CN102978286A

CN102978286A - Method for paternity test through utilizing specific single nucleotide polymorphism (SNP) combination

Info

Publication number: CN102978286A
Application number: CN 201210522691
Authority: CN
Inventors: 王健; 其他发明人请求不公开姓名
Original assignee: SHANGHAI DIDAO TECHNOLOGY Co Ltd
Current assignee: SHANGHAI DIDAO TECHNOLOGY Co Ltd
Priority date: 2012-12-08
Filing date: 2012-12-08
Publication date: 2013-03-20

Abstract

The invention discloses a method for a paternity test through utilizing a specific single nucleotide polymorphism (SNP) mark combination which comprises 60 SNP marks. The nucleotide sequences of the forward primers of the SNP marks are shown as SEQ ID NO.1-60; the nucleotide sequences of downstream primers are shown as SEQ ID NO.61-120; and the nucleotide sequences of single base extension primers are shown as SEQ ID NO.121-180. The method utilizes a flight mass spectrum method, the SNP mark combination is used for human paternity tests, and the method has the advantages of accurate result, quickness and convenience in method and high flux.

Description

The method of utilizing specific SNP combination to carry out paternity test

Technical field

The present invention relates to use the SNP mark to carry out the detection system of human paternity test, be specifically related to 60 marker combination and primer sequence and detection method.

Background technology

Whether the method that modern sibship is identified is DNA analysis, utilize genetic marker on the human chromosomal to identify between two individualities and have relationship by blood.Between the akin individuality, share the probability of same tag, than there not being the individual large of sibship.At present, the genetic marker that is used for paternity test is s-generation genetic marker STR (STR), and it has widely distributed, is easy to detect, and contains much information, and the height polymorphism is arranged and follow the characteristics such as Mendelian's codominant inheritance.When STR carries out paternity test, generally detect tens STR sites, if there is the site more than 3 different, then can get rid of parentchild relationship.But the not homoallelic sequence length of STR is similar, and distinguishing and declaring type has certain difficulty, and generally each PCR reaction declares the type error rate and can reach 1% ~ 5%(bonin et al. 2004; Weller et al. 2004), declare accuracy and the repeatability of the paternity test of type erroneous effects.

Along with the development of science and technology, single nucleotide polymorphism (SNP) more and more enters people's the visual field as third generation genetic marker.In the numerous areas such as heredity drawing, linksystem analysis, disease gene location and the research of species polymorphism, SNP has replaced STR gradually, becomes first-selected genetic marker.With respect to STR, the distribution of SNP in karyomit(e) is more extensive, more, and detection method is also more convenient reliable.Identify the field in sibship, the SNP somatotype also can be given full play to its advantage:

1) as genetic marker, SNP is more more stable than STR, and the mutation rate of STR sequence is higher than the average mutation rate of Human genome, and exists the phenomenons such as a plurality of core sequences repeat, the non-multiple of core sequence repeats among the STR, and then there is not this class problem in SNP;

2) SNP detects and detects lower to the requirement of dna sample than STR, adapt to more particular surroundings, SNP is single base picture, the general length that only needs to increase about 100bp can be finished detection in the PCR process, 9.11 the wrecked personnel identity identification of disaster, because much sample is under the impact of extreme temperature and humidity, its DNA degrades, be difficult to collect enough STR data, so some utilizes SNP to finish;

3) with respect to STR, the detection of SNP is more cheap, and flux is higher, and the experience of the developed countries such as the U.S. in the events such as 9.11 shows, it is thousands of dollar that the average cost of utilizing the STR authenticate technology successfully to identify remains can reach, and the SNP authenticate technology is expected to the identification cost reduced by half even is lower;

4) aspect the sibship between the evaluation parent and child is such, institute is widely used at present, the STR identification systems in tens sites can provide sufficient information, but when the demand of identifying extends to farther sibship, such as working as a disaster disaster victim, when only having a cousin can provide identity to differentiate required sample, such STR identifies unable to do what one wishes with regard to some, and the SNP site is owing to have the quantity that surpasses the STR site far away, more information can be provided, can also provide believable judgement to far kinship in theory;

In sum, it is high that the SNP mark has stability, measure accurately, and the advantages such as the easy and high-throughput of detection method, the prospect that is applied to paternity test is very wide, has represented following developing direction.

Summary of the invention

The objective of the invention is to utilize SNP mark high-throughput, easily detect, make things convenient for having a few of automatization, set up a cover and be used for the marker combination of human paternity test, and set up the technology that easy accurately flight mass spectrum method detects this cover SNP mark polymorphism.

For achieving the above object, technical scheme of the present invention provides a kind of SNP marker combination for human paternity test, 60 the SNP marks of its protection shown in subordinate list: above-mentioned marker combination preferably utilizes flight mass spectrum (MALDI-TOF) method to detect, detection method may further comprise the steps (1) and extracts genomic dna, comprises extracting anticoagulation DNA and mouth epithelial cells DNA; (2) it is polymorphic that flight mass spectrum detects SNP; (3) utilizing software to carry out the parent-offspring infers.

The present invention detects the polymorphic and parent-offspring of SNP through flight mass spectrum and infers experimental verification, no matter is that set membership is inferred or parents infer that parentchild relationship all is extremely significant, and infer that relation and actual sibship fit like a glove; SNP marker combination provided by the invention is used for human paternity test, and the result is accurate.Utilize the flight mass spectrum method, can be fast, high-throughput, easy carry out human paternity test.

Embodiment

Below in conjunction with embodiment, the specific embodiment of the present invention is described in detail, embodiment is used for explanation the present invention, but is not used for limiting the scope of the invention.

Embodiment 1: adopt the flight mass spectrum method that the crowd is carried out SNP somatotype and parent-offspring's deduction:

One) extracts genomic dna

1) sample of this test is the oral mucosa cell that uses scraper to gather, room temperature preservation.The concrete steps of scraper sampling DNA extraction are as follows:

2) cast-off cells on the scraper of oral cavity are dissolved in the 400uL 1X lysate, add the 1uL Proteinase K, 56 degree water-baths 30 minutes;

3) take out sample and put ice-water bath 1 minute, add 6mol/L NaCl 150uL, thermal agitation 15-20 second, 13000 left the heart 10 minutes;

4) get supernatant liquor to new centrifuge tube, add the 1.1mL dehydrated alcohol, turn upside down 10 times to the appearance of cotton-shaped DNA precipitation, room temperature was placed 10 minutes, and 13000 leave 2 minutes precipitations of heart DNA;

6) abandoning supernatant adds 0.25mL70% washing with alcohol DNA precipitation again, and room temperature was placed after 1 minute, and 13000 left the heart 5 minutes;

7) carefully draw ethanol with pipettor, then put into stink cupboard and ventilated 10 minutes, then use 35uL TE dissolving DNA; 8) DNA can preserve 1-2 month at 4 degree, perhaps left-20 degree prolonged preservation in;

9) use 2uL DNA sample, 1.5% sepharose, 120V electrophoresis 15 minutes is transferred in the PCR pipe after observations is qualified;

10) ultraviolet spectrophotometer SMA3000 measures concentration and the A260/A280 ratio of DNA in the solution, measures and averages for 3 times, and concentration should be at 30ng/uL, and A260/A280 ratio should be between 1.7-2.0, and 4 ℃ of qualified DNA take or-20 ℃ of preservations;

Two) it is polymorphic that flight mass spectrum detects SNP:

1. primer and DNA dilution and quality inspection:

1) general primer being diluted to final concentration is 100pmol/uL, and according to the dilution formula of Sequenom, single-basic extension is stopped primer, and to be diluted to final concentration be between the 135.3-284.8pmol/uL;

2) 13000 leave heart 2.5min, slightly shake with vibrator, every 1h concussion once, put at normal temperatures to 3-4 hour and get final product, take or-20 ℃ of preservations for 4 ℃;

3) use the concentration that has that it's too late of 1.5% agarose and ultraviolet spectrophotometer SMA3000 working sample DNA, keep qualified samples, failed sample extracts again, and the DNA concentration dilution to 10-20ng/uL, is taken or-20 ℃ of preservations for 4 ℃;

2. 384 hole multi-PRC reactions

1) multiplex PCR process and regular-PCR process are identical, add successively system in the table 2 in 384 hole PCR plates, according to table 3 PCR instrument program are set, and extra increase by 38% when mixing PCR reaction solution Mix is to prevent windfall loss;

Table 2 5uLPCR reaction system

Moiety	Consumption (μ L)
		H ₂O	0.95
PCR Buffer (10 *, contain 15mM MgCl ₂)	0.625
		MgCl ₂(25mM)	0.325
dNTP(2.5 mM each)	1.0
		Primer uses liquid	1.0
HotstarTaq（5U/μL）	0.1
		Template DNA (50ng/uL)	1.0
Final volume	5.0

Table 3 multi-PRC reaction loop parameter

Figure 2012105226912100002DEST_PATH_IMAGE001

3. 384 hole SAP(shrimp alkaline phosphotases) digestion reaction

Purpose is to digest remaining dNTP, the PCR Sptting plate is put into the centrifugal several seconds of whizzer 1000rpm, SAP digestive process and regular-PCR process are similar, in 384 hole PCR plates, add successively system in the table 4, according to table 5 PCR instrument program is set, mixing SAP enzyme Mix is extra increase by 38%, to prevent windfall loss;

Table 4 SAP digestion reaction system

Moiety	Concentration	Consumption (μ L)
			H ₂O	NA	1.53*480
PCR Buffer	10X	0.17*480
			The SAP enzyme	1.7U/uL	0.3*480
Amount to	?	2*480

Table 5 SAP digestion reaction loop parameter

Temperature (℃)	Time (minute)	Cycle number
			37	40	1
85	5	1
			4	∞	1

4. single-basic extension termination reaction

The SAP Sptting plate is put into whizzer 1000 leave calculation second; Single-basic extension termination reaction process and regular-PCR process are similar, add successively system in the table 6 in 384 hole PCR plates, according to table 7 PCR instrument program are set, and extra increase by 38% during single-basic extension Mix is to prevent windfall loss;

Table 6 single-basic extension termination reaction system

Moiety	Concentration	Consumption (μ L)
			H ₂O	NA	0.619*480
The iPLEX damping fluid	10X	0.2*480
			The iPLEX terminator	NA	0.2*480
The single-basic extension primer	0.6-1.4uM	0.94*480
			The iPLEX enzyme	NA	0.041*480
Amount to	?	2*480

Table 7 single-basic extension stops loop parameter

Figure 2012105226912100002DEST_PATH_IMAGE002

5. resin purification and data gathering

Reaction product (altogether 9uL) is added 25uL water dilute, use resin to carry out desalination; With the sample spot after the desalting treatment on the chip target spot, spontaneous nucleation, upper machine carries out mass spectrometric detection, and collects data;

Three) carrying out the parent-offspring infers

To the SNP somatotype data analysis that flight mass spectrum produces, extrapolate the paternity index (PI) in each SNP site, the formula in the basis table 2 of PI value carries out (Kaiser, L. and Sever, G, 1983)

Table 2. PI calculation formula table

Child	Mother	Father to be confirmed	PI
				q	pq	q	1/q
pq	p or pr	q	1/q
				q	q	q	1/q
pq	p or pr or ps	qr (or pq)	1/2q
				q	pq	qr (or pq)	1/2q
q	q	qr	1/2q
				pq	pq	pq	1/(p+q)
pq	pq	q	1/(p+q)
				pq	pq	qr	1/(2p+2q)
q	pq	r	0
				q	q	r	0
q	The maternal gene type is unknown	q	1/q
				pq	The maternal gene type is unknown	q	1/2q
q	The maternal gene type is unknown	qr	1/2q
				pq	The maternal gene type is unknown	pq	(p+q) / 4pq
pq	The maternal gene type is unknown	qr	1/4q
				q	The maternal gene type is unknown	r	0

Annotate: the p of first three columns in the table, q, the SNP allele that the r representative is different, the p of last row, q, r represent the frequency of occurrences of this allele in the crowd

Final PI value (composite PI between two samples, CPI), multiplied each other by the PI value in all SNP sites and to obtain, the CPI value〉two samples of 1000, can determine real set membership, the SNP site number of CPI value=0 and PI=0 is more than or equal to 3, can paternity exclusion, the SNP site number of CPI value=0 and PI=0 is 1 or 2, for set membership be can not determine and can not be got rid of.

Reference

Kaiser,?L.?and?Sever,?G.?(1983).?Paternity?testing:?I.?Calculation?of?paternity?indexes.?American?Journal?of?Medical?Genetics.?15(2):?323-329.

Claims

1. a SNP marker combination that is used for human paternity test is characterized in that comprise 60 SNP marks, the nucleotide sequence of the upstream primer of these SNP marks is successively shown in SEQ ID NO. 1 ~ 60; The nucleotide sequence of downstream primer is successively shown in SEQ ID NO. 61 ~ 120; The nucleotide sequence of single-basic extension primer is successively shown in SEQ ID NO. 121 ~ 180.

2. the application of paternity test SNP marker combination claimed in claim 1 in human paternity test.

3. one kind is utilized paternity test marker combination claimed in claim 1 to carry out the method that human paternity test detects, and may further comprise the steps: (1) extracts genomic dna, comprises and extracts anticoagulation DNA and mouth epithelial cells DNA; (2) it is polymorphic that flight mass spectrum detects SNP; (3) utilizing software to carry out the parent-offspring infers.