of Beijing China.In table 1,
List detection probe and the corresponding SNP detection site of single-stranded derivative boot sequence.Probe label is routinely marked using this field
Note technology.
Table 1: detection probe and the corresponding SNP site of single-stranded derivative boot sequence
The extraction that leucocyte is carried out from blood, EDTA anticoagulated blood sample to be checked is mixed by inversion for several times；Newly extract
EDTA anticoagulated blood sample, is stored in 4 DEG C, should handle in 24 hours.Erythrocyte cracked liquid (ammonium chloride) and aqua sterilisa press 1:9 ratio
Example is diluted to working solution.1.5ml centrifuge tube is taken, is added 1mL erythrocyte cracked liquid (working solution), 150uL sample to be checked is taken to be added
In centrifuge tube, mixing of turning upside down is stored at room temperature 5min；After blood sample and erythrocyte cracked liquid mix, liquid color becomes clarifying
Red.Centrifuge tube is put into a centrifuge, 5min is centrifuged；Revolving speed: 3000 turns/700g.Centrifuge tube is taken out after the completion of centrifugation, is used
Pipette tips suck supernatant；After centrifugation, it is centrifuged bottom of the tube visible white grain of rice size shape leucocyte.100uL PHARM-GENE is added
01 snp analysis saves liquid, spare after mixing.
By the first/bis- single-stranded derivative boot sequence and the first/bis- sequencing probe dry powder centrifugation, 3000rpm, 1min add super
Pure water dissolution, is diluted to 20 μM, measures the OD value under 260nm using ultraviolet specrophotometer SMA1000,
Referring to formula:
ODSEQ ID NO:2/LSEQ ID NO:2×V SEQ ID NO:2=ODSEQ ID NO:1/L SEQ ID NO:1×V SEQ ID NO:1
× 4, i.e. boot sequence one: two=1:4 of boot sequence,
And formula:
ODSEQ ID NO:2/LSEQ ID NO:2×VSEQ ID NO:2=ODSEQ ID NO:5/LSEQ ID NO:5×VSEQ ID NO:5=
ODSEQ ID NO:6/LSEQ ID NO:6×VSEQ ID NO:6, i.e., boot sequence two: the first be sequenced probe: second sequencing probe 2=1:1:
1, calculate the volume of single-stranded derived sequence and the first/bis- sequencing probe in reaction system.
Reaction system
The configuration of above-mentioned reaction system is also applied for other combined sequences.
It is reacted on the grand TL988A fluorescence detector in day, single stranded derivative and sequencing by hybridization reaction condition: process 1:
95 DEG C of initial denaturation, 10min, 1 cyclic process 2: 95 DEG C, 30s, 62 DEG C annealing of denaturation are hybridized, and 75s, progress 55 is miscellaneous altogether
Hand over sequencing procedure circulation.
It is detected on fluorescence detector (the grand TL988A in day), during entire single stranded derivative and sequencing by hybridization,
First probe and the second probe in conjunction with template, issue fluorescence signal respectively after matched specific bond, unmatched non-specific
In conjunction with that may issue very faint signal or not issue signal, judged according to fluorescence intensity.For the ease of machine
Automatic interpretation converts threshold value elaboration, so as to setting program, by software automatic interpretation sequence for the principle in Figure 1B.
The present invention to the interpretation analysis of testing result is carried out by " snp analysis system is sequenced in fluorescence in situ hybridization ",
The system comprises working control module, backstage setup module, sample setup module, temperature control setup module, operation monitoring modules
And data analysis module." snp analysis system is sequenced in fluorescence in situ hybridization " is the limited public affairs of Beijing China epoch bioengineering
Department's design is write.
Working control module mainly includes newly-built program, opens program, saves result, beginning or terminate program and opening
The functions such as backstage set interface.
Backstage setup module is mainly parameter setting, and major parameter includes gene type, the channel 1St value upper limit, channel 1St
It is worth lower limit, the channel 2St value upper limit, channel 2St value lower limit, the St value difference upper limit, St value difference lower limit, 1 genotype of channel, 2 base of channel
Because type and temperature-controlled conditions select.
Sample setup module mainly includes sample names, gene type and result.
Temperature control setup module is then different temperature-controlled conditions set interfaces, and different temperature programs is set according to actual conditions.
Operation monitoring module mainly includes that temperature control monitoring plate and fluorescence read plate, and real-time display is with sequencing by hybridization
It carries out, the change curve of fluorescent value.Data assay surface mainly shows the fluorescence detection curve after coupling, while reading and showing
Show the St value of binary channels fluorescence curve, the gene order and genotype results of the sample are shown after progress operation.
Fluorescence detection curve after data analysis module display coupling, while reading and showing the St of binary channels fluorescence curve
Value shows the genotype results of the sample after the operation of progress St value difference.St value difference be one St value of channel subtract two St of channel be worth
The numerical value arrived, software backstage can according to standard sequence sequencing by hybridization as a result, setting one standard St value, it is miscellaneous to will test sample
It hands over the resulting St value of sequencing to compare with standard St value, that is, can determine whether the gene order of sample to be tested.
Fig. 1 C shows the operation schematic diagram of the present invention " snp analysis system is sequenced in fluorescence in situ hybridization ".
After the completion of program operation, at data analysis module interface, showing binary channels, (wild-type probe of flag F AM is logical
The saltant type probe channel in road and label HEX) fluorescence curve St value, according to the parameter that backstage setup module is arranged, software can be certainly
The dynamic gene order and genotype calculated and provide detection sample.In sample set interface, selection needs to print testing result
Sample name, then clicking printing can be obtained final testing result.
According to the size of the fluorescence data acquisition in two channels, difference is worth according to ST, system provides SNP site automatically
Gene order and genotype.Software analyzes there are two ways to interpretation result: 1. judge according to fluorescence intensity: system detection two
Fluorescence channel, channel one (channel FAM) and channel two (channel HEX), system calculate the fluorescence intensity of detection fluorescence first.Such as
The fluorescence intensity that fruit is calculated is greater than the numerical value for the noisy fluoroscopic being arranged in software, and system thinks the fluorescence data in this channel
It is effective, and then calculates ST value；It is considered invalid if the fluorescence intensity calculated is less than the numerical value of the noisy fluoroscopic of setting
Data, skip over, ST can be shown as 0 or -1.If the fluorescence intensity in two channels is respectively less than set numerical value, software is aobvious
It is shown as " resurveying ".2. being judged according to ST difference: software default is that channel one (channel FAM) ST value subtracts channel and (HEX is logical
Road) ST value.When the ST difference obtained is between the heterozygous genes sequence ST threshold value bound that software is arranged, as the result is shown for
Heterozygosis.It is as the result is shown gene representated by that small channel of ST value when ST difference is when except the ST value bound of setting
Type.Entire program operation, which finishes, needs 2 hour 31 minutes.
Fig. 2-Figure 13 is the judging result of specific SNP detection.
Fig. 2 is the detection homozygosis GG genotype results figure of GSTP1 I105/V gene rs1695, there is two straight lines in figure
Two passes are represent, each probe for carrying fluorescent marker, (black wire tag) probe of channel one marks GG genotype, by preceding
Principle is stated, is compared with the St threshold value of standard form, the genotype that can calculate this sample is GG genotype.
Fig. 3 is the detection homozygosis AA genotype results figure of GSTP1 I 105/V gene rs1695, there is two straight lines in figure
Two passes are represent, each probe for carrying fluorescent marker, (light gray wire tag) probe of channel two marks AA genotype, by preceding
Principle is stated, is compared with the St threshold value of standard form, the genotype that can calculate this sample is AA genotype.
Fig. 4 is the detection heterozygosis GA genotype results figure of GSTP1 I 105/V gene rs1695, there is two straight lines in figure
Two passes are represent, each probe for carrying fluorescent marker, channel one (black wire tag), channel two (light gray wire tag) probe
Label GG and AA genotype is compared by aforementioned principles with the St threshold value of standard form, and the genotype that can calculate this sample is
GA genotype.
When carrying out oncotherapy using fluorouracil, AA type patient outcomes are poor, and therapeutic response reduces, low total existence
Phase, mortality risk increase, and AG type is slightly good, and GG type is more preferable.
Fig. 5 is the detection homozygosis CC genotype results figure of GSTP1 A114V gene rs1138272, have in figure two it is straight
Line represents two passes, and each probe for carrying fluorescent marker, (black wire tag) probe of channel one marks CC genotype, passes through
Aforementioned principles are compared with the St threshold value of standard form, and the genotype that can calculate this sample is CC genotype.
Fig. 6 is the detection homozygosis TT genotype results figure of GSTP1 A114V gene rs1138272, have in figure two it is straight
Line represents two passes, and each probe for carrying fluorescent marker, (light grey line label) probe of channel two marks TT genotype, leads to
Aforementioned principles are crossed, are compared with the St threshold value of standard form, the genotype that can calculate this sample is TT genotype.
Fig. 7 is the detection heterozygosis CT genotype results figure of GSTP1 A114V gene rs1138272, have in figure two it is straight
Line represents two passes, and each probe for carrying fluorescent marker, channel one (black wire tag), channel two (light gray wire tag) is visited
Needle label CC and TT genotype compares with the St threshold value of standard form by aforementioned principles, can calculate the genotype of this sample
For CT genotype.
GSTP1 A114V gene C c-type patient receive its life span after fluorouracil and plus cisplatin in treatment be longer than CT type or
TT type.
Fig. 8 is the detection homozygosis GG genotype results figure of DPYD*2A gene rs3918290, there is two straight line generations in figure
Table two passes, each probe for carrying fluorescent marker, (black wire tag) probe of channel one marks GG genotype, by aforementioned
Principle is compared with the St threshold value of standard form, and the genotype that can calculate this sample is GG genotype.
Fig. 9 is the detection homozygosis AA genotype results figure of DPYD*2A gene rs3918290, there is two straight line generations in figure
Table two passes, each probe for carrying fluorescent marker, (light gray wire tag) probe of channel two marks AA genotype, by aforementioned
Principle is compared with the St threshold value of standard form, and the genotype that can calculate this sample is AA genotype.
Figure 10 is the detection heterozygosis GA genotype results figure of DPYD*2A gene rs3918290, there is two straight line generations in figure
Table two passes, each probe for carrying fluorescent marker, channel one (black wire tag), channel two (light gray wire tag) probe mark
Note GG and AA genotype is compared by aforementioned principles with the St threshold value of standard form, and the genotype that can calculate this sample is GA
Genotype.
The GA type of DPYD*2A gene and AA type patient DPYD activity lack, when receiving fluorouracil drug and treating
Serious or even lethal toxic reaction probability is apparently higher than GG type.
Figure 11 is the detection homozygosis CC genotype results figure of DPYD*13 gene rs55886062, there is two straight lines in figure
Two passes are represent, each probe for carrying fluorescent marker, (black wire tag) probe of channel one marks CC genotype, by preceding
Principle is stated, is compared with the St threshold value of standard form, the genotype that can calculate this sample is CC genotype.
Figure 12 is the detection homozygosis AA genotype results figure of DPYD*13 gene rs55886062, there is two straight lines in figure
Two passes are represent, each probe for carrying fluorescent marker, (light grey line label) probe of channel two marks AA genotype, passes through
Aforementioned principles are compared with the St threshold value of standard form, and the genotype that can calculate this sample is AA genotype.
Figure 13 is the detection heterozygosis AC genotype results figure of DPYD*13 gene rs55886062, there is two straight lines in figure
Two passes are represent, each probe for carrying fluorescent marker, channel one (black wire tag), channel two (light gray wire tag) probe
Label CC and AA genotype is compared by aforementioned principles with the St threshold value of standard form, and the genotype that can calculate this sample is
CA genotype.
DPYD*13 Gene A c-type DPYD active part inactivation, then DPYD activity divides inactivation to CC type completely, with wild type AA type
It compares, when tumor patient receives the chemotherapy based on fluorouracil, the relative risk and severity that drug toxicity occurs are bright
It is aobvious to increase.
Sequence table
<110>Beijing China epoch Gene science Development Co., Ltd
<120>for the nucleic acid molecule combination of fluorouracil metabolism related gene SNP detection
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 30
<212> DNA
<213> Homo sapiens
<400> 1
ttstccstca tgcagatgst cacatagttg 30
<210> 2
<211> 17
<212> DNA
<213> Homo sapiens
<400> 2
ggststatgg gaaggac 17
<210> 3
<211> 31
<212> DNA
<213> Homo sapiens
<400> 3
tcstcstgcg agagtaggat gatacatggt g 31
<210> 4
<211> 19
<212> DNA
<213> Homo sapiens
<400> 4
tcccacaatg aaggtsttg 19
<210> 5
<211> 34
<212> DNA
<213> Homo sapiens
<400> 5
ttaagtgtga taacttatgc caattctctt gttt 34
<210> 6
<211> 22
<212> DNA
<213> Homo sapiens
<400> 6
ctgaatattg agctcatcag tg 22
<210> 7
<211> 26
<212> DNA
<213> Homo sapiens
<400> 7
acgaagagct tgagggcaaa acccca 26
<210> 8
<211> 18
<212> DNA
<213> Homo sapiens
<400> 8
aaatggccgg attgaagt 18
<210> 9
<211> 16
<212> DNA
<213> Homo sapiens
<400> 9
tgtatttgca gcggag 16
<210> 10
<211> 15
<212> DNA
<213> Homo sapiens
<400> 10
cgtatttgca gcgga 15
<210> 11
<211> 16
<212> DNA
<213> Homo sapiens
<400> 11
cgggcaagga tgasta 16
<210> 12
<211> 17
<212> DNA
<213> Homo sapiens
<400> 12
tgggcaagga tgastat 17
<210> 13
<211> 18
<212> DNA
<213> Homo sapiens
<400> 13
cgttgtctgg aaagtcag 18
<210> 14
<211> 18
<212> DNA
<213> Homo sapiens
<400> 14
tgttgtctgg aaagtcag 18
<210> 15
<211> 19
<212> DNA
<213> Homo sapiens
<400> 15
aatcattgat gtgctggtg 19
<210> 16
<211> 18
<212> DNA
<213> Homo sapiens
<400> 16
actcattgat gtgctggt 18
Claims (10)
1. one group of nucleic acid molecule combination for fluorouracil metabolism related gene SNP detection, the combination is former for fluorescence
The SNP of position sequencing by hybridization detection fluorouracil and its derivative drug metabolism related gene GSTP1 and DPYD, the combination include
It is defined as the oligonucleotide molecules of the first boot sequence and the second boot sequence used in single stranded is derivative and is defined as the
The sequencing by hybridization probe of one sequencing probe and the second sequencing probe, first boot sequence, the second boot sequence, the first sequencing
Probe and the second sequencing probe are following combination:
SEQ ID NO:1,2,9 and 10；
SEQ ID NO:3,4,11 and 12；
SEQ ID NO:5,6,13 and 14；With
SEQ ID NO:7,8,15 and 16.
2. the fluorescence in situ hybridization of the SNP of a kind of fluorouracil and its derivative drug metabolism related gene GSTP1 and DPYD are surveyed
Sequence detection method, includes the following steps:
(1) DNA of sample to be tested is extracted；
(2) DNA obtained using step (1) is template, in the presence of dNTP, reaction buffer, DNA cutting agent, in widow
It is derivative that single stranded is carried out under the guidance of nucleic acid molecule, the oligonucleotide molecules are divided into the first boot sequence and the second guidance sequence
Column, while the different sequencing probe of two sequences is added, it is respectively defined as the first sequencing probe and the second sequencing probe, in institute
5 ' the ends for stating two probes are marked with fluorescent molecule different from each other respectively, are marked with quenching group respectively at its 3 ' end,
First boot sequence, the second boot sequence, the first sequencing probe and the second sequencing probe are selected from following combination:
SEQ ID NO:1,2,9 and 10；
SEQ ID NO:3,4,11 and 12；
SEQ ID NO:5,6,13 and 14；Or
SEQ ID NO:7,8,15 and 16；
(3) results of hybridization to the single stranded derivative of step (2) respectively with the first sequencing probe and the second sequencing probe carries out
Interpretation.
3. according to the method described in claim 2, it is characterized in that, the fluorescent molecule of the first sequencing probe label is 5- carboxylic
The fluorescent molecule of base fluorescein, the second sequencing probe label is chlordene fluorescein, and the quenching group is BHQ.
4. according to the method described in claim 2, it is characterized in that, sample to be tested described in step (1) is blood, body fluid, divides
Secretion, metabolin, cast or tissue samples.
5. according to the method described in claim 4, it is characterized in that, the sample to be tested DNA template concentration is 103A/uL with
On.
6. according to the method described in claim 2, it is characterized in that, the first boot sequence and second in step (2) reaction system
The ratio of boot sequence is 1:2-1:6, and the second boot sequence: the first sequencing probe: the second sequencing probe ratio is 1:1:1.
7. according to the method described in claim 6, it is characterized in that, the first boot sequence and second in step (2) reaction system
The ratio of boot sequence is 1:4.
8. according to the method described in claim 2, it is characterized in that, the dNTP in step (2) reaction system is 5mM, buffer
For 10 ×, the cutting agent is the exonuclease of 2.5U/ μ l.
9. according to the method described in claim 2, it is characterized in that, step (3) derivative and the circulation step of hybridization are as follows:
(1) 95 DEG C of initial denaturation, 10 minutes, 1 circulation；
(2) 95 DEG C are denaturalized, 30 seconds, 60 DEG C~64 DEG C annealing were hybridized, and 75 seconds, carried out 50-60 hybridization circulation altogether.
10. according to the method described in claim 2, it is characterized in that, the method for step (3) described interpretation is to read two kinds of sequencings
The difference of the St value of fluorescence intensity after probe and single stranded derivative sequencing by hybridization.
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