CN105176992B

CN105176992B - The micro-deleted detection primer in the areas Y chromosome AZF

Info

Publication number: CN105176992B
Application number: CN201510679999.1A
Authority: CN
Inventors: 费嘉
Original assignee: Beijing Zhongyi Kangwei Medical Instrument Co Ltd
Current assignee: Beijing Zhongyi Kangwei Medical Instrument Co Ltd
Priority date: 2015-10-19
Filing date: 2015-10-19
Publication date: 2018-07-31
Anticipated expiration: 2035-10-19
Also published as: CN105176992A

Abstract

The present invention provides a kind of Primer compositions that the areas detection Y chromosome AZF are micro-deleted.Primer sequence is devised for the regions Y chromosome AZF and other primer sites selected as the specific regions of internal reference, the characteristics of these primer sequences is：（1）Every group of primer is to design three continuous primers using genome the preceding paragraph sequence as template, and pcr template is formed after this three primers connections；（2）In normal person's chromosomal target region, there is known and constant copy number in primer sites sequence, and the sequence is not present in other chromosomal regions except target area；（3）These aligning primers have close annealing temperature, fluctuation range<5℃；（4）There is no complementarity with universal amplification primer sequence；（5）Internal reference site only uniquely occurs in target area, and the site is not present in other regions of genome.Additionally provide the purposes of the Primer composition.

Description

The micro-deleted detection primer in the areas Y chromosome AZF

Technical field

The invention belongs to biotechnologies, and in particular to a kind of to be contaminated using gDNA or peripheral blood dissociative DNA detection male Y The micro-deleted primer system in the areas colour solid AZF and its application.

Background technology

There is research report to point out, infertile couples account for the 15% of Childbearing Mr. and Mrs, and wherein male factor accounts for about half, and male Property sterility 35% is since inherent cause causes.Micro-deleted Y chromosome is the main of the male sterility in addition to gram formula syndrome Inherent cause.The a large amount of repetitive sequence of Y chromosome and palindrome also make Y contaminate while maintaining Y chromosome Evolution Stability Colour solid is easier recurring structure rearrangement, leads to partial deletion of chromosome or repetition.Y chromosome is micro-deleted to occur mainly in Y dyeing Area AZF (azoospermia factor) on body.The areas AZF include tri- subprovinces AZFa, AZFb, AZFc, these positions include Many genes related with spermiogenesis tail, these regions it is complete lack or excalation can cause mankind spermatozoon generation obstacle, Few essence, weak essence even azoospermatism shape.

The micro-deleted routine inspection for having become male sterility training now in the areas detection Y chromosome AZF, can be for male not It educates patient and finds out the cause of disease, foundation is provided for genetic counselling；Also guidance is provided for the diagnosis of clinician and successive treatment.So one Kind is simple and quick, accuracy is high and inexpensive detection method is of great significance to clinic.

The micro-deleted method in the areas detection Y chromosome AZF has much at present, such as multiplex PCR, real-time fluorescence PCR, chip are miscellaneous The technologies such as friendship, MLPA, the most commonly used is use multiplexed PCR amplification sequence tagged site (Sequence Tagged Site, STS).This method selects the STS sequences in the area AZFa, AZFb, AZFc to carry out PCR amplification, electrophoresis is carried out to amplified production, according to item The presence or absence of band judges whether each areas AZF lack.European association of andrology (European Academy of Andrology, EAA) join with European molecular genetic Quality Control net (European Molecular Genetics Quality Network, EMQN) Close publication《The micro-deleted molecule of Y chromosome diagnoses practice guideline》It recommends multiplex PCR and detects micro-deleted 6 in the areas Y chromosome AZF A STS (AZFa：SY84, sY86；AZFb：SY127, sY134；AZFc：SY254, sY255).On this basis, each detection is real Test the more accurate detection of quantity progress that room optionally increases STS.This method is simple and practicable, and current use is most extensive. But the sites STS of this method amplification are less (6 or more), and electrophoresis can only qualitative detection purpose product whether deposit , the variation of copy number cannot be detected, this may detect inaccurately the case where Y chromosome AZF area's excalations, so this Kind method need to be improved in accuracy in detection and sensitivity.One of method is to increase the STS quantity of detection, but this is needed Multiple multi-PRC reaction is carried out, to greatly increase experimental work amount and testing cost.

The areas detection Y chromosome AZF are micro-deleted can also to use multiple real time fluorescence PCR, this method easy to be quickly more accurate Really, but probe designs cumbersome and reporter group is needed to modify, expensive, is not suitable for clinical promotion and application.

There is reported in literature to use the areas chip hybridization technology detection Y chromosome AZF micro-deleted, it, can compared to multiple PCR technique To greatly improve the accuracy and sensitivity of detection.But the disadvantage is that this method needs to design a large amount of probe, cost is higher, separately Outer detection signal analysis is also complex, is not suitable for clinician's interpretation.

MRC-Holland companies release a SALSA MLPA probe-mix kit P360-A1 kits, are contaminated for Y The micro-deleted detection in the areas colour solid AZF obtains probe Relative copy number signal by hybridization, connection means, and according to product length The corresponding probe of identification, test result is provided eventually by probe signals.The technological means is faced with as chip hybridization technology The complicated defect of signal analysis is detected, probe is identified according to product length additionally, due to needs, is had on probe is designed and identified Certain limitation.

In conclusion defect existing for the areas Y chromosome AZF micro-deleted detection methods mainly has accuracy and sensitive at present Spend low, of high cost, testing result interpretation complexity etc..Therefore there is an urgent need for develop a kind of new side of the micro-deleted detection in the areas Y chromosome AZF Method, to solve deficiencies of the prior art.

Invention content

It is micro-deleted using gDNA or the areas peripheral blood dissociative DNA detection Y chromosome AZF that the object of the present invention is to provide a kind of Primer system, detection reagent, kit and detection method, to overcome the shortcomings of present in above-mentioned art methods.

The first aspect of the present invention provides a kind of Primer composition that the areas detection Y chromosome AZF are micro-deleted, primer position Point includes the internal reference site of at least six Y chromosome AZF position points and at least one other specific regions of Y chromosome.Its In, three primers are swum according to the sequence design upper, middle and lower of each primer sites, every primer grows 10~50bp, preferably 20bp；It is continuous that three primers from 5 ' to 3 ' directions in reference gene group sequence are swum in the upper, middle and lower of each primer sites 's.The reference gene group sequence is hg18 or hg19 versions.

Preferably, the G/C content of the primer is 30%~70%, and more preferable 50%.

Preferably, 5 ' ends of the midstream and downstream primer of each primer sites carry out phosphorylation modification respectively.

Preferably, 5 ' ends of the sense primer of each primer sites extend the protection sequence of 15~30bp, more preferably For 20bp.

Preferably, 5 ' ends of the sense primer of each primer sites and 3 ' ends of downstream primer each extend over general expansion Increase primer sequence.

The present invention primer system primer sites according to the areas AZF can be divided into the area AZFa, AZFb and AZFc b1, b2, B3, b4, g1, g2, g3, r1, r2, r3, r4, y1, y2 etc. different subprovinces (as shown in Figure 2).The wherein subprovinces such as b, g, r are this hairs The bright important references region for being used for judging the detailed missing information in the areas AZFc.

In a specific embodiment, for the regions Y chromosome AZF and other specific areas as internal reference 148 selected primer sites of domain (including sry gene, ZFY genes and TBL1Y genes etc.), devise 148 groups of primer sequences altogether Row.The characteristics of these primer sequences is：(1) every group of primer is continuous to design three using genome the preceding paragraph sequence as template Primer, form pcr template after this three primers connection；(2) in normal person's chromosomal target region, primer sites sequence exists Known and constant copy number, and the sequence is not present in other chromosomal regions except target area；(3) these sequences are drawn Object has close annealing temperature, fluctuation range<5℃；(4) there is no complementarity with universal amplification primer sequence；(5) internal reference position Point only uniquely occurs in target area, and the site is not present in other regions of genome.

The sequence that three primers are swum in the upper, middle and lower of the primer sites is respectively selected from SEQ ID NO:n、SEQ ID NO: (n+148)、SEQ ID NO:(n+296), the natural number that wherein n is 1~148.

In optimal embodiment, the Primer composition includes that sequence is respectively SEQ ID NO:Whole primers of 1-444.

The second aspect of the present invention provides the purposes of above-mentioned Primer composition composition.

Above-mentioned Primer composition can be used for preparing the diagnostic reagent or kit micro-deleted for detecting the areas Y chromosome AZF.

It is micro-deleted that above-mentioned Primer composition can be used for the areas vitro detection Y chromosome AZF.The detection can be diagnostic purpose (such as diagnosis male sterility etc.), can also be non-diagnostic purpose (such as laboratory research etc.).

The Part III of the present invention provides a kind of detection reagent that the areas detection Y chromosome AZF are micro-deleted comprising above-mentioned Primer composition.

The Part IV of the present invention provides a kind of kit that the areas detection Y chromosome AZF are micro-deleted comprising above-mentioned to draw Compositions.Preferably, the kit further include DNA extracts reagents, general PCR primer, archaeal dna polymerase, DNA ligase, Terminal enzyme (DNA), dNTPs, reaction buffer, magnetic bead and buffer solution for purifying PCR reaction products；It is highly preferred that further including Negative quality-control product and positive quality control product；Most preferably, further include reagent for high-flux sequence.The high-flux sequence side Method is selected from Hiseq, Miseq of PGM, Proton or Illumina of Ion Torrent.

The fifth aspect of the present invention provides a kind of method that the areas vitro detection Y chromosome AZF are micro-deleted, and feature exists In including the following steps：

(1) gDNA or peripheral blood dissociative DNA are extracted as template；

(2) Primer composition of the present invention and DNA profiling are subjected to hybridization reaction；

(3) hybrid product is attached reaction；

(4) connection product is purified；

(5) product after purification is subjected to PCR amplification, builds library；

(6) pcr amplification product is subjected to high-flux sequence；

(7) sequencing data is analyzed.

Preferably, include the following steps：

(1) it detaches, extract the histocyte gDNA of sample to be detected or the dissociative DNA segment of peripheral blood；

(2) sample DNA template end is used into biotin labeling, the DNA profiling and magnetic bead after label combine；

(3) Primer composition that the first aspect of the present invention defines hybridized with DNA profiling, connect reaction, reacted Product is expanded, and amplified production constitutes library；

(4) amplified production is subjected to high-flux sequence；

(5) data analysis：By sequencing data carry out target area positioning, i.e., with the reference gene group for design primer Sequence fragment is compared, and the sequencing sequence item number of each target area is calculated according to comparison result, according to the survey of target area Sequence sequence item number calculates the copy number information of each zonule in the areas AZF, passes through the change with normal person's theoretical value comparative analysis copy number Change, to judge the micro-deleted detection information in the areas AZF.

The criterion of test result is as follows：

(1) if all or part of site copy numbers in the areas AZFa are 0, remaining regional sites copy number is constant, then test result For the areas AZFa missing or excalation；

(2) if the subprovince y3, y4, b5, the b6 and/or u1 site copy number in the areas AZFb become 0 or copied part number become Change, the areas AZFa and the positions AZFc point copy number are constant, then test result mutually should be the areas AZFb missing or part according to change information Missing；

(3) if the position g1, g2, g3 point copy number reduces 1/3~2/3, the position r1, r2, r3, r4 point copy number reduces 1/2 More than, the position b1, b2, b3, b4 point copy number reduces 1/4, and the position y1, y2 point copy number reduces 1/2, remaining regional sites copy Number is constant, then test result lacks for gr/gr；

(4) if the position b1, b2, b3, b4 point copy number reduce 1/2 or so, g1, g2, g3, r1, r2, r3, r4, y1, y2, The position t1, t2 point copy number is 0, remaining regional sites copy number is constant, then test result lacks for b2/b4, also referred to as AZFc Area lacks；

(5) if existing simultaneously the copy number change information of (2) and (4), test result lacks for the areas AZFb+c, specific to lack The subprovince region of mistake can change according to site copy number to judge.

The detection can be diagnostic purpose (such as diagnosis male sterility etc.), can also be that non-diagnostic purpose is (such as real Test room research etc.).

Description of the drawings

The design flow diagram of Fig. 1 present invention.DNA profiling is extracted first, is then hybridized, connects reaction, and to product It is purified, carry out PCR amplification later and library is sequenced using high throughput sequencing technologies, ultimate analysis sequencing data.

The detailed SUBNUCLEAR DIVISION of the object detection areas Fig. 2.The areas Y chromosome AZF can be divided into the area AZFa, AZFbc in detail Y, b, g, r, t, grey etc. different subprovince, some subprovinces there may be cis- repetition or palindrome repeat region, such as g1, g2, Tri- duplicate blocks g3.

The copy number change information of Fig. 3 embodiments 1.

The copy number change information of Fig. 4 embodiments 2.

The copy number change information of Fig. 5 embodiments 3.

The copy number change information of Fig. 6 embodiments 4.

Specific implementation mode

The scheme in the embodiment of the present invention clearly will completely be described below, but the present invention is not intended to be limited thereto, Described embodiment is only a part of the embodiment of the present invention, based on the embodiment of the present invention, those skilled in the art institute The every other embodiment obtained, belongs to protection scope of the present invention；Similarly, the attached drawing of embodiment is only the present invention one The attached drawing of section Example, the other accompanying drawings that those skilled in the art are obtained according to these attached drawings also belong to the present invention's Protection domain.

The hybridized primer used in the following example is that Primer composition shown in table 1 (wherein, distinguish by upstream primer sequence For SEQ ID NO:1-148, middle reaches primer sequence are respectively SEQ ID NO:149-296, downstream primer sequence are respectively SEQ ID NO:297-444), test method without specific conditions, usually according to normal condition, or according to proposed by manufacturer Condition.

1 hybridized primer sequence of table

1 areas hair follicle pattern detection AZF of embodiment are micro-deleted

One, material：

1. sample：Samples sources are the hair follicle sample for the male aspermia or oligospermia patient that chain hospital is seen and treated patients, and are adopted through hospital It is detected as the areas Y chromosome AZFc missing with multiple PCR technique.

2. reagent：Minigene group DNA extraction kit, end-filling kit,DsDNA HS analytical reagents Box, marker enzyme, label enzyme reaction solution, ultra-pure water, hybridization buffer, hybridized primer, DNA lysates (Low TE), connects biotin Connect enzyme, connection enzyme reaction solution, PCR reaction solution, label connector, cleaning solution, XP magnetic beads, isopropanol, absolute ethyl alcohol,MyOneTM Streptavidin C1 beads (Myone C1 magnetic beads), 1M NaOH, Nuclease-free Water(not DEPC-treated)、Ion PGMTM Template OT2 200Kit。

3. equipment：High speed freezing centrifuge, water-bath, ultrasound interrupt instrument Covaris M220, magnetic frame, PCR instrument, Ion OneTouch^TM 2 Instrument、Ion OneTouch^TMES, Ion torrentPGM sequenators, Ion 316^TM Chip V2, Four-dimensional rotary mixer, mini centrifuge, vortex oscillator, Qubit2.0, pipettor (1000/200/100/10/2.5 μ L)。

Two, operating procedure：

1. extracting the genomic DNA in hair follicle sample.Ultrasound is broken into 150bp size segments, and system is prepared according to following table, It is stored at room temperature 20 minutes filling-in ends.

Ingredient	Volume
		Fragmented gDNA	158μL
5×End Repair Buffer	40μL
		End Repair Enzyme	2μL
Total system	200μL

360 μ LXP magnetic beads are added in the system for completing filling-in, 5 minutes are placed at room temperature for after mixing, magnetic is after liquid clarification Supernatant is abandoned, centrifuge tube is not removed, 75% ethyl alcohol, 500 μ L are added, magnetic frame is tilted back and forth and rinses magnetic bead surfaces 5~10 times, suction is abandoned Cleaning one time is repeated after liquid.Centrifuge tube brief centrifugation is removed, magnetic frame is reapposed over and is discarded after liquid clarification Clear liquid dries magnetic bead, and DNA lysates are added, and magnetic collects supernatant after mixing.

2.DNA is marked

Reaction system is prepared according to following table, 37 DEG C of warm bath 1 hour after mixing.

Reagent	Volume
		DNA	38.75μL
Biotin	0.25μL
		Marker enzyme	1μL
Mark enzyme reaction solution	10μL
		Total system	50μL

3. the purifying of marked product

Add isometric isopropanol (precooling -20 DEG C), -70 DEG C are placed 1 hour or more or -20 DEG C stand overnight, 4 DEG C 13000rpm centrifuges 40min.Precipitation is washed with 300 μ L, 75% ethyl alcohol newly prepared, 4 DEG C of 13000rpm centrifuge 8min, abandon supernatant. It washes altogether twice.Room temperature is dried after precipitation with 13 μ LDNA lysate dissolving DNAs.

4. hybridization

Prepare Myone C1 magnetic beads：It is thorough that (30min takes out the incubation at room temperature of Myone C1 magnetic beads in advance) vortex oscillation is more than 30s Bottom mixing takes 10 μ L magnetic beads to move on in a new PCR pipe, magnetic 3min, abandons supernatant 5 μ L, spare.According to following table hybrid reaction System, gently mixing (avoid generate bubble), 95 DEG C of reaction 5min in PCR instrument, after reaction rapid ice set 10min.

Reagent	Volume
		Mark purified product	12.5μL
Hybridization buffer	22.5μL
		Hybridized primer	10μL
Total system	45μL

After ice is set, reaction solution is added in ready Myone C1 magnetic beads brief centrifugation, and vortex oscillation 30s is mixed It is even, bubble is avoided, gently rotation mixes 2h to 22~25 DEG C of room temperature (room temperature cannot be below 20 DEG C, and highest is no more than 27 DEG C).

5. the purifying of hybrid product

PCR pipe is placed in magnetic 3min on magnetic frame, supernatant is abandoned after liquid clarification, PCR pipe is removed and (is sure not to keep magnetic bead dry It is dry).Add 50 μ L cleaning solutions, pressure-vaccum mixing abandons supernatant after magnetic, removes centrifuge tube (being sure not that magnetic bead is made to dry).It washs 3 times altogether. 45 μ L DNA lysates are added, magnetic bead is resuspended.

6. connection

The linked system of following table is placed in PCR instrument, 37 DEG C are reacted 1 hour.

Reagent	Volume
		Hybridize purified product	45μL
Ligase	0.1μL
		Connect enzyme reaction solution	5μL
Total system	50.1μL

7. the purifying of connection product

The PCR pipe for completing connection reaction is placed in magnetic 3min on magnetic frame, supernatant is abandoned after liquid clarification, removes PCR pipe (being sure not that magnetic bead is made to dry).Add 50 μ L cleaning solutions, pressure-vaccum mixing abandons supernatant after magnetic, removes centrifuge tube.It washs 2 times altogether.It is added Magnetic bead is resuspended in 23 μ LDNA lysates, is denaturalized 3min for 95 DEG C in PCR instrument, is put into magnetic on magnetic frame immediately, Aspirate supernatant body is extremely In new PCR pipe.

8.PCR

According to following table plus reaction system：

It is reacted according to following programs：

The purifying of 9.PCR products

XP magnetic beads are shifted to an earlier date into 30min and take out balance to room temperature, vortex oscillation 30s or more respectively takes 20 μ L, 97 μ L to be put into new In EP pipes and mark.It takes 45 μ LPCR products to be added in 20 μ LXP magnetic beads, 5min is placed at room temperature for after mixing.Magnetic 3~ 5min takes in 62 μ L of supernatant to 97 μ LXP magnetic beads after liquid thoroughly clarification, 5min is placed at room temperature for after mixing.Magnetic waits for that liquid is clear Supernatant is abandoned after clear, does not remove centrifuge tube, 75% ethyl alcohol, 150 μ L are added, tilts magnetic frame back and forth, makes the table of liquid wash XP magnetic beads Face 5~10 times, and abandon liquid in 1min interior suctions.It washes altogether twice.Centrifuge tube brief centrifugation is removed, then is reapposed over magnetic frame On, until liquid discards supernatant when clarifying.Magnetic bead is dried, 15 μ L DNA lysates are added, 3~5min, magnetic are placed at room temperature for after mixing Supernatant (i.e. library) is transferred in new EP pipes after suction.

10. library is sequenced

2 libraries μ L are taken to carry out concentration mensuration with Qubit2.0.Library is diluted to 18pmol/ μ L through with Ion PGM^TM Template OT2 200Kit are prepared into emulsion-based PCR sample, are carried out on Ion OneTouchTM ES after product enrichment in Ion It is sequenced on Torrent PGM (life technology).

11. data analysis

Low quality sequencing sequence is filtered first, removes the sequencing segment less than 70bp, by filtered sequence alignment to 148 A reference gene group sequence fragment for design primer, calculate this 148 target areas total sequencing sequence item number N and respectively Sequencing sequence item number Ni.

Using the mean value N0 of the primer sequence item number in 10 internal reference regions, by the primer sequence item in 138 regions AZF Number is uniformed, and formula is：Ci=Ni/N0.

According to the Ci values of each primer, the mean value of each cell of AZF Divisions is calculated to get to sample AZF to be detected The true copies number information of each cell in area, such as it is respectively g1, g2, g3 ... g10 that, which there are 10 primer sites, corresponding Ci values in the areas g, The then areas g copy number：

By the true copies number information of each cell in the areas sample AZF to be detected compared with the variation of theoretical value, AZFc is found The area g, r copy number becomes the areas 0, b copy number reduction half in area, and the areas AZFa and AZFb copy number is constant (as shown in Figure 3).Inspection It is that b2/b4 missings occur for the areas sample Y chromosome AZF to survey conclusion.

It is micro-deleted that 2 peripheral blood sample of embodiment detects the areas AZF

One, material：

1. sample：Samples sources are the peripheral blood sample for the male aspermia or oligospermia patient that chain hospital is seen and treated patients, through hospital The areas Y chromosome AZFc excalation is detected as using multiple PCR technique.

2. reagent：Plasma dna extracts kit,DsDNA HS assay kits, biotin, marker enzyme, label Enzyme reaction solution, ultra-pure water, hybridization buffer, hybridized primer, DNA lysates (Low TE), ligase, connection enzyme reaction solution, PCR Reaction solution, label connector, cleaning solution, XP magnetic beads, isopropanol, absolute ethyl alcohol,MyOneTM Streptavidin C1beads (Myone C1 magnetic beads), 1M NaOH, Nuclease-free Water (not DEPC-treated), Ion PGMTM Template OT2 200Kit。

3. equipment：High speed freezing centrifuge, water-bath, magnetic frame, PCR instrument, Ion OneTouch^TM 2Instrument、 Ion OneTouch^TMES, Ion torrentPGM sequenators, Ion 316^TMChip v2, Four-dimensional rotary mixer, it is mini from Scheming, vortex oscillator, Qubit2.0, pipettor (1000/200/100/10/2.5 μ L).

Two, operating procedure：

1. extracting plasma dna with plasma dna extracts kit.

2.DNA is marked

3. the purifying of marked product

Add isometric isopropanol (precooling -20 DEG C), -70 DEG C are placed 1 hour or more or -20 DEG C stand overnight, 4 DEG C 13000rpm centrifuges 40min.Precipitation is washed with 75% ethyl alcohol of 300 μ L Fresh, 4 DEG C of 13000rpm centrifuge 8min, abandon Clearly.It washes altogether twice.Room temperature is dried after precipitation with 13 μ LDNA lysate dissolving DNAs.

4. hybridization

5. the purifying of hybrid product

6. connection

7. the purifying of connection product

The PCR pipe for completing connection reaction is placed in magnetic 3min on magnetic frame, supernatant is abandoned after liquid clarification, removes PCR pipe (being sure not that magnetic bead is made to dry).Add 50 μ L cleaning solutions, pressure-vaccum mixing abandons supernatant after magnetic, removes centrifuge tube.It washs 2 times altogether.It is added Magnetic bead is resuspended in 23 μ LDNA lysates, is denaturalized 3min for 95 DEG C in PCR instrument, is put into magnetic on magnetic frame immediately, Aspirate supernatant moves to In new PCR pipe.

8.PCR

According to table plus reaction system.

Reagent	Volume
		Connect purified product	23μL
PCR reaction solution	25μL
		PCR primer	1μL
Label connector	1μL
		Total system	50μL

It is reacted according to following programs.

The purifying of 9.PCR products

XP magnetic beads are shifted to an earlier date into 30min and take out balance to room temperature, vortex oscillation 30s or more respectively takes 20 μ L, 97 μ L to be put into new In EP pipes and mark.It takes 45 μ LPCR products to be added in 20 μ LXP magnetic beads, 5min is placed at room temperature for after mixing.Magnetic 3~ 5min takes in 62 μ L of supernatant to 97 μ L XP magnetic beads after liquid thoroughly clarification, 5min is placed at room temperature for after mixing.Magnetic waits for liquid Supernatant is abandoned after clarification, does not remove centrifuge tube, and 75% ethyl alcohol, 150 μ L are added, tilts magnetic frame back and forth, makes liquid wash XP magnetic beads Surface 5~10 times, and abandon liquid in 1min interior suctions.It washes altogether twice.Centrifuge tube brief centrifugation is removed, then is reapposed over magnetic frame On, until liquid discards supernatant when clarifying.Magnetic bead is dried, 15 μ L DNA lysates are added, 3~5min, magnetic are placed at room temperature for after mixing Supernatant (i.e. library) is transferred in new EP pipes after suction.

10. library is sequenced

2 libraries μ L are taken to carry out concentration mensuration with Qubit2.0.Library is diluted to 18pmol/ μ L through with Ion PGM^TM Template OT2 200Kit are prepared into emulsion-based PCR sample, Ion OneTouch^TMIn Ion after progress product enrichment on ES It is sequenced on Torrent PGM (life technology).

11. data analysis

By the true copies number information of each cell in the areas sample AZF to be detected compared with the variation of theoretical value, AZFc is found The areas g copy number becomes 0 in area, and there are nearly 1/4 copy number, judgement b2/b3 generation inversions in the areas r；Find the areas b copy number simultaneously Half is reduced, copy number constant test result (as shown in Figure 4) in the areas AZFa and AZFb is that b2/ occurs for the areas sample Y chromosome AZF The areas g1/b4 lack after b3 inversions.

It is micro-deleted that 3 peripheral blood sample of embodiment detects the areas AZF

One, material：

1. sample：Samples sources are the peripheral blood sample for the male aspermia or oligospermia patient that chain hospital is seen and treated patients, through hospital It is normal that the areas Y chromosome AZF are detected as using multiple PCR technique.

2. reagent：Plasma dna extracts kit,DsDNA HS assay kits, biotin, marker enzyme, label Enzyme reaction solution, ultra-pure water, hybridization buffer, hybridized primer, DNA lysates (Low TE), ligase, connection enzyme reaction solution, PCR Reaction solution, label connector, cleaning solution, XP magnetic beads, isopropanol, absolute ethyl alcohol,MyOneTM Streptavidin C1 beads (Myone C1 magnetic beads), 1M NaOH, Nuclease-free Water (not DEPC-treated), Ion PGMTM Template OT2 200Kit。

Two, operating procedure：

1. extracting plasma dna with plasma dna extracts kit.

2.DNA is marked

3. the purifying of marked product

4. hybridization

5. the purifying of hybrid product

6. connection

7. the purifying of connection product

8.PCR

According to table plus reaction system.

It is reacted according to following programs.

The purifying of 9.PCR products

10. library is sequenced

11. data analysis

By the true copies number information of each cell in the areas sample AZF to be detected compared with the variation of theoretical value, discovery AZFa, The area AZFb, AZFc copy number is constant (as shown in Figure 5).Test result is that the areas sample Y chromosome AZF are normal, causes patient few The reason of smart symptom is the inherent cause or non-genetic factor except the areas AZF.

It is micro-deleted that 4 peripheral blood sample of embodiment detects the areas AZF

One, material：

1. sample：Samples sources are the peripheral blood sample for the male aspermia or oligospermia patient that chain hospital is seen and treated patients, through hospital The areas Y chromosome AZFb+c missing is detected as using multiple PCR technique.

2. reagent：Poba gene group DNA extraction kit, end-filling kit,DsDNA HS analytical reagents Box, marker enzyme, label enzyme reaction solution, ultra-pure water, hybridization buffer, hybridized primer, DNA lysates (Low TE), connects biotin Connect enzyme, connection enzyme reaction solution, PCR reaction solution, label connector, cleaning solution, XP magnetic beads, isopropanol, absolute ethyl alcohol,MyOneTM Streptavidin C1 beads (Myone C1 magnetic beads), 1M NaOH, Nuclease-free Water(not DEPC-treated)、Ion PGMTM Template OT2 200Kit。

3. equipment：High speed freezing centrifuge, water-bath, ultrasound interrupt instrument Covaris M220, magnetic frame, PCR instrument, Ion OneTouch^TM 2Instrument、Ion OneTouch^TMES, Ion torrentPGM sequenators, Ion 316^TMChip v2、 Four-dimensional rotary mixer, mini centrifuge, vortex oscillator, Qubit2.0, pipettor (1000/200/100/10/2.5 μ L).

Two, operating procedure：

1. extracting the genomic DNA in peripheral blood sample.Ultrasound is broken into 150bp size segments, and body is prepared according to following table System, is stored at room temperature 20 minutes filling-in ends.

2.DNA is marked

3. the purifying of marked product

4. hybridization

5. the purifying of hybrid product

6. connection

7. the purifying of connection product

8.PCR

According to table plus reaction system.

It is reacted according to following programs.

The purifying of 9.PCR products

10. library is sequenced

11. data analysis

By the true copies number information of each cell in the areas sample AZF to be detected compared with the variation of theoretical value, AZFb is found To become 0, AZFa copy numbers constant (as shown in Figure 6) for areas' copy number such as b, g, r in the areas such as b5, b6, u1 and the areas AZFc in area.Inspection It is that the areas AZFb+c missing occurs for the areas sample Y chromosome AZF to survey conclusion.

Claims

1. a kind of Primer composition that the areas detection Y chromosome AZF are micro-deleted, which is characterized in that primer sites include at least six Y The internal reference site of the positions chromosome AZF point and at least one other specific regions of Y chromosome；The areas the AZF primer position The subprovince y, b, g, r, t, grey in the point covering areas AZFa and the areas AZFbc；Wherein, according to the sequence design of each primer sites Three primers are swum in upper, middle and lower, and three primers are swum in reference gene group sequence from 5 ' in the upper, middle and lower of each primer sites It is continuous to 3 ' directions；The Primer composition includes that sequence is respectively SEQ ID NO:Whole primers of 1-444.

2. the Primer composition of claim 1 is respectively SEQ ID NO by sequence:The primer of 1-444 forms.

3. the Primer composition of claim 1,5 ' ends of the sense primer of each primer sites further extend 15 ~ The protection sequence of 30bp, 5 ' ends of the sense primer and 3 ' ends of the downstream primer further extend universal amplification and draw respectively Object sequence.

4. the Primer composition of claim 2,5 ' ends of the sense primer of each primer sites further extend 15 ~ The protection sequence of 30bp, 5 ' ends of the sense primer and 3 ' ends of the downstream primer further extend universal amplification and draw respectively Object sequence.

5. the Primer composition of any one of claim 1-4, wherein the 5 ' of the midstream and downstream primer of each primer sites End carries out phosphorylation modification respectively.

6. the Primer composition of any one of claim 1-4, wherein the reference gene group sequence is hg18 or hg19 versions.

7. the Primer composition of claim 3 or 4, wherein the sequence of the primer sites is not deposited with universal amplification primer sequence In complementarity.

8. the Primer composition of any one of claim 1-4, wherein known to the areas the AZF primer sites exist in the region The position is not present in copy number and copy number immobilizes in the regions normal person AZF, other regions of the genome except the regions AZF Point.

9. the Primer composition of any one of claim 1-4, wherein the internal reference site be selected from sry gene, ZFY genes and TBL1Y gene regions.

10. the Primer composition of any one of claim 1-4, wherein the internal reference site only uniquely occurs in target area, The site is not present in other regions of genome.

11. the Primer composition of any one of claim 1-10 is preparing the diagnosis examination micro-deleted for detecting the areas Y chromosome AZF Application in agent.

12. the Primer composition of any one of claim 1-10 is preparing the kit micro-deleted for detecting the areas Y chromosome AZF In application.

13. the Primer composition of any one of claim 1-10 is during the areas non-diagnostic purpose detection Y chromosome AZF are micro-deleted in vitro Application.