CN106636435A - Method for genetic testing in single cells by HRM (high resolution melting) and pyrosequencing - Google Patents
Method for genetic testing in single cells by HRM (high resolution melting) and pyrosequencing Download PDFInfo
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Abstract
The invention belongs to the technical field of medical science and discloses a method for genetic testing in single cells by HRM (high resolution melting) and pyrosequencing. By high-throughput sequencing for haplotype analysis of a single-gene inheritance disease family, an analysis method for mutation detection and SNP (single nucleotide polymorphism) by adoption of a single-cell whole genome amplification product as a template on the basis of HRM and pyrosequencing is established. The method can be applied clinically directly to provide fast detection means for PGD (preimplantation genetic diagnosis) diagnosis of patients suffering from single gene inheritance diseases.
Description
Technical field
The invention belongs to medicine technology field, more particularly to a kind of carried out in unicellular using HRM and pyrosequencing
The method of Genetic Detection.
Background technology
PGD (preimplantation genetic diagnosis, PGD) is referred to embryo or ovum
Sub-line blastomere or blastaea trophoblastic cell biopsy, make chromosome and (or) genetics detection, then by the embryo without hereditary
Tire is implanted into maternal uterine, is that one kind that a kind of foundation is derived on assisted reproduction, molecular biology and genetic base is new
Diagnostic techniquess.The girl baby that nineteen ninety, in the world the first Jing PGD were diagnosed is born in Britain.Hereafter PGD technologies are developed rapidly, are entered
The later stage nineties 20th century, PGD starts popularization, and the PGD cycles of 7693 monogenic diseases have been reported in the current whole world.It is walked substantially
Suddenly it is by external fertilization (in vitro fertilization, IVF) or intracytoplasmic sperm injection
(intracytoplasmic sperm injection, ICSI) obtains germ cell, and Jing embryo biopsies art obtains 1-2 embryo's ovum
Schistocyte, after genetic analysis cleavage-cell, determines the genetic constitution of embryo, selects fetal tissues to transplant in utero.
PGD technological difficulties are to detect limited material and the allele dropout rate (allele dropout, ADO) that causes
Increase, i.e., with unicellular substrate diagnosis single-gene defect disease inch, such as heterozygote it is unicellular in, one of two allele
It is not amplified.This is the main cause for causing PGD mistaken diagnosis.Grifo reports PGD because ADO causes the case of mistaken diagnosis within 1994.From
And clinically cause the attention to ADO phenomenons.ADO can be with chance mechanism in two allele any one.Normal
In autosomal dominant heredopathia, it is fetal tissues that the ADO of mutation allele may cause ill embryo's mistaken diagnosis.The finger of ESHRE
Lead suggestion and think that ADO rates are more preferably less than 10%.
At present monogenic disease PGD (single gene defect PGD, SGD-PGD) is most uses nest-type PRC or multiple
Nested PCR amplification is directed to the special target sequence amplification in mutational site, then is sequenced or follow-up molecular biological analysis；Or
First unicellular DNA is carried out using unicellular whole genome amplification (whole genome amplification, WGA) technology pre-
Amplification, then follow-up analysis is carried out, because WGA products can carry out multiple, many Locus Analysis in Shoots, it is increasingly becoming the weight of SGD-PGD
Want approach.WGA is a kind of to be intended to completely equably expand whole base with minimum amplification bias (amplification bias)
Because of the technology of group sequence, full genome amplification technique includes the degenerate oligonucleotide primed PCR (DOP-PCR) based on thermal cycle
Not multiple displacement amplification (the multiple of the use random primer constant-temperature amplification based on PCR
Displacementamplification, MDA) technology.In recent years, Zong etc. reports repeatedly annealing ring-type cyclic amplification
(multiple annealing and loopingbased amplification cycles, MALBAC) technology carries out full base
Because of group amplification and a follow-up high-flux sequence, MALBAC employs linear amplification and unconventional exponential amplification mode, thus energy
The homogeneity of amplification is increased substantially, is that a kind of PGD that can be applicable to analyzes more good full genome amplification technique.It is up till now
Only, unicellular gene amplification diagnosis still can not completely avoid ADO problems, in order to reduce singe-cell PCR in ADO and the mistake that causes
Examine, existing laboratory starts to introduce hereditism's haplotyping (preimplantationgenetic before Embryonic limb bud cell
Haplotyping, PGH) strategy, by the multiple STRs (short tandem repeat, STR) to embryo
Or single nucleotide polymorphism (single nucleotide polymorphism, SNP) site primer connects with reference to family haplotype
Lock analysis, can solve the problems, such as that in theory unicellular amplification template allelic is lost.The appearance of high throughput sequencing technologies
The technological innovation of PGH is greatly accelerated, using target sequence capture chip the pathogenic base of high throughput sequencing technologies rapid screening has been combined
Because of the SNP haplotype informations in mutational site, with reference to the SNP information by inspection embryo and mutation point analysiss information, list can be effectively eliminated
The impact of ADO during cell whole genome amplification, improves the accuracy rate of SGD-PGD, it is anticipated that flat based on high-flux sequence
The PGD that the PGH of platform and the detection of gene mutation point combine is the development trend of following SGD-PGD.
The present invention is the methodology and clinical application research based on SGD-PGD.At present many reproductive centers are using jelly embryo
Transplant with blastaea, although there are some researches show fresh embryo and freeze the transplanting succeed rate no significant difference of embryo, and the pregnancy rate of blastaea is higher than
Division stage embryo, but expose the factor that can not ignore in really ART safety evaluations in vitro for a long time.According to ESHRE to complete
The statistics in ball SGD-PGD cycles, in all biopsy embryos, 90% is division stage biopsy, and more than 98% is all fresh embryo transfer.It is fresh
Embryonic implantation needs accurately to judge the genotype of embryo within 24-48h that this needs possess sequencing inside inspection center
Platform sets up quick service agreement with company, but practical operation gets up to acquire a certain degree of difficulty.
High-resolution fusion curve analytical technology (high-resolution melting analysis, HRM) and pyrophosphoric acid
Sequencing (pyrosequeneing) technology is a kind of brand-new mutation scanning and the heredity of gene type point that developed recently gets up
Analysis method.Round pcrs of the HRM based on Efficient robust, HRM is not limited to by mutating alkali yl site with type, without the need for sequence-specific
Probe, directly operation high-resolution melts after PCR terminates, you can complete paired samples mutation, SNP, methylate, HLA distribution type etc.
Analysis.Because easy to operate quick, use cost is low, as a result accurately, by common concern.And pyrosequencing by nucleotide and
Template combines the pyrophosphoric acid of rear release and causes enzyme cascade, promotes fluorescein luminescence and is detected.Pyrosequencing is one
Individual preferable genetic analyses technology platform, can both carry out DNA sequence analysis, can carry out being detected and being waited based on sequence analysis SNP again
Position gene frequency measure etc., the technology has been widely used in the every field such as medical biotechnology.At present, this two technologies exist
Application in PGD detections has not been reported, this invention address that develop application of this two technologies in SGD-PGD, in the hope of can
Accelerate PGD result detection times in the range of control.
Status both at home and abroad and development trend
With the progress and the rising of Disease-causing gene number of reports of genetic diagnosises technology, family's coefficient of gene pathogenic sites is determined
Amount also occur it is larger rise, the demand of SGD-PGD is presented and continues ascendant trend, it is desirable to carried out the monogenic disease patient of PGD
Become an important crowd of clinical assisted reproduction treatment.
Treff etc. carries out chip target acquistion sequence and high-flux sequence in Ion Torrent microarray datasets, to carrying
Cystic fibrosises (cystic fibrosis, CF), Wo Kehuabao syndromes (Walker-Warburg syndrome, WWS), family
Race's property vegetative dystonie (familial dysautonomia, FD), the chain phosphopenic ricketses (X-linked of X-
Hypophosphatemicrickets, XHR) or neurofibromatosiss (neurofibromatosis1, NF1) the totally 6 couples of Mr. and Mrs
Embryo's whole genome amplification product has carried out high-flux sequence, the detection knot of its PGD result and laboratory another two conventional method
Fruit is all consistent.Because there is the monogenic disease such as polycystic kidney and adrenal cortical hyperplasia of pseudogene at some in secondary sequencing detection
Mutation recall rate it is relatively low, the error rate of SNP haplotype linkage analysts is higher, thus using it is secondary sequencing simplifying and speeding up
Still need to while SGD-PGD processes rely on a generation sequencing verified, the present invention application precisely in order to accelerate PGD detect into
Journey, to realize embryo's cleavage stage biopsy, the purpose of fresh cycle transplanting.
Flourishing recently as secondary sequencing technologies so that high-throughout gene sequencing technology is ripe day by day, by
Gradually clinical medical application is proceeded to from conventional base R＆D units, especially under the historical background that test-tube baby is born in a large number,
The advantage for how playing secondary sequencing carries out the task that the gene before implantation differentiates and becomes the emphasis of research.
The content of the invention
It is an object of the invention to provide using high-resolution fusion curve analytical technology (high-resolution
Melting analysis, HRM) and pyrosequencing method that Genetic Detection is carried out in unicellular, it is intended to solve PGD diagnosis
During generation sequencing analysis mutational site and SNP time length problem.
The present invention is achieved in that a kind of utilization HRM and pyrosequencing carry out the side of Genetic Detection in unicellular
Method, the utilization HRM and pyrosequencing carry out the method for Genetic Detection in unicellular to be included：
Haplotyping is carried out to single gene inheritance disease family using high-flux sequence；
Set up carries out abrupt climatic change with unicellular whole genome amplification product based on HRM and pyrosequencing techniques as template
With the analysis method of single nucleotide polymorphism (single nucleotide polymorphism, SNP) typing.
Further, the utilization HRM and pyrosequencing carry out the method for Genetic Detection in unicellular and specifically include：
(1) DNA extraction：
5ml peripheral bloods are taken, in being stored in EDTA anticoagulant tubes, is fully mixed；
Using QIAamp DNA Blood Mini Kit test kits, peripheral blood DNA extraction is completed；
Concentration of specimens is detected：Quantitative using Nano drop2000 detections, DNA total amounts are not less than 3ug, and concentration is not less than
30ng/ul, OD260/280 are 1.7-2.0；
(2) high-flux sequence is combined using target sequence capture, to there is fertility to want in clearly pathogenic monogenic disease family
Another one patient (or carrier) peripheral blood DNA sample carries out capture sequencing and bioinformatics SNP in the Mr. and Mrs for asking and family
Analysis, obtains the chain haplotype information of this pair of Mr. and Mrs' Disease-causing gene；
(3) generation sequence verification：The SNP site of the provided information that high-flux sequence is detected, on mutational site
Select 3～4 sites within the 1M of downstream respectively, primer is designed, to obtain the peripheral blood of three persons under inspection of haplotype information
DNA is template PCR amplifications, sequencing；
(4) single lymphocyte is separated：
5ml peripheral bloods (three persons under inspection for carrying out haplotyping) are taken, in being stored in anticoagulant heparin pipe, is fully mixed；
Take 2ml peripheral bloods and separate single lymphocyte；
(5) whole genome amplification
Using REPLI-g Single Cell kit test kits, complete unicellular or blastomere whole genome amplification and extract
DNA；
Concentration of specimens is detected：Quantitative using Qubit Fluorometer detections, DNA total amounts are not less than 2ugM<；
Electrophoresis detection：, in 300bp～2000bp, negative control is without band for amplification scope；
(6) PGD preliminary experiments：With single lymphocyte whole genome amplification product as template, abrupt climatic change and SNP point are carried out
Type, sequencing calculates ADO rates；
With single lymphocyte whole genome amplification product as template, with HRM abrupt climatic change and SNP typings are carried out；
With single lymphocyte whole genome amplification product as template, with pyrosequencing abrupt climatic change and SNP are carried out
Typing.
Further, the employing target sequence capture combines high-flux sequence, in clearly pathogenic monogenic disease family
There is another one patient (or carrier) peripheral blood DNA sample in the Mr. and Mrs for giving birth to requirement and family to carry out bioinformatic analysis,
The chain haplotype information of this pair of Mr. and Mrs' Disease-causing gene is obtained, is specifically included：
GDNA fragmentations, fragment ends reparation adds joint, builds library；
Enter the ligation-mediated PCR method amplification of rearrangement before capture to library；
Sample after amplification and chip are hybridized；
Chip is cleaned, and removes uncombined sequence, then elutes capture sequence；
LM-PCR amplifications are carried out again to sample after capture；
The relative enrichment times of target area are estimated by qPCR, to be reached carried out after Quality Control requirement front preparation is sequenced；
High-flux sequence is analyzed.
Further, HRM gene types include：
Enter performing PCR reaction to sample, need 40min～2h；
Thermal denaturation and gathered data, thermal denaturation time 5min～10min are carried out to PCR primer by dissolution equipment；
Analyzed by data analysis software, draw the information of mutation or SNP.
Further, pyrosequencing gene type includes：
Design and synthetic pcr primer thing, one of them labelling biotin；
PCR reacts；
The annealing of the separation of DNA double chain, the single stranded DNA containing biotin and sequencing primer；
Single stranded DNA containing biotin is sequenced；
Abrupt climatic change and snp analysis based on pyrosequencing.
The present invention carries out accurate PGD detections to monogenic disease family with reference to secondary sequencing technologies, is SGD-PGD clinic works
The new thinking that the development of work is provided, the generation to preventing disease is improved the quality of the population and is had great importance.
Clinic is present invention can be directly applicable to, for SGD-PGD diagnosis new more quick detection meanss are provided.
Description of the drawings
Fig. 1 is the side that utilization HRM provided in an embodiment of the present invention and pyrosequencing carry out Genetic Detection in unicellular
Method flow chart.
Fig. 2 is the DMD patient PGD diagnosis that high-flux sequence haplotype analysis method provided in an embodiment of the present invention is completed
Figure；
Fig. 3 is that RET genes provided in an embodiment of the present invention have C634Y heterozygous mutant figures.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that specific embodiment described herein is not used to only to explain the present invention
Limit the present invention.
The method that utilization HRM provided in an embodiment of the present invention and pyrosequencing carry out Genetic Detection in unicellular, bag
Include：
Haplotyping is carried out to single gene inheritance disease family using high-flux sequence；
Set up carries out abrupt climatic change with unicellular whole genome amplification product based on HRM and pyrosequencing techniques as template
With the analysis method of SNP typings；
The application principle of the present invention is described in detail with reference to specific embodiment.
As shown in figure 1, utilization HRM provided in an embodiment of the present invention and pyrosequencing carry out Genetic Detection in unicellular
Method, specifically include：
1st, the patient with genetic diseasess and its Family data are collected from genetic counselling outpatient service；Carry out gene test it
Afterwards, informed consent is signed, obtains another ill or carrier peripheral blood in couple and family.
(1) DNA extraction
Take adult person under inspection and be not less than 5ml peripheral bloods, in being stored in EDTA anticoagulant tubes, fully mix；
Using the QIAamp DNA Blood Mini Kit test kits of Qiagen companies, in strict accordance with kit specification
Complete peripheral blood DNA extraction；
Concentration of specimens is detected：Quantitative using Nano drop2000 detections, DNA total amounts are not less than 3ug, and concentration is not less than
30ng/ul, OD260/280 are 1.7-2.0.
(2) single lymphocyte is separated
Take adult person under inspection and be not less than 5ml peripheral bloods, in being stored in anticoagulant heparin pipe, fully mix；
Take 2ml peripheral bloods and separate single lymphocyte；
(3) whole genome amplification
It is complete in strict accordance with kit specification using the REPLI-g Single Cell kit test kits of Qiagen companies
DNA is extracted into unicellular or blastomere whole genome amplification；
Concentration of specimens is detected：Quantitative using Qubit Fluorometer detections, DNA total amounts are not less than 2ug；；
Electrophoresis detection：Main band is clear, size 10K or so or master tape disperse, is distributed mainly on 1K area above, and clearly
It can be seen that, negative control is without band.
2nd, the system high throughput sequencing technologies of HiSeq 2500 are combined using target sequence capture, to family sample (two kinds of lists
Times type sperm whole genome amplification product and wife's peripheral blood DNA) capture sequencing and bioinformatics snp analysis are carried out, obtain
Mr. and Mrs and the chain haplotype information of Disease-causing gene.
Basic step：
GDNA fragmentations, fragment ends reparation adds joint, builds library；
The ligation-mediated PCR method (Ligation-Mediated PCR, LM-PCR) for entering rearrangement before capture to library expands
Increase；
Sample after amplification and chip are hybridized；
Chip is cleaned, and removes uncombined sequence, then elutes capture sequence；
LM-PCR amplifications are carried out again to sample after capture；
The relative enrichment times of target area are estimated by qPCR, carries out that front preparation is sequenced by reaching after Quality Control requirement；
High-flux sequence is analyzed.
3rd, generation sequence verification
The SNP site of the provided information detected to high-flux sequence, selects respectively within upstream and downstream 1M of mutational site
Select 3-4 site, design primer, with obtain haplotype information three persons under inspection peripheral blood DNA as template PCR amplifications, survey
Sequence.
4th, HRM gene types
Enter performing PCR reaction to sample, need 40min～2h；
Thermal denaturation and gathered data are carried out to PCR primer by dissolution equipment, 5-10min is needed；
By expert data analysis software, the information of mutation or SNP is drawn.
5th, pyrosequencing gene type
One of design and synthetic pcr primer thing, primer labelling biotin；
PCR reacts；
The annealing of the separation of DNA double chain, the single stranded DNA containing biotin and sequencing primer；
Single stranded DNA containing biotin is sequenced；
Abrupt climatic change and snp analysis based on sequencing.
6th, PGD preliminary experiments
With single lymphocyte whole genome amplification product as template, abrupt climatic change and SNP typings are carried out, be sequenced, calculated
ADO rates；
With single lymphocyte whole genome amplification product as template, with HRM abrupt climatic change and SNP typings are carried out；
With single lymphocyte whole genome amplification product as template, with pyrosequencing abrupt climatic change and SNP are carried out
Typing；
Grope HRM consistent in the hope of the testing result for reaching three kinds of methods with pyrosequencing experiment condition.
8th, PGD diagnosis
The condition set up using preliminary experiment carries out PGD diagnosis for patient's single blastomere.
The application principle of the present invention is further described with reference to specific embodiment.
The present invention has successfully realized high-flux sequence haplotype analysis method in progressive muscular dystrophy at present
(Duchenne Muscular Dystrophy, DMD) (Fig. 2), myeloid muscular dystrophy (spinal muscular
Atophy, SMA), MEA (Fig. 3), ichthyosiss, the inspection in hemophilia and part polycystic kidney disease PGD
Survey.
Experimental work of the present invention will rely on our unit's reproductive center, central laboratory and Ministry of Education's reproduction heredity laboratory.
Possess LightCycler480 II (Roche LightCycler480 quantitative fluorescent PCR system), Qiagen PyroMark Q24 burnt
Phosphoric acid sequenator,System (Qiagen) capillary electrophoresis system, Roche MagNA Pure LC 2.0 are complete certainly
It is cold that kinetonucleus acid isolates and purifies sample adding system, BioRad-s1000PCR instrument, Bio-Rad Labworks image acquisition and analysis softwares, eppendorf
Freeze centrifuge, MilliQ ultrapure water machines, NanoDrop2000 ultramicrospectrophotometers and3.0 fluorescent quantitation instrument
Deng.The instrument and equipment and technology used from needed for unicellular separation, PCR, HRM and pyrosequencing can be met.
Presently preferred embodiments of the present invention is the foregoing is only, not to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (5)
1. a kind of method that utilization HRM and pyrosequencing carry out Genetic Detection in unicellular, it is characterised in that the utilization
HRM and pyrosequencing carry out the method for Genetic Detection in unicellular to be included：
Haplotyping is carried out to single gene inheritance disease family using high-flux sequence；
Set up carries out abrupt climatic change and list with unicellular whole genome amplification product based on HRM and pyrosequencing techniques as template
The analysis method of nucleotide polymorphisms typing.
2. the method for as claimed in claim 1 Genetic Detection being carried out in unicellular using HRM and pyrosequencing, its feature
It is that the utilization HRM and pyrosequencing carry out the method for Genetic Detection in unicellular and specifically include：
(1) DNA extraction：
5ml peripheral bloods are taken, in being stored in EDTA anticoagulant tubes, is fully mixed；
Using QIAamp DNA Blood Mini Kit test kits, peripheral blood DNA extraction is completed；
Concentration of specimens is detected：Quantitative using Nano drop2000 detections, DNA total amounts are not less than 3ug, and concentration is not less than 30ng/
Ul, OD260/280 are 1.7-2.0；
(2) high-flux sequence is combined using target sequence capture, to having what fertility was required in clearly pathogenic monogenic disease family
Another one patient or carrier peripheral blood DNA sample carry out capture sequencing and bioinformatics snp analysis in Mr. and Mrs and family, obtain
Obtain the chain haplotype information of this pair of Mr. and Mrs' Disease-causing gene；
(3) generation sequence verification：The SNP site of the provided information that high-flux sequence is detected, in mutational site upstream and downstream
Select 3～4 sites within 1M respectively, design primer, the peripheral blood DNA with three persons under inspection for obtaining haplotype information is
Template PCR amplifications, sequencing；
(4) single lymphocyte is separated：
5ml peripheral bloods are taken, in being stored in anticoagulant heparin pipe, is fully mixed；
Take 2ml peripheral bloods and separate single lymphocyte；
(5) whole genome amplification
Using REPLI-g Single Cell kit test kits, complete unicellular or blastomere whole genome amplification and extract DNA；
Concentration of specimens is detected：Quantitative using Qubit Fluorometer detections, DNA total amounts are not less than 2ug；
Electrophoresis detection：, in 300bp～2000bp, negative control is without band for amplification scope；
(6) PGD preliminary experiments：With single lymphocyte whole genome amplification product as template, abrupt climatic change and SNP typings are carried out,
Sequencing, calculates ADO rates；
With single lymphocyte whole genome amplification product as template, with HRM abrupt climatic change and SNP typings are carried out；
With single lymphocyte whole genome amplification product as template, with pyrosequencing abrupt climatic change and SNP typings are carried out.
3. the method for as claimed in claim 2 Genetic Detection being carried out in unicellular using HRM and pyrosequencing, its feature
It is that the employing target sequence capture combines high-flux sequence, to there is fertility to require in clearly pathogenic monogenic disease family
Mr. and Mrs and family in another one patient or carrier peripheral blood DNA sample carry out bioinformatic analysis, obtain this couple of Mr. and Mrs
The chain haplotype information of Disease-causing gene, specifically includes：
GDNA fragmentations, fragment ends reparation adds joint, builds library；
Enter the ligation-mediated PCR method amplification of rearrangement before capture to library；
Sample after amplification and chip are hybridized；
Chip is cleaned, and removes uncombined sequence, then elutes capture sequence；
LM-PCR amplifications are carried out again to sample after capture；
The relative enrichment times of target area are estimated by qPCR, to be reached carried out after Quality Control requirement front preparation is sequenced；
High-flux sequence is analyzed.
4. the method for as claimed in claim 2 Genetic Detection being carried out in unicellular using HRM and pyrosequencing, its feature
It is that HRM gene types include：
Enter performing PCR reaction to sample, need 40min～2h；
Thermal denaturation and gathered data, thermal denaturation time 5min～10min are carried out to PCR primer by dissolution equipment；
Analyzed by data analysis software, draw the information of mutation or SNP.
5. the method for as claimed in claim 2 Genetic Detection being carried out in unicellular using HRM and pyrosequencing, its feature
It is that pyrosequencing gene type includes：
Design and synthetic pcr primer thing, wherein a primer mark biotin；
PCR reacts；
The annealing of the separation of DNA double chain, the single stranded DNA containing biotin and sequencing primer；
Single stranded DNA containing biotin is sequenced；
Abrupt climatic change and snp analysis based on pyrosequencing.
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| CN108823330B (en) * | 2018-07-09 | 2022-01-14 | 安徽农业大学 | Soybean HRM-SNP molecular marker point marking method and application thereof |
| CN109086571A (en) * | 2018-08-03 | 2018-12-25 | 国家卫生计生委科学技术研究所 | A kind of method and system that monogenic disease hereditary variation is intelligently interpreted and reported |
| CN111321207A (en) * | 2020-02-15 | 2020-06-23 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Detection method for congenital bone marrow failure disease genotyping |
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