CN101914625A - Kit and method for determining sex by detecting enamel protein gene with pyrosequencing method - Google Patents

Abstract

The invention discloses a kit and a method for determining sex by detecting enamel protein gene with a pyrosequencing method. The kit comprises a PCR buffer solution, TaqDAN polymerase, a primer, a template DNA, a pyrosequencing kit, magnetic beads, a combination buffer solution, a washing butter solution, an annealing buffer solution and a degeneration buffer solution, wherein the primer consists of a PCR amplification primer and a sequencing primer, the PCR amplification primer has the following base sequences: upstream primer ATTGAATCCCAAGTAATC and downstream primer GACTAGAAGTAATC, and the sequencing primer has the base sequence of ATTGAATCCCAAGTAATC. The test method is carried out by the following steps of DNA extraction, PCR amplification, determination of sprayed base sequence of CTGATCTGAC, single stranded separation and purification, pyrosequencing reaction and reaction product determination. The method for sex determination is valuable complement to the traditional determination method.

Description

The tetra-sodium sequencing detects test kit and the method that enamel protein gene carries out sex identification

Technical field

The present invention relates to Human genome and detect, the sequencing analysis of particularly short-movie section sequence specifically adopts the tetra-sodium sequencing to detect test kit and method that short-movie section enamel protein gene carries out sex identification.

Background technology

In medicolegal practice, often run into mutilated body, highly corruption and decompose to bony skeleton corpse; Normal many people are murdered in extensive disaster accident, and the corpse appearance is seriously damaged, and all individual's identification and sex identification are caused certain difficulty.Owing in this class case, can't use morphology methods to carry out the judgement of sex, so the method for applied molecular biology is carried out sex identification to the biology sample from human body, and vital role has been played in the detection of case.In addition, in some clinical diagnosises,, also all need to distinguish sex as the discriminating of fetus early diagnosis and archeology decompose to bony skeleton corpse.

Enamel protein gene (amelogenin) is positioned at human X chromosome p22, coding enamel albumen, and near the sequence with enamel protein gene of distributing the Y chromosome centriole has the gene of 90% homology.In these two sections homologous sequences, there are a lot of pleomorphism sites, sufficiently long homologous sequence design primer is arranged again simultaneously, promptly with increase simultaneously in a PCR reacts enamel protein gene on X, the Y chromosome of a pair of primer, realizability is not judged.1991, Nakahori etc. carried out sex identification with regard to reporting a pair of homology primer with enamel protein gene.In addition, Akane (1991), Bailey (1992), Sullivan (1993) etc. also successively carry out the gender typing with pcr amplification list copy X, Y homology zone.The detection method of at present the most frequently used enamel protein gene is exactly the Sullivan method---uses with a pair of primer amplification X, Y homologous enamel protein gene, primer side homologous region introne 1 has the disappearance of 6-bp, the amplified production X chromosome is 106bp, and Y chromosome is 112bp.Yet this method also has its limitation, suddenlys change when test sample exists at the enamel protein gene PBR, and perhaps for the individuality of sex chromosome variation, the Sullivan method detects and easily causes invalid amplification.And for the height degraded sample of some difficult cases,, the fragment length of this method can not make the correct type of declaring to such an extent as to still showing long slightly.Studies show that in a large number the power that is detected as of height degradation of dna sample increases with the shortening of pcr amplification product fragment.

Summary of the invention

The technical problem to be solved in the present invention is the sex identification at conventional somatotype failure, provide a kind of tetra-sodium sequencing to detect test kit and method that enamel protein gene carries out sex identification, make the fragment that detects DNA foreshorten to 44/45bp, and comprise 3 mutational sites and an insertion deletion segment, improve success ratio height degradation of dna somatotype.

For solving the problems of the technologies described above, the technical solution used in the present invention is: the tetra-sodium sequencing detects the test kit that enamel protein gene carries out sex identification, it comprises: PCR buffered soln, TaqDNA polysaccharase, primer, template DNA, tetra-sodium sequencing kit, magnetic bead, binding buffer solution, washing buffer solution, annealing buffer solution and sex change buffered soln; Described primer is made up of pcr amplification primer and sequencing primer; The base sequence of described pcr amplification primer is: upstream primer ATTGAATCCCAAGTAATC, downstream primer GACTAGAATAGCCAATGAT, the base sequence of described sequencing primer are ATTGAATCCCAAGTAATC, described downstream primer mark vitamin H.

Utilize the mentioned reagent box to carry out the testing method of sex identification, carry out according to following step:

A) extract DNA, get DNA extraction liquid;

B) pcr amplification

Add the DNA extraction liquid that obtains in the step a) and carry out pcr amplification in the pcr amplification system, the pcr amplification condition is: 95 ℃, the pre-sex change of 5min; Then successively at 95 ℃, 15s, 50 ℃, 30s, 72 ℃, 15s carry out 45 circulations; Keep 5min at 72 ℃ again, finally remain on 4 ℃, get amplified production;

C) determine that spray base sequence is CTGATCTGAC;

D) strand separation and purification

The amplified production of step b) gained is added binding buffer solution and the magnetic bead that contains even streptavidin, hatch 20min at normal temperatures; Be to wash 15 seconds in sex change 15 seconds in 70% alcohol washing 5 seconds, the sex change damping fluid, the lavation buffer solution in volume ratio respectively with amplified production then, above-mentioned amplified production becomes the single stranded DNA template, again described single stranded DNA template is changed in the annealing buffer solution that contains sequencing primer, at 80 ℃ of hybridization 3min, be cooled to room temperature, must separate the single stranded DNA template behind the purifying;

E) tetra-sodium sequencing reaction

Be ready to the used enzyme mixture of tetra-sodium sequencing reaction, substrate mixture and dNTP, add the reagent cabin, single stranded DNA template after the separation and purification of gained in the step d) is put into the tetra-sodium sequenator, carry out the tetra-sodium sequencing reaction according to the spray base sequence that designs in the step c);

F) determine reaction product

Instrument carries out interpretation to sequencing result automatically, obtains sex information according to order-checking collection of illustrative plates and result.

The target sequence that generates according to above-mentioned steps is respectively:

Target sequence X:ATTGAATCCCAAGTAATCGGT-GCCTATCATTGGCTATTCTAGTC;

Or target sequence Y:ATTGAATCCCAAGTAATCTGACGACTATCATTGGCTATTCTAGTC.

Its relevant information is: the Genbank of AMELX number is NG_012040 on the X chromosome, is positioned at 11637-11686; The Genbank of AMELY number is NG_008011 on the Y chromosome, is positioned at 12395-12445.The choice criteria of described target sequence: (1) is less than 50bp; (2) sufficiently long homologous sequence design primer is arranged, promptly the upstream and downstream homologous region is greater than 18bp; (3) a plurality of point mutation and insertion deletion segment exist jointly; (4) be fit to design pcr amplification primer and sequencing primer.

Above-mentioned method and test kit can be used to analyze the DNA sample of blood, bone, hair, muscle or histoorgan from the mankind.

Adopt aforesaid method to the irrelevant individual specimen of the health of 100 parts of known sexes (each 50 parts of men and women) below, carry out sequencing analysis, verify the accuracy of this method: the blood sample that extracts 100 parts of individualities according to ordinary method, primer with the present invention's design increases, use the tetra-sodium sequencing technologies again amplified production is checked order, can directly declare type from sequencer map.

Detected result can find out that from the sequencing result of Fig. 1-1 and Fig. 1-2 the distinguished sequence of AMELX is referring to Fig. 1-1, Fig. 1-2: GGTGC, and the distinguished sequence of AMELY is: TGACGA; The difference of the two is: 3 point mutation sites---G/T, and T/A, C/A and an insertion deletion segment---... / C.Therefore, women's (XX) sequencing result is: G/G, T/T ... / ..., C/C (referring to Fig. 1-1), the male sex's (XY) sequencing result is: G/T, T/A ... / C, C/A (referring to Fig. 1-2); Fig. 1-1 is consistent with the expection model of Fig. 2-1 and 2-2 order-checking software design respectively with Fig. 1-2.These 100 parts known sex dna samples are checked order, all obtain correct somatotype, the somatotype success ratio is 100%.

Adopt aforesaid method to carry out accurate somatotype, finish sex identification Different Individual to the sample of highly degraded.Below by testing data advantage of the present invention is described.

Simultaneous test 1

Extract the DNA that places corrupt blood sample of degrading of 26 weeks under 8 parts of outdoor natural condition according to ordinary method, adopt Ampf/STR Identifiler test kit and test kit of the present invention that the DNA that extracts is carried out the detection of enamel protein gene then respectively.Adopt test kit of the present invention and detection method, 8 parts of sample standard deviations are somatotype correctly, and detected result is referring to Fig. 3-1a and Fig. 3-1b; And adopt Ampf/STR Identifiler test kit wherein to have 2 increments originally can't somatotype, referring to Fig. 3-2a and Fig. 3-2b.

Comparison test 2

With the artificial degradation of dna of 9 different time points of DNase I preparation (5min, 10min, 15min, 20min, 25min, 30min, 40min and 45min), adopt the method for Ampf/STR Identifiler test kit and test kit of the present invention and method artificial degradation of dna to be carried out the detection of enamel protein gene then respectively.

Adopt the detected result of test kit of the present invention and method to see Fig. 4-1～Fig. 4-10, wherein Fig. 4-1 is the contrast collection of illustrative plates; As can be seen, DNA to enzymic digestion 5min, 10min, 15min, 20min, 25min, 30min, 35min and 40min, the method that the present invention sets up all can be carried out correct somatotype to degradation of dna, and to the DNA of enzymic digestion 45min, this method detects and do not obtain the product peak.The detected result that adopts AmpF/STR Identifiler test kit is referring to Fig. 5-1～Fig. 5-10, and wherein Fig. 5-1 is the contrast collection of illustrative plates; As can be seen, only there is the lower peak of relative fluorescence unit (Rfu) in the enamel protein gene zone at the dna sample of enzymic digestion 5min and 10min, and enzymic digestion 15min and above DNA are not all had the product peak.

Method of the present invention also has reasonable human species specificity, illustrates below by testing data.

Non-human sample to obtained comprises intestinal bacteria, faecalis, candidiasis, rhesus monkey, dog, pig, chicken, ox, rabbit, fish, detects with present method.

Detected result is seen Fig. 6-1～Fig. 6-10, can find out and have only monkey in this fragment the product peak to be arranged, and all the other sample standard deviations do not have amplified production, shows that this method has human preferably species specificity.Analyze the sequencing result of monkey, find that it both had been different from human male's sequencing result, also be different from human women's sequencing result, human women's (XX) sequencing result is: G/G, T/T ... / ..., C/C, the male sex's (XY) sequencing result is: G/T, T/A ... / C, C/A, and the sequencing result of monkey be G/T, T/A ... / C, C/?Use the human difference with monkey enamel protein gene nucleotide sequence of BLAST comparison, find the fragment that checks order in this experiment, the sequence of monkey AMELY is TGACGT, and only last base is different with the sequence TGACGA of human AMELY; Monkey is GGT-GC with the mankind's AMELX in this section sequence.In view of the above, can determine that tested monkey is male in this experiment, its sequencing result and human male there are differences, and show last SNP site: human is C/A, and monkey is C/T.This results suggest for male, is used this method and can be distinguished primates and human DNA.

Adopt aforesaid method also can carry out accurate somatotype, finish sex identification Different Individual to sample bone.Illustrate below by testing data.

Extract the DNA of 5 parts of bone samples, the primer that designs with present method increases, and uses the tetra-sodium sequencing technologies again amplified production is checked order, and can directly declare type from sequencer map.Detected result can find out directly that referring to Fig. 7-1～Fig. 7-5 these 5 parts of sample bones all can obtain somatotype result clearly.

Adopt beneficial effect of the present invention to be: to adopt present method that the sample of highly degrading is detected, aspects such as its distinguishing ability, sensitivity, specific amplification, accuracy, stability can satisfy the requirement of forensic dna check real work, are useful the replenishing of existing authentication method; And its cost is low, performance good, because the high-throughput of this method is more suitable for being applied to the research of extensive disaster event and medical jurisprudence mass survey, can be widely used in the sample that DNA such as Old Bones, tooth, putrid muscle, tissue highly degrade, and the trace blood stain, refer to that the sex of low levels DNA samples such as (toe) first, hair detects.

Description of drawings

The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.

Fig. 1-1 and Fig. 1-2 are respectively the standard women that detects of the present invention and the sequencing result of standard male sex sample;

Fig. 2-1 and 2-2 are respectively the women that sets up of the present invention and the male sex's order-checking model;

Fig. 3-1a and Fig. 3-1b adopt the sequencing result of test kit of the present invention to the blood sample DNA enamel protein gene of the corruption degraded in 26 weeks of placement under the outdoor natural condition;

Fig. 3-2a and Fig. 3-2b adopt the sequencing result of Ampf/STR Identifiler test kit to the blood sample DNA enamel protein gene of the corruption degraded in 26 weeks of placement under the outdoor natural condition;

Fig. 4-1～Fig. 4-10 carries out the somatotype result that enamel protein gene detects for adopting test kit of the present invention and method to the sample that DNase I digests 9 different time points;

Fig. 5-1～Fig. 5-10 carries out the somatotype result that enamel protein gene detects for adopting Ampf/STR Identifiler test kit to the sample of 9 different time points of DNase I digestion;

Fig. 6-1～Fig. 6-10 is respectively and adopts the somatotype result of method of the present invention to the enamel Protein Detection of candidiasis, intestinal bacteria, faecalis, chicken, pig, rabbit, dog, rhesus monkey, ox, fish;

Fig. 7-1～Fig. 7-5 is for adopting the detected result of method of the present invention to 5 parts of bone DNA enamel protein genes.

Embodiment

The present invention is described in detail below in conjunction with embodiment.

The tetra-sodium sequencing detects the test kit that enamel protein gene carries out sex identification, it comprises: PCR buffered soln, TaqDNA polysaccharase, primer, template DNA, tetra-sodium sequencing kit, magnetic bead, binding buffer solution, washing buffer solution, annealing buffer solution and sex change buffered soln; Described primer is made up of pcr amplification primer and sequencing primer; The base sequence of described pcr amplification primer is: upstream primer ATTGAATCCCAAGTAATC, downstream primer GACTAGAATAGCCAATGAT, the base sequence of described sequencing primer are ATTGAATCCCAAGTAATC, described downstream primer mark vitamin H.

Below in conjunction with embodiment mentioned reagent box and detection method are described in detail.

Embodiment one

8 parts of fresh blood samples are positioned over airtight preservation in the anticoagulant tube respectively, place under the outdoor natural condition (June-November) and placed for 26 weeks, it is example that simulation is prepared into the corrupt degraded of nature sample, illustrates that using tetra-sodium sequencing detection short-movie section enamel protein gene carries out the application of the method for sex identification

A) extract DNA

Step is as follows:

1) getting an amount of blood stain sample puts in the 1.5ml Eppendorf pipe;

2) add the 1ml sterilized water and soak soaking at room temperature 15-30min;

3) the centrifugal 3min of 13000rpm removes supernatant liquor, stays 100 μ L liquid;

4) add 100 μ L 10%Chelex-100;

5) 56 ℃ of insulation 30min, thermal agitation 10s;

6) 100 ℃ of insulation 8min, thermal agitation 10s;

7) the centrifugal 3min of 13000rpm gets DNA extraction liquid first;

8) directly adding 200 μ L 10%Chelex-100 mixings in the DNA extraction liquid first;

9) 56 ℃ of insulation 30min, thermal agitation 10s;

10) 100 ℃ of insulation 8min, thermal agitation 10s;

11) the centrifugal 3min of 13000rpm gets secondary DNA extraction liquid, is respectively applied for pcr amplification.

B) pcr amplification

Present embodiment adopts PCR increase (polymerase chain reaction, polymerase chain reaction).Amplification system sees Table 1.

Component, concentration and the content of table 1PCR amplification

The component title	Concentration	Content
				10 * PCR buffered soln	-	4.0μL
The pcr amplification primer	10μmol/L	0.8μL
			The Taq archaeal dna polymerase	1U/μL	1.2μL
d?NTP	10mmol/L	0.8μL
			Template DNA	-	2.0μL
MgCl 2	25mmol/L	2.4μL

Amplification system also comprises deionized water, and its cumulative volume is 40 μ L.

The base sequence of described pcr amplification primer: upstream primer ATTGAATCCCAAGTAATC, downstream primer GACTAGAATAGCCAATGAT, its middle and lower reaches amplimer mark vitamin H.Primer is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized, the method for the PAGE purifying that upstream primer all adopts, and the primer of downstream mark vitamin H then adopts the method for high back voltage liquid chromatography (LC) (HPLC) purifying.

The pcr amplification step:

In ABI 9700PCR amplification instrument, increase thermal circulation parameters: 95 ℃, the pre-sex change of 5min; Then at 95 ℃, 15s, 50 ℃, 30s, 72 ℃, 15s carry out 45 circulations altogether; 72 ℃, keep 5min, finally remain on 4 ℃, get amplified production.

Before carrying out the tetra-sodium reaction, need carry out simple authentication with gel electrophoresis.Generally, only need carry out agarose gel electrophoresis and get final product, but the PCR product of this experiment has only 45bp, can not obtain band more clearly with agarose gel electrophoresis.So this experiment most of selection before order-checking verified with polyacrylamide gel electrophoresis.If electrophoresis result obtains purpose band more clearly, and non-specific band is less does not influence order-checking, can carry out the tetra-sodium sequencing reaction.

Carry out the tetra-sodium reaction subsequently.

C) spray base sequence (Dispensation order)

Before checking order, need to obtain earlier Dispensation order.Open PyroMark ID software, click Simplex Entries, the primer catalogue of this experiment of being preserved in the primer-design software is introduced, program can generate the tetra-sodium sequence chart (Pyrogram) of a Dispensation order and expection automatically.Simply adjusting back gained Dispensation order according to the needs of this experiment then is: CTGATCTGAC.Determine that spray base sequence is CTGATCTGAC;

D) strand separation and purification

At first get amplified production 30 μ L, add the magnetic bead (beads) that 33 μ L washing binding buffer liquid (binding buffer) and 2 μ l contain even streptavidin (avidin), hatched at normal temperatures 20 minutes, make beads combine with vitamin H.Be fixed on pcr amplification product washing 5 seconds, sex change damping fluid (the Denaturation buffer of 120mL in 70% (volume ratio) alcohol of 180mL respectively on the magnetic bead, 0.2mol/L 1 * washings (Washing Buffer of sex change 15 seconds, 180mL NaOH), 10mM Tris-Acetate) washing is 15 seconds in, makes it to become the single stranded DNA template.The single stranded DNA template is changed in the annealing buffer solution (Annealing buffer) that 29 μ L contain 1.5 μ L sequencing primers (10 μ mol/L), 80 ℃ of hybridization 3min, be cooled to room temperature, must separate the single stranded DNA template behind the purifying, can carry out the tetra-sodium sequencing reaction then, wherein said sequencing primer is ATTGAATCCCAAGTAATC.

E) tetra-sodium reaction

Open PyroMark ID software, click the SNP Runs under the SNP master menu district, the right button of clicking the mouse is selected New SNP Run, the information of reactions such as input Run Name, selective reaction hole.Switch to design interface subsequently, select suitable Entry, input sample information etc.In the View menu, click run, can show the concrete consumption of enzyme mixture, substrate mixture and various dNTPs that this experiment will be used.With enzyme mixture, comprise archaeal dna polymerase, ATP sulfurylase, luciferase and apyrase, and substrate mixture APS and fluorescein are dissolved in the 650 μ L nuclease free waters.The mixture of enzyme mixture, each 3.6 μ l of substrate mixture and 1.8 μ l Annealing Buffer is added in the reacting hole, and four class Nucleotide (A, T, C, G) are added fixed position, reagent cabin respectively according to the amount that system shows, click operation key Run at software interface and can check order.

F) determine reaction product

Instrument carries out interpretation to sequencing result automatically, obtains sex information according to order-checking collection of illustrative plates and result.

The DNA that places corrupt blood sample of degrading of 26 weeks under outdoor natural condition is checked order, referring to accompanying drawing 3-1a and Fig. 3-1b.As can be seen, the method that adopts the present invention to set up can be carried out somatotype well to the blood sample of highly degraded, can realize sex identification.

Embodiment two

Present embodiment is that sample checks order with artificial 9 different time points of degraded of DNase I (5min, 10min, 15min, 20min, 25min, 30min, 40min and 45min) back DNA.

The extraction step of DNA:

1) get 25 μ g DNA, 10 * DNase I reaction buffer, 100 μ L adding distil waters are made into the system of 275 μ L, shift out 10 μ L and make blank, add 2.5 μ L DNase I (1U/ μ L) in remaining 265 μ L.

2) place it under 25 ℃ of conditions and digest, when digesting to 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min and 45min, shift out 30 μ L samples in each time point, the EDTA that in the sample that shifts out, adds 2 μ L 25mM, hatch 15min for 70 ℃, stop the effect of DNaseI.

3) get 10 μ samples, carry out agarose gel electrophoresis, tetra-sodium sequencing technologies detection enamel protein gene respectively.

Pcr amplification, tetra-sodium reaction and detection step are with embodiment one.The somatotype result adopts the present invention to carry out somatotype well by the artificial DNA that degrades of DNase I referring to Fig. 4-2～Fig. 4-10, finishes the identification to individuality.

To sum up, the target sequence that the present invention is selected, and use high-throughout tetra-sodium sequencing technologies to the sample of highly degraded can be accurately, stablize somatotype, can satisfy the requirement of forensic dna check real work, can carry out sex identification accurately to each individuality, be useful the replenishing of existing sex appraisal method.

Claims (2)

1. a tetra-sodium sequencing detects the test kit that enamel protein gene carries out sex identification, it comprises: PCR buffered soln, TaqDNA polysaccharase, primer, template DNA, tetra-sodium sequencing kit, magnetic bead, binding buffer solution, washing buffer solution, annealing buffer solution and sex change buffered soln; It is characterized in that described primer is made up of pcr amplification primer and sequencing primer; The base sequence of described pcr amplification primer is: upstream primer ATTGAATCCCAAGTAATC, downstream primer GACTAGAATAGCCAATGAT, the base sequence of described sequencing primer are ATTGAATCCCAAGTAATC, described downstream primer mark vitamin H.

2. utilize the described test kit of claim 1 to carry out the testing method of sex identification, it is characterized in that, carry out according to following step:

A) extract DNA, get DNA extraction liquid;

B) pcr amplification

Add the DNA extraction liquid that obtains in the step a) and carry out pcr amplification in the pcr amplification system, the pcr amplification condition is: 95 ℃, the pre-sex change of 5min; Then successively at 95 ℃, 15s, 50 ℃, 30s, 72 ℃, 15s carry out 45 circulations; Keep 5min at 72 ℃ again, finally remain on 4 ℃, get amplified production;

C) determine that spray base sequence is CTGATCTGAC;

D) strand separation and purification

The amplified production of step b) gained is added binding buffer solution and the magnetic bead that contains even streptavidin, hatch 20min at normal temperatures; Be to wash 15 seconds in sex change 15 seconds in 70% alcohol washing 5 seconds, the sex change damping fluid, the lavation buffer solution in volume ratio respectively with amplified production then, above-mentioned amplified production becomes the single stranded DNA template, again described single stranded DNA template is changed in the annealing buffer solution that contains sequencing primer, at 80 ℃ of hybridization 3min, be cooled to room temperature, must separate the single stranded DNA template behind the purifying;

E) tetra-sodium sequencing reaction

Be ready to the used enzyme mixture of tetra-sodium sequencing reaction, substrate mixture and dNTP, add the reagent cabin, single stranded DNA template after the separation and purification of gained in the step d) is put into the tetra-sodium sequenator, carry out the tetra-sodium sequencing reaction according to the spray base sequence that designs in the step c);

F) determine reaction product

Instrument carries out interpretation to sequencing result automatically, obtains sex information according to order-checking collection of illustrative plates and result.

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