CN108384843A - A method of identifying single copy gene mutated-genotype using digital pcr - Google Patents
A method of identifying single copy gene mutated-genotype using digital pcr Download PDFInfo
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Abstract
The present invention provides a kind of method for identifying single copy gene mutated-genotype using digital pcr, the method includes the single copy gene mutational sites to be measured to sample to design digital pcr amplimer and probe, to the single copy house-keeping gene design digital pcr amplimer and probe of sample, twin-channel dual digital pcr is carried out to mutational site and house-keeping gene and is reacted；It is augmented with the digital pcr reaction by single copy gene mutational site and whether there is the single copy gene mutational site without determining sample to be tested；If there is the single copy gene mutational site, the genotype of single copy gene to be measured is identified by comparing the copy number of single copy gene to be measured and single copy house-keeping gene.It is accurate, convenient, fast and at low cost that the method for the present invention has the characteristics that, can be used for genetic screening, prevention and gene diagnosis.
Description
Technical field
The present invention relates to microlayer model digital pcr technical fields, and in particular to a kind of to identify single copy gene using digital pcr
The method of mutated-genotype.
Background technology
Genome (Genome) is commonly defined as whole base sequences of all chromosomes in haploid cell, i.e., single times
Body genome.Single copy gene refers to the gene that copy is only existed in haploid genome, and single copy gene is in gene
50-80% is accounted in group, in human haploid genome, about the gene order of 60-65% belongs to single copy gene, such as more
The gene order of number encoder protein is single copy gene.Such as human leucocyte antigen (HLA) HLA genes, hereditary hearing impairment gene
GJB2 etc..Huge hereditary information is stored in single copy gene, encodes the protein of various different function.With single copy gene
Corresponding is multi-copy gene, that is, refers to the gene there are multiple copies in haploid genome, such as rRNA genes, tRNA
Gene etc., there are about 200 copy tRNA genes in the haploid genome of people, certain histone genes are also multi-copy gene.
Housekeeping gene (house-keeping genes), also known as house-keeping gene refer to being intended to stablize expression in all cells
A genoid, product be to maintain radical cellular activities necessary to.Such as microtubule protein gene, glycolysis enzyme system base
Cause, ribosomal protein gene etc..House-keeping gene is that one kind remains that low-level methylates and is constantly in active transcription
The gene of state.House-keeping gene expression is smaller by such environmental effects, and is almost complete in each growth phase of individual
Continuous expression in portion's tissue, therefore be normally present in the euchromatin of biological cell core.Its expression only by initiating sequence or is opened
The influence of mover and RNA polymerase interaction, without being adjusted by other mechanism.Single copy house-keeping gene refers in monoploid base
Because there is the house-keeping gene that individually copies in group, most of house-keeping genes are single copy genes, such as Gene A CTB, ALDOA,
GAPD、NPGK1、LDHA、RPS27A、RPL19、RPL11、NONO、ARHGDIA、RPPH1、GAPDH、NAGK、TUFMP1、
LINE1, UBBP4, EFTUD2 etc..
Single nucleotide polymorphism (SNP) is primarily referred to as not influencing individual function and group's frequency of the base of change is not low
In 1% single sequence change.If the change of DNA affects the structure and function of product (RNA or albumen), certain disease is caused
Sick or specific phenotype, then referred to as gene mutation.Polymorphism and mutation are in itself without difference, and only frequency is different.By
Cause the adaptability of individual very low in mutation majority, so being caused frequency very low by the effect of negative selection.For polymorphic
Property, frequency is influenced by several factors, including genetic drift, group factor, natural selection etc..The SNP of people is usually showed
For polymorphic allele, allele (allele) refers to being located in pair of homologous chromosome same position to control relativity
The one pair of genes of shape.If two members are identical in pairs of allele, which is homozygous for this character
Son.If two allele differ, which is heterozygote for the character.Therefore, the homozygote of allele or
Heterozygote genotype determines the character of biology, and many diseases and the genotype of allele are closely related.Quickly and accurately examine
The genotype for surveying allele is of great importance to disease, clinical detection and molecule diagnosis etc..
The present invention illustrates a kind of new method of quick detection list copy allele genic homozygote and heterozygote genotype, the party
Method have the characteristics that accuracy height, high sensitivity, operation it is simple and can quantitative analysis, can be used for genetic screening, prevention and base
Because of diagnosis.
Invention content
In one embodiment, the present invention provides a kind of identifying single copy gene mutated-genotype using digital pcr
Method the described method comprises the following steps：Step 1：The single copy gene mutational site to be measured design digital pcr of sample is expanded
Increase primer and probe；Step 2：To the single copy house-keeping gene design digital pcr amplimer and probe of sample；Step 3：It is right
The mutational site of step 1 and the house-keeping gene of step 2 carry out twin-channel dual digital pcr reaction；And step 4：Pass through list
The digital pcr reaction of copy gene mutation site is augmented with is mutated position without determining sample to be tested with the presence or absence of the single copy gene
Point；If there is the single copy gene mutational site, copying for house-keeping gene is copied by comparing single copy gene to be measured and list
Shellfish number identifies the genotype of single copy gene to be measured.Detectable sample source is various in the present invention, it is preferred that sample source
It can be blood sample, can also be buccal swab, expand the use scope of the present invention.Relative to the minimally invasive materials of blood,
Buccal swab is a kind of simple and quick, cheap and noninvasive sample collection method, is the ideal sample for substituting blood.Oral cavity
It can extract the complete genome of people in swab, there is ideal detection result in multiple research fields.Sample collection is without special
Sample collection can be completed with reference to buccal swab acquisition operations regulation in industry technical staff, detected person.It can pass through after sample collection
Mailing way is transported, and room temperature preservation is placed in.
Preferably, the method includes using micro-fluidic chip to divide digital pcr reaction system by microlayer model technology
At individual unit, PCR amplification is carried out to each unit and collects the fluorescence signal in each unit, obtains fluorescence signal value.
Preferably, each microlayer model is a digital PCR amplification system in method provided by the present invention, is used
PCR amplification instrument carries out PCR amplification, by detecting the fluorescence signal of each drop, to obtain the scatter plot of all drops.
In one embodiment, the method is further comprising the steps of：If the copy number of single copy gene to be measured and
The ratio of the copy number of single copy house-keeping gene then judges that single copy gene to be measured is homozygous in 1 ± 15% range；If
The copy number ratio of the copy number of single copy gene to be measured and single copy house-keeping gene then judges list in 0.5 ± 15% range
Copy gene is heterozygous.
In one embodiment, the method is further comprising the steps of：If the copy number of single copy gene to be measured and
The ratio of the copy number of single copy house-keeping gene then judges single copy to be measured in 90% or more confidence interval of theoretical value 1
Gene is homozygous；If the copy number ratio of the copy number of single copy gene to be measured and single copy house-keeping gene is in theoretical value
The 90% of 0.5 is in upper confidence interval, then to judge single copy gene for heterozygous.
In one embodiment, the method is further comprising the steps of：If the copy number of single copy gene to be measured and
The ratio of the copy number of single copy house-keeping gene then judges single copy gene to be measured in 95% confidence interval of theoretical value 1
It is homozygous；If the copy number ratio of the copy number of single copy gene to be measured and single copy house-keeping gene is in theoretical value 0.5
In 95% confidence interval, then judge single copy gene for heterozygous.
In one embodiment, the single copy gene to be measured be selected from GJB2, SLC26A4, MTRNR1, GJB3, HBA1,
Any of HBA2, HBB, PAH, HLA, TRIO, SMN1, DMD；And/or single copy house-keeping gene be selected from ACTB, ALDOA,
GAPD、NPGK1、LDHA、RPS27A、RPL19、RPL11、NONO、ARHGDIA、RPPH1、GAPDH、NAGK、TUFMP1、
Any of LINE1, UBBP4 and EFTUD2.
In one embodiment, the single copy gene to be measured is HLA-B27 and/or single copy house-keeping gene
It is RPPH1, GAPDH or NAGK.
In one embodiment, the single copy gene to be measured is the upper of the digital pcr amplification in the mutational sites HLA-B27
It swims primer and is selected from SEQ ID NOs：Any of 1-9；Downstream primer is selected from SEQ ID NOs：Any of 10-12；With
Detection probe is selected from SEQ ID NOs：Any of 13-16.
In one embodiment, the detection probe 5 ' holds mark fluorescent dyestuff, preferably FAM or VIC fluorescence to contaminate
Material；With 3 ' end label MGB.
In one embodiment, the sense primer of the digital pcr amplification of single copy house-keeping gene is selected from SEQ ID
NOs：17,19, any of 21；Downstream primer is selected from SEQ ID NOs：18,20, any of 22；It is selected with detection probe
From SEQ ID NOs：Any of 23-25.
In one embodiment, the detection probe 5 ' holds mark fluorescent dyestuff, preferably FAM or VIC fluorescence to contaminate
Material；With 3 ' end label MGB.
In one embodiment, the sense primer of the digital pcr amplification of single copy house-keeping gene is selected from SEQ ID
NOs：17,19, any of 21；Downstream primer is selected from SEQ ID NOs：18,20, any of 22；It is selected with detection probe
From SEQ ID NOs：Any of 23-25.
In one embodiment, the detection probe 5 ' holds mark fluorescent dyestuff, preferably VIC or TET fluorescence to contaminate
Material and 3 ' end label MGB or BHQ1.
The present invention provides a kind of digital pcr method of detection human leukocyte antigen HLA-B 27 gene type, including following step
Suddenly：1) reaction system containing oligonucleotides described in table 1 is prepared；2) reaction system is prepared as microlayer model with sample preparation instrument；
3) microlayer model containing reaction system is put into PCR instrument, carries out PCR amplification；4) it is measured with chip reading instrument glimmering after PCR reactions
Optical signal；With the ratio that B27 gene copy numbers and reference gene copy number 5) are calculated by software, according to gene copy number and interior
The ratio judgement HLA-B27 genes for joining gene copy number are homozygous or heterozygous.
The program of PCR amplification described in above-mentioned digital pcr method is：95 DEG C, 10min；95 DEG C of 30sec, 60 DEG C of 1min are carried out
40 recycle, then 98 DEG C of 10min.
Table 1 is used to detect the primer and probe sequence of human leukocyte antigen HLA-B 27 gene type
The quantitative detection to HLA-B27 genotype may be implemented using digital pcr detection method provided by the present invention, spirit
Sensitivity can reach 0.1%.In addition, this method step simplicity is easily operated, it is a kind of effective ways carrying out Genotyping.
Compared with art methods, the present invention has the advantages that：
1. providing the method for identifying human leukocyte antigen HLA-B 27 gene type using digital pcr technology for the first time；
2. avoiding through flow cytometry, real-time fluorescence quantitative PCR (qPCR) quantitative analysis is inaccurate, error is big, knot
The problem of fruit difficult judgment.The present invention is that one kind is quick, accurately and reliably identifies HLA-B27 homozygotes or heterozygote genotype
And the method for carrying out absolute quantitation.
The present invention illustrates a kind of new method of quick detection list copy allele genic homozygote and heterozygote genotype, the party
It is accurate, convenient, fast and at low cost that method has the characteristics that, can be used for genetic screening, prevention and gene diagnosis.
Description of the drawings
It in order to more clearly explain the technical solutions in the embodiments of the present application, below will be to needed in the embodiment
Attached drawing is briefly described, it should be apparent that, the accompanying drawings in the following description is only some embodiments described in the application, right
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings
Its attached drawing.
Fig. 1 is HLA-B27 digital pcr results amplification scatter plot.
Specific implementation mode
In order to make art technology field personnel more fully understand the technical solution in the application, below in conjunction with embodiment
The invention will be further described, it is clear that and described embodiments are only a part of embodiments of the present application, rather than whole
Embodiment.Based on the embodiment in the application, those of ordinary skill in the art are obtained without making creative work
The all other embodiment obtained, shall fall within the protection scope of the present application.The present invention is made with reference to the accompanying drawings and embodiments
It further describes.
Embodiment identifies human leukocyte antigen HLA-B 27 gene type by digital pcr
Microlayer model digital pcr reaction condition example for detection primer probe performance is as follows：Pass through nucleic acid extraction first
Kit extracts clinical sample nucleic acid, then prepares PCR amplification system, and pcr amplification reaction mixture includes：1×
MasterMix premixed liquids (new Yi, a legendary monarch of Youqiong State in the xia Dynasty manufacture), 1 × stabilizer (new Yi, a legendary monarch of Youqiong State in the xia Dynasty manufacture), each 200-1000nM of primer (B27-F1, B27-R1,
RPPH1-F, RPPH1-R, each 100-800nM of detection probe (B27-Pb1, RPPH1-Pb), template DNA 10-100ng, moisturizing are arrived
30ul, by reagent mixing.Microlayer model is carried out according to specification with sample preparation instrument (new Yi, a legendary monarch of Youqiong State in the xia Dynasty manufactures scientific and technological (Beijing) Co., Ltd)
It prepares.Then the 8 townhouse pipes containing microlayer model are put into PCR instrument and are expanded, amplification condition setting is as follows：
After PCR amplification, with chip reading instrument (new Yi, a legendary monarch of Youqiong State in the xia Dynasty manufactures scientific and technological (Beijing) Co., Ltd) with reference to instrument operation instructions
Carry out drop detection and data analysis.B27 genotype is detected by fluorescence signal, and absolute quantitation is directly carried out to it.It calculates
The ratio of the copy number of B27 genes and reference gene in homozygote and heterozygosis subsample determines that sample HLA-B27 genes are homozygosis
Son or heterozygote genotype.
Fig. 1 is 1 digital pcr of sample as a result, can see from the scatter plot of fluorescence signal, and B27 genes (channels FAM) are sun
Property, by being compared with reference gene (channels VIC) copy number and (being shown in Table 2), obtain the ratio of FAM and the channels VIC copy number
It is 0.49, then the interpretation sample is heterozygote B27 genotype, (B*27 consistent with sequencing result:05:02B*44:03:02).
Table 2 includes the testing result of 28 parts of clinical samples, passes through the homozygote or heterozygote genotype of ddPCR identifications and survey
The result of sequence identification is completely the same, illustrates that the method for the present invention is a kind of allele genic homozygote and miscellaneous accurately and rapidly identified
The method of zygotic genotype, kit of the invention can to human leukocyte antigen HLA-B 27 homozygote or heterozygote genotype into
Row precise Identification.
Table 2 includes the testing result of 28 parts of clinical samples, passes through the homozygote or heterozygote genotype of ddPCR identifications and survey
The result of sequence identification is completely the same, wherein 8 parts of samples of homozygote, and 20 parts of samples of heterozygote are for statistical analysis to these samples,
95% confidence interval formula of normal distribution is CI=[+1.96 σ of μ -1.96 σ, μ], and wherein μ is the FAM repeatedly measured and the channels VIC
The average value of the ratio of copy number；σ is the variance repeatedly measured.Result of calculation is as follows：Homozygote FAM and the channels VIC copy number
95% confidence interval of ratio be [0.85,1.11], 95% confidence area of the ratio of heterozygote FAM and the channels VIC copy number
Between be [0.47,0.53].With further increasing for sample size, for judging heterozygote or homozygote genotype in the present invention
Its 95% confidence interval will be closer to actual value to FAM with the ratio of the channels VIC copy number, and as a result judging will be more acurrate.
By calculating the ratio of B27 gene copy numbers and reference gene copy number, if HLA-B27 gene copy numbers and interior
Join the ratio of gene copy number in 95% confidence interval of theoretical value 1 (present invention is measured as 0.85-1.11), then can determine that
HLA-B27 genes are homozygous；If the ratio of HLA-B27 gene copy numbers and reference gene copy number is in theoretical value 0.5
95% confidence interval in (present invention is measured as 0.47-0.53), then judge HLA-B27 genes for heterozygous.
The HLA-B27 digital pcr testing results of 2. clinical sample of table
It should be understood that the present invention disclosed is not limited only to specific method, scheme and the substance of description, because these
It is alterable.It will also be understood that purpose of the terminology used here just for the sake of the specific embodiment scheme of description, rather than
It is intended to limit the scope of the invention, the scope of the present invention is limited solely by the attached claims.
Those skilled in the art, which will also be appreciated that or be able to confirm that, uses no more than routine experiment, institute herein
Many equivalents of the specific embodiment of the present invention stated.These equivalents are also contained in the attached claims.
Sequence table
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Claims (10)
1. a method of utilize digital pcr identify single copy gene mutated-genotype, which is characterized in that the method includes with
Lower step：
Step 1：Digital pcr amplimer and probe are designed to the single copy gene mutational site to be measured of sample；
Step 2：To the single copy house-keeping gene design digital pcr amplimer and probe of sample；
Step 3：The house-keeping gene in mutational site and step 2 to step 1 carries out twin-channel dual digital pcr reaction；With
Step 4：It is augmented with by the digital pcr reaction in single copy gene mutational site and whether there is institute without determining sample to be tested
State single copy gene mutational site；If there is the single copy gene mutational site, by comparing single copy gene to be measured and
List copies the copy number of house-keeping gene to identify the genotype of single copy gene to be measured.
2. according to the method described in claim 1, it is characterized in that, the method is further comprising the steps of：If to be measured singly copy
The ratio of the copy number of the copy number of shellfish gene and single copy house-keeping gene then judges single copy to be measured in 1 ± 15% range
Gene is homozygous；If the copy number ratio of the copy number of single copy gene to be measured and single copy house-keeping gene 0.5 ±
In 15% range, then judge single copy gene for heterozygous.
3. according to the method described in claim 1, it is characterized in that, the method is further comprising the steps of：If to be measured singly copy
The copy number and list of shellfish gene copy the ratio of the copy number of house-keeping gene in 90% or more confidence interval of theoretical value 1,
Then judge that single copy gene to be measured is homozygous；If the copy of the copy number of single copy gene to be measured and single copy house-keeping gene
Ratio is counted the 90% of theoretical value 0.5 in upper confidence interval, then to judge single copy gene for heterozygous.
4. according to the method described in claim 3, it is characterized in that, the method is further comprising the steps of：If to be measured singly copy
The ratio of the copy number of the copy number of shellfish gene and single copy house-keeping gene is then sentenced in 95% confidence interval of theoretical value 1
Fixed single copy gene to be measured is homozygous；If the copy number ratio of the copy number of single copy gene to be measured and single copy house-keeping gene
Value then judges single copy gene for heterozygous in 95% confidence interval of theoretical value 0.5.
5. according to the method described in claim 1, it is characterized in that, the single copy gene to be measured be selected from GJB2, SLC26A4,
Any of MTRNR1, GJB3, HBA1, HBA2, HBB, PAH, HLA, TRIO, SMN1, DMD；And/or single copy house-keeping gene
Selected from ACTB, ALDOA, GAPD, NPGK1, LDHA, RPS27A, RPL19, RPL11, NONO, ARHGDIA, RPPH1, GAPDH,
Any of NAGK, TUFMP1, LINE1, UBBP4 and EFTUD2.
6. according to the method described in claim 5, the single copy gene to be measured is HLA-B27 and/or single copy house keeper
Gene is RPPH1, GAPDH or NAGK.
7. according to the method described in claim 6, the digital pcr that the single copy gene to be measured is the mutational sites HLA-B27 expands
The sense primer of increasing is selected from SEQ ID NOs：Any of 1-9；Downstream primer is selected from SEQ ID NOs：Any in 10-12
It is a；It is selected from SEQ ID NOs with detection probe：Any of 13-16.
8. according to the method described in claim 7, the detection probe 5 ' holds mark fluorescent dyestuff, preferably FAM or VIC are glimmering
Photoinitiator dye；With 3 ' end label MGB.
9. according to the method described in claim 6, the sense primer of the digital pcr amplification of single copy house-keeping gene is selected from
SEQ ID NOs：17,19, any of 21；Downstream primer is selected from SEQ ID NOs：18,20, any of 22；And inspection
Probing needle is selected from SEQ ID NOs：Any of 23-25.
10. according to the method described in claim 9, the detection probe 5 ' holds mark fluorescent dyestuff, preferably VIC or TET
Fluorescent dye；With 3 ' end label MGB or BHQ1.
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