CN109988826A - It is a kind of to calculate the method and system that mixing sample is fitted into ratio based on STR - Google Patents
It is a kind of to calculate the method and system that mixing sample is fitted into ratio based on STR Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 51
- 238000002156 mixing Methods 0.000 title claims abstract description 47
- 108010007570 Amelogenin Proteins 0.000 claims abstract description 21
- 238000012408 PCR amplification Methods 0.000 claims abstract description 18
- 108020004414 DNA Proteins 0.000 claims description 36
- 108700028369 Alleles Proteins 0.000 claims description 13
- 108090000623 proteins and genes Proteins 0.000 claims description 13
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- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
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- 238000004519 manufacturing process Methods 0.000 claims description 2
- 210000001519 tissue Anatomy 0.000 description 9
- 238000001179 sorption measurement Methods 0.000 description 8
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- 238000004458 analytical method Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
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- 238000005336 cracking Methods 0.000 description 2
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- 238000001962 electrophoresis Methods 0.000 description 2
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- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
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- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 description 1
- 102100039088 Amelogenin, X isoform Human genes 0.000 description 1
- 102100039109 Amelogenin, Y isoform Human genes 0.000 description 1
- 206010008805 Chromosomal abnormalities Diseases 0.000 description 1
- 208000031404 Chromosome Aberrations Diseases 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 101000959114 Homo sapiens Amelogenin, X isoform Proteins 0.000 description 1
- 101000959107 Homo sapiens Amelogenin, Y isoform Proteins 0.000 description 1
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
It is a kind of to calculate the method and system that mixing sample is fitted into ratio based on STR.The method and system that mixing sample is fitted into ratio are calculated based on AMELOGENIN gene the present invention relates to a kind of.This method comprises: calculating the chimeric ratio of sample to be tested based on AMELOGENIN gene, its step includes: the PCR amplification for utilizing the primer with fluorescence for expanding the AMELOGENIN gene to carry out the sample to be tested, to obtain the data-oriented for the sample to be tested to calculate the chimeric ratio of the sample to be tested.
Description
Technical field
The method and system that mixing sample is fitted into ratio are calculated based on Short tandem repeatSTR the present invention relates to a kind of.
Background technique
In the detection case for carrying out fetal chromosomal abnormalities using two generation sequencing technologies (NGS), it is mixed in fetus sample
The sample of source of parents happens occasionally, the especially abortion tissue sample pollution that is highly prone to source of parents.In this case, information analysis people
Member is difficult to determine that the truth of sample is chromosomal mosaic or has source of parents pollution, and concentration of specimens ratio is more complex etc.
Problem makes the analysis of inspection result be commonly present uncertainty.Therefore there is an urgent need to one kind can identify pollution, calculate chimeric ratio
System and method as supplementary means, to assist NGS to make correct judgement.
In addition, generally requiring to judge whether the abortion tissue is mixed with source of parents pollution when detecting abortion tissue, such as
Fruit judges that the abortion tissue is mixed with source of parents and pollutes the reliability that then will affect to the outcome evaluation of abortion tissue.
STR (Short tandem repeatSTR (short tandem repeats)) as a kind of maturation generation sequencing technologies in parent-offspring
Identification field is used widely, and the basic principle of STR fragment analysis is that the str locus type half of same individual comes from father,
Half comes from mother.I.e. in everywhere STR, from father, another allelotype is lost for an allelotype heredity of filial generation
It passes from mother.In addition to enzygotic twins, the DNA sequence dna that everyone or biology include is different, unique.Based on STR's
Testing principle is the primer using fluorescent marker in PCR amplification str locus seat, makes fluorescence mark on a chain band of PCR product
Note.This DNA fragmentation with fluorescent molecule in gel from the scanning window of the laser scanner of cathode terminal, fluorescent dye by
To excitation, the light of certain wavelength is issued, is recorded by fluorescence intensity, each DNA fragmentation electrophoresis track with fluorescent dye
It is recorded by the real time each by laser scanning window, each segment is indicated with fluorescent absorption peak.Peak value is got over
Height indicates that the fragment amount is more；The more early appearance in peak, segment are smaller.Therefore it can use the principle according to different size segment
Peak value height determines whether sample is mixing sample with different loci peak number amount.
Meanwhile it can use the peak value height of AMELOGENIN gene or peak area ratio determines sample mixing or chimeric ratio
Example.
Bibliography
Non-patent literature
Gill P,Sparke R,Pinchin R,et al.Interpreting sipmle STR mixtures
using allele peak areas.Forensic Science International,1998,91:41
Prinz M,Boll K,Baum,et al.Multiplexing of Y chromosome specific STRs
and performance for minxed samples.Forensic sicience Inte,trnational,1997,85:
209
Zheng Xiufen, Ji Guijin, Liu is superfine, two component hybrid dna sample STR time profiles, Chinese Journal of Forensic Sciences,
2000,15,4: 203-207
Summary of the invention
Mixing sample is fitted into ratio computational problem in NGS and in the process for judging that whether abortion tissue is polluted by source of parents
In be the frequent problem, since not only time-consuming for technology preferences problem, but also be difficult to tell the true source of sample
With proportion.
The mixing of sample can be simply and rapidly obtained using the analysis system of the invention based on AMELOGENIN gene
Situation.It can solve following problems using the present invention: determining the substantially ratio of each component in mixing sample；According to standard items etc.
The peak area ratio of position gene makes standard curve, then fits regression equation, by the peak area of sample to be tested allele
Than substituting into the regression equation, the ratio of component can be obtained.
As described above, the present invention relates to following contents:
1. a kind of calculate the method that mixing sample is fitted into ratio based on Short tandem repeatSTR comprising:
Judge that sample to be tested is chimeric with the presence or absence of sample based on Short tandem repeatSTR；And
After judgement is chimeric there are sample, the chimeric ratio of sample to be tested is calculated based on AMELOGENIN gene.
2. according to method described in item 1, wherein judge that sample to be tested is chimeric with the presence or absence of sample based on Short tandem repeatSTR
The step of include:
The PCR amplification of the sample to be tested, base are carried out using the primer with fluorescence for expanding the Short tandem repeatSTR
It is fitted into the result of PCR amplification to determine whether existing.
3. the method according to item 1 or 2, wherein calculate the chimeric ratio of sample to be tested based on Short tandem repeatSTR
Step includes:
Expanded using the PCR that the primer with fluorescence for expanding the AMELOGENIN gene carries out the sample to be tested
Increase, to obtain the data-oriented for the sample to be tested to calculate the chimeric ratio of the sample to be tested.
4. the method according to item 2 or 3, wherein the result of based on PCR amplification is to determine whether exist chimeric specific
Are as follows: if the number that allele occurs in same gene seat is 3 or more, and sample to be tested is the aggregate sample of more than two individuals
This.
5. the method according to item 2 or 3, wherein the result of based on PCR amplification to determine whether include: in the presence of chimeric
If the number that allele occurs in same gene seat is 3,4, sample to be tested is 2 individual mixing samples；
If the number that allele occurs in same gene seat is 5,6, sample to be tested is 3 individual mixing samples；
If the number that allele occurs in same gene seat is greater than 6, sample to be tested is 3 or 3 or more individual mixed
Close sample.
The number that allele occurs in usual sample same gene seat is 1 or 2.
6. the method according to item 3 or 4, wherein the data-oriented is being expanded by PCR from the sample to be tested
Increase obtained band and the ratio between the peak area of electrophoretic band obtained by Capillary Electrophoresis fluorescent quantitation.
7. the method according to any one of item 1~6, further include:
DNA is extracted and concentration control, obtains the DNA from sample to be tested from sample to be tested, controls the dense of the DNA for making to extract
Degree is more than 2ng/ μ L.
8. the method according to any one of item 1~7, wherein
The Short tandem repeat is selected from following any three or three or more: D21S11, D13S317,
D8S1179, D18S51, D5S818, VWA, PENTA E, D7S820, TPOX, D3S1358, D16S539, FGA, TH01, CSFIPO
With penta d.
9. the method according to any one of item 1~7, further include: production standard curve makes a series of according to giving
The standard items mixing sample for determining mixed proportion mixing, is marked using the primer with fluorescence for expanding AMELOGENIN gene
The PCR amplification of quasi- product mixing sample, and the data-oriented for the standard items mixing sample of preparation is obtained to make standard curve；
Calculated result calculates the embedding of the sample to be tested using the data-oriented of standard curve and the sample to be tested
Composition and division in a proportion example.
10. according to method described in item 8, wherein the standard items mixing sample is to utilize the base from male and female
It is made because group DNA sample is mixed according to given ratio.
11. a kind of system for calculating mixing sample and being fitted into ratio comprising:
Judge that sample to be tested whether there is the module that sample is fitted into based on Short tandem repeatSTR, and
The module of the chimeric ratio of sample to be tested is calculated based on AMELOGENIN gene.
12. according to system described in item 11, wherein calculate the chimeric ratio of sample to be tested based on AMELOGENIN gene
Module include: utilize the primer with fluorescence for expanding the AMELOGENIN gene carry out the sample to be tested PCR expand
Increase, to obtain the data-oriented for the sample to be tested to calculate the chimeric ratio of the sample to be tested.
13. according to system described in item 12, wherein the data-oriented is being expanded by PCR from the sample to be tested
Increase obtained band and the ratio between the peak area of electrophoretic band obtained by Capillary Electrophoresis fluorescent quantitation.
14. the system according to any one of item 11~13, further include:
DNA is extracted and concentration control module, obtains the DNA from sample to be tested from sample to be tested, control makes the DNA extracted
Concentration more than 2ng/ μ L.
15. the system according to any one of item 11~14, further include:
Standard curve making module makes a series of standard items mixing samples mixed according to given mixed proportion, utilizes
The primer with fluorescence for expanding the AMELOGENIN gene carries out the PCR amplification of standard items mixing sample, and obtains and be directed to
The data-oriented of the standard items mixing sample of preparation makes standard curve；
As a result computing module calculates the sample to be tested using the data-oriented of standard curve and the sample to be tested
Chimeric ratio.
16. according to system described in item 15, wherein the standard items mixing sample is to utilize the base from male and female
It is made because group DNA sample is mixed according to given ratio.
The effect of invention
Have following benefit using the method and system that STR calculates chimeric ratio: this method and system time-consuming are short,
The result of chimeric ratio can be provided in for 24 hours.The technology finds peak area ratio by the corresponding peak area of measurement allele
There is close relationship with mixing sample component ratio, the sample of different proportion mixing will appear the imbalance of peak area, calculate mixed
Sample allele peak area ratio is closed, finds the ratio approximation sample mixed proportion of peak area, therefore can be by peak area ratio
Speculate the mixed proportion of sample.
Specific embodiment
Experimental procedure of the invention is as follows.
Step 1: obtaining the DNA from sample to be tested from sample to be tested.
Step 2: the concentration for the DNA that control is extracted, so that the concentration of the DNA extracted is more than 2ng/ μ L.
Step 3: the making step of standard items mixing sample.
Step 4: PCR amplification step.
Step 5: result calculates step.
In the extraction step of first step DNA, extracting the method for DNA for sample to be tested, there is no particular limitation, can be with
Mechanical Method, object are segmented into according to the difference of cracking mode using DNA extraction method known to anyone skilled in the art
Logos and chemical method.Mechanical Method for example has homogenate method, smashs method, polishing to pieces；Physical method for example has ultrasonic method, multigelation
Method, hypotonic splits method at hot and cold alternation method；Chemical method for example has the method using organic solvent, the method using detergent and benefit
With the method for enzymatic hydrolysis.Adsorbent material combined techniques, strong salty method, You Jirong can be divided into according to the difference for the mode that nucleic acid isolates and purifies
Agent extraction process and density-gradient centrifugation method.
In the specific embodiment of the present invention, sample to be tested is, for example, amniotic fluid or abortion tissue.
Following method is used in a specific embodiment of the invention, wherein the method that cracking uses enzymatic hydrolysis, and
Separation is utilized adsorbent material and is separated.Certain those skilled in the art can be suitable using other according to their own needs
DNA extraction method.
It, can be first using the means of detection DNA concentration come the concentration of the DNA of Detection and Extraction, if inspection in second step
Survey has met as the result is shown more than 2ng/ μ L, then is used directly for subsequent step, needs if being unsatisfactory for above-mentioned condition
The sample is handled to meet above-mentioned concentration requirement.For example, if the excessive concentration of sample it can then be carried out it is dilute
It releases, if the concentration of sample is too low needs that sample is concentrated.
In the third step, it will be mixed from the standard DNA sample (standard items) of mixing sample component.Not by two kinds
It can according to need detection when being mixed with the standard items genomic DNA of sex (for example, derive from) in source
Range is mixed, and usually for example according to 1:1,2:1,4:1,6:1,8:1,10:1 are mixed.
In the 4th step, to DNA points extracted in the third step according to the standard items DNA and the first step of different proportion mixing
It Jin Hang not be with the PCR amplification of the primer of fluorescent marker.It usually can be according to target gene to be amplified (i.e. for the amplification of PCR
Short tandem repeatSTR) it is expanded to set amplification program.
In a specific embodiment of the invention, the target gene for calculating chimeric ratio is
AMELOGENIN, i.e. amelogenin gene, in the area X chromosome Amelogenin gene (enamel protein gene) introne l
The characteristic design primer of 6bp missing, amplification X chromosome specific fragment (AMELX) length are 106bp, the Y dyeing homologous with it
Body specific fragment (AMELY) is 112bp.
In the 5th step, using the 4th step PCR's as a result, using under various concentration standard items proportionate relationship make standard
Curve is fitted regression equation, then brings the peak area ratio that sample to be tested can distinguish pollution condition into the regression equation and count
It calculates, the mixed proportion of available sample to be tested.
Using method of the invention, the mixing sample for preparing different mixing proportion carries out DNA typing inspection, by result
Analysis, accurately calculate mixing sample each component content.
Using the homology of AMELOGENIN locus on the sex chromosome of normal male and women sample, single male's sample
Should the allele peak area ratio of locus be about 1, and women and male DNA detect ratio about according to 1:1 is mixed
It for 2:1, detects that ratio is about 3:1 according to 2:1 is mixed, detects that ratio is about 5:1 according to 4:1 is mixed, according to
6:1 is mixed to detect that ratio is about 7:1, detects that ratio is about 9:1 according to 8:1 is mixed, after mixing according to 10:1
It is difficult to detect femaleness segment, therefore is fitted the standard curve of 5 ratios and obtains regression equation, by sample to be tested feature
Peak area ratio brings equation calculation into, it can be deduced that mixing sample component ratio be obviously closer in sample mixed component it is true
Ratio.
Embodiment
Step 1: the extraction of DNA
Extracts kit: blood/tissue/cell genomic dna extracts kit (Tiangeng biochemical technology (Beijing) limited public affairs
Department)
According to the specification of the kit, nothing first please is added in the buffer GD of the kit and rinsing liquid PW using preceding
Water-ethanol.
A. amniotic fluid being transferred to 1.5ml centrifuge tube, 12000rpm is centrifuged 2 minutes, and precipitating is resuspended with 200 μ L GA and is mixed,
It carries out in next step；Experiment specific step is as follows:
B. 20 μ L Proteinase K solution are added, mix；
C. 200 μ L buffer GB are added, are sufficiently mixed by inversion, 70 DEG C of placement 10min, solution strains limpid, brief centrifugation
To remove droplet when 70 DEG C of placements (the general buffer GB that is added can generate white precipitate, can disappear) of cap wall；
D. 200 μ L dehydrated alcohols are added, fullys shake and mixes 15sec, be likely to occur flocculent deposit at this time, brief centrifugation with
Remove the droplet of cap wall；
E. previous step acquired solution and flocculent deposit are all added in an adsorption column CB3,12000rpm (13400xg)
It is centrifuged 30sec, waste liquid is outwelled, adsorption column CB3 is put back in collecting pipe；
F. 500 μ L buffer GD (dehydrated alcohol is added using preceding first check whether) is added into adsorption column CB3,
12000rpm (13400xg) is centrifuged 30sec, outwells waste liquid, adsorption column CB3 is put back in collecting pipe；
G. 600 μ L buffer PW (dehydrated alcohol is added using preceding first check whether) is added into adsorption column CB3,
12000rpm (13400xg) is centrifuged 30sec, outwells waste liquid, adsorption column CB3 is put back in collecting pipe；
H. repetitive operation step g；
I. adsorption column CB3 is put back in collecting pipe, 12000rpm (13400 × g) is centrifuged 2min, outwells waste liquid, will adsorb
Column CB3, which is placed in, to be placed at room temperature for several minutes, thoroughly to dry rinsing liquid remaining in adsorbent material；
J. adsorption column CB3 is transferred in a clean centrifuge tube, 50 μ L is vacantly added dropwise to the intermediate position of adsorbed film and wash
De- buffer TE (can select EB or water according to actual needs) is placed at room temperature for 2-5min, 12000rpm (13400 × g) centrifugation
Solution is collected into centrifuge tube by 2min；
K., the solution collected in centrifuge tube is vacantly added drop-wise to the intermediate position of adsorbed film again, is placed at room temperature for 2-5min,
12000rpm (13400 × g) is centrifuged 2min, and solution is collected into centrifuge tube, -20 DEG C of preservations are placed in.
Step 2: the concentration for the DNA that control is extracted
Quantitatively extract the DNA concentration of tissue using Qubit 2.0BR, testing result display density is 12.9ng/ μ L, into
Before row PCR, sample is diluted to 2ng/ μ L.
Step 3: the preparation of standard items mixing sample
By two groups of standard items DNA, that is, buys from the 2800M and K562 of Promega and quantified respectively with Qubit3.0HS, then
It is diluted to 2ng/ μ L, the mixed proportion according to two groups of standard items is 1:1,2:1,4:1,6:1,8:1, and 10:1 is mixed, so
It is used for the PCR amplification step of next step afterwards.
Step 4: PCR amplification
Using16HS System kit (being purchased from promega) is to str locus seat and Amelogenin
Gene carries out the amplification of PCR, the amplification program that amplification program uses kit to provide.Wherein the mixture of PCR is to use 5 μ L'sThe PowerPlexR 16HS10X Primer Pair Mix of HS 5X Master Mix, 2.5 μ L (are band
The primer of fluorescent marker) and 1 μ L DNA sample, the DNA sample be respectively above-mentioned standard product mixing sample and to
The 16.5 μ L of DNA and ultrapure water extracted in sample.It can be synchronous with the amplification of Amelogenin gene to str locus seat
Completion can also be completed step by step.
The program of PCR are as follows: at 96 DEG C, 2 minutes, ramp is set as 4 (indicating 4 DEG C/s of cooling rate), then carries out 10
Circulation below: 94 DEG C, 30 seconds, ramp was set as 4,60 DEG C, and 30 seconds, ramp, which is set as 0.6, (indicated cooling rate 0.6
DEG C/s), 72 DEG C, 45 seconds, ramp was set as 0.3 (indicating 0.3 DEG C/s of cooling rate), then carry out 22 circulations below:
90 DEG C, 30 seconds, ramp was set as 4 (indicating 4 DEG C/s of cooling rate), and 60 DEG C, 30 seconds, ramp, which is set as 0.6, (indicated cooling
0.6 DEG C/s of speed), 72 DEG C, 45 seconds, ramp was set as 0.3 (indicating 0.3 DEG C/s of cooling rate), then it is maintained at 60 DEG C, 20
Minute, ramp is set as 4 (indicating 4 DEG C/s of cooling rate), is finally maintained at 4 DEG C.
PCR product is protected from light -20 degrees Celsius of preservations, and 4 DEG C are kept when sending detection outside.
Step 5: calculating step
Using K562 genomic DNA under various concentration and 2800M genomic DNA mixing sample standard items X Y chromosome
Proportionate relationship makes standard curve, is fitted regression equation, then the peak area ratio that sample to be tested can distinguish pollution condition is brought into
Equation calculation, the mixed proportion of available sample to be tested.
Table 2 gives the peak area ratio that standard items detect in PCR amplification, which is by expanding in PCR
Band obtained in increasing carries out Capillary Electrophoresis fluorescent quantitation, then uses the peak face of GeneMarker software analytical electrophoresis band
Product, and the ratio between peak area for analyzing absorption peak and ask calculating.It can be by concentration ratio and peak area according to the data provided in table 2
Than being fitted, the standard curve of mixed proportion and peak area ratio is obtained.
2 K562 and 2800M mixing sample component ratio of table and peak area ratio
The mixing sample component ratio to be measured of table 3 and peak area ratio
According to the data fit standard curve of table 2, and obtain regression equation y=1.082x-1.038 (R2=0.996),
In, y indicates that peak area ratio, x indicate mixed proportion.Bring the peak area ratio 2.175582 of the sample to be tested locus into equation meter
It calculates, it can be deduced that mixing sample component ratio is 1.30994, result 56.7%, more close with NGS detection ratio.
Claims (10)
1. a kind of calculate the method that mixing sample is fitted into ratio based on Short tandem repeatSTR comprising:
Judge that sample to be tested is chimeric with the presence or absence of sample based on Short tandem repeatSTR；And
After judgement is chimeric there are sample, the chimeric ratio of sample to be tested is calculated based on AMELOGENIN gene.
2. according to the method described in claim 1, wherein, judging that sample to be tested is embedding with the presence or absence of sample based on Short tandem repeatSTR
The step of conjunction includes:
The PCR amplification of the sample to be tested, based on PCR are carried out using the primer with fluorescence for expanding the Short tandem repeatSTR
The result of amplification is to determine whether exist chimeric.
3. method according to claim 1 or 2, wherein calculate the chimeric ratio of sample to be tested based on Short tandem repeatSTR
The step of include:
The PCR amplification that the sample to be tested is carried out using the primer with fluorescence for expanding the AMELOGENIN gene, from
And the data-oriented for the sample to be tested is obtained to calculate the chimeric ratio of the sample to be tested.
4. according to the method in claim 2 or 3, wherein the result of based on PCR amplification is to determine whether exist chimeric specific
Are as follows: if the number that allele occurs in same gene seat is 3 or more, and sample to be tested is the aggregate sample of more than two individuals
This.
5. according to the method in claim 2 or 3, wherein the result of based on PCR amplification is to determine whether have chimeric packet
It includes:
If the number that allele occurs in same gene seat is 3,4, sample to be tested is 2 individual mixing samples；
If the number that allele occurs in same gene seat is 5,6, sample to be tested is 3 individual mixing samples；
If the number that allele occurs in same gene seat is greater than 6, sample to be tested is 3 or 3 or more individual aggregate samples
Product.
6. the method according to claim 3 or 4, wherein the data-oriented is to pass through PCR from the sample to be tested
Expand obtained band and the ratio between the peak area of electrophoretic band obtained by Capillary Electrophoresis fluorescent quantitation.
7. method described according to claim 1~any one of 6, further include:
DNA is extracted and concentration control, obtains the DNA from sample to be tested from sample to be tested, control makes the concentration of the DNA extracted exist
2ng/ μ L or more.
8. method according to any one of claims 1 to 7, wherein
The Short tandem repeat is selected from following any three or three or more: D21S11, D13S317, D8S1179,
D18S51, D5S818, VWA, PENTA E, D7S820, TPOX, D3S1358, D16S539, FGA, TH01, CSFIPO and penta
d。
9. method according to any one of claims 1 to 7, further include: production standard curve, make it is a series of according to
The standard items mixing sample of given mixed proportion mixing, is carried out using the primer with fluorescence for expanding AMELOGENIN gene
The PCR amplification of standard items mixing sample, and the data-oriented for the standard items mixing sample of preparation is obtained to make standard song
Line；
Calculated result calculates the chimeric ratio of the sample to be tested using the data-oriented of standard curve and the sample to be tested
Example.
10. according to the method described in claim 8, wherein, the standard items mixing sample is using from male and female
Genome DNA sample is mixed to make according to given ratio.
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