CN107502668A - The detection method of people's Y chromosome label site sY1291 a kind of and its application - Google Patents
The detection method of people's Y chromosome label site sY1291 a kind of and its application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention belongs to beyond body nucleic acid detection technique field, specially a kind of people's Y chromosome label site sY1291 detection method and its application.The invention provides a kind of new sY1291 site primer strategies, specific PCR amplimer is redesigned in the EAA/EMQN sY1291 site primer region upstream 38bp large fragments repeat regions recommended, 38bp is held in new pcr amplification primer analyte detection region I 5 ' ends away from the EAA/EMQN sY1291 site primers regions 3 ' recommended, and new pcr amplification primer thing can detect tetra- regions of I, II, III, IV simultaneously.Inspection policies combination fine jade sugar electrophoresis, fluorescent dye primer Capillary Electrophoresis or fluorescence probe method of the present invention etc. form the different detection methods in sY1291 sites.People's Y chromosome label site provided by the invention sY1291 detection methods, high specificity, detection Y chromosome region are wide, are more suitable for the micro-deleted expansion detections of people's Y chromosome AZF.
Description
Technical field
The invention belongs to people's beyond body nucleic acid detection technique field, and in particular to people's Y chromosome label site sY1291 inspection
Survey method and application.
Background technology
The Y chromosome of people is peculiar for male.In genetic development, Y chromosome can only be entailed son, i.e. Y by father
Chromosome shows the feature of paternal inheritance.Meanwhile have decisive other gene such as SRY on Y chromosome, generated with mankind spermatozoon
Related gene or key area, such as Azoospermia factor (Azoospermia Factor, AZF).However, due to Y chromosome
A large amount of very high homology sequences itself be present, in genetic process, the missing of big chromosomal region can occur for Y chromosome, the most
Commonly AZF areas is micro-deleted.
Due to this feature of the paternal inheritance of Y chromosome in itself, often by the polymorphism on Y chromosome in forensic DNA analysis
Genetic marker is used for interindividual same paternal identification, such as half-blooded two boys by the Y chromosome of shared his father,
The two is also by the haplotype with identical Y chromosome polymorphic markers in theory.However, work as one of boy
Exist the micro-deleted and another boy of Y chromosome do not occur Y chromosome missing when, will cause this judgment rule fail.Therefore, exist
When being examined using the close source of the same paternal line of Y chromosome polymorphic markers progress, micro-deleted detection is carried out to Y chromosome and also must
Want, especially provide exclude this same paternal conclusion when the micro-deleted inspection of Y chromosome it is more important.Deposited for consideration
In the micro-deleted individual of Y chromosome, it is also desirable to understand its missing scope have it is much, if cover it is near it, for father
It is the Y chromosome pleomorphism site examined.
Male sterility field equally is concerned with to the micro-deleted exception of Y chromosome.AZF is related to mankind spermatozoon generation
Key area.AZF is to be proposed by Tiepolo and Zuffardi in 1976 earliest, and they think that AZF is to be present in Y dyeing
The gene loci of the long-armed control Sperm specific enzyme of body.Substantial amounts of research is confirmed Y chromosome is long-armed 5th, at least exists in 6 spacer regions
The region of 3 non-overlapping copies is relevant with Sperm specific enzyme, i.e. AZFa, AZFb and AZFc.Dyszoospermia caused by its missing is to cause
The major reason of male sterility, Primary Azoospermia (Azoospermia) or aspermia or oligospermia (Oligospermia) are shown as, its
In, most common absent region is AZFc areas.In view of this, in version Europe association of andrology in 2013（EAA）Lost with European molecule
Pass experiment Quality Control coorporative network（EMQN）On in the micro-deleted molecule diagnosis best practice guideline of Y chromosome, recommending for AZFa
The inspection micro-deleted with method and AZF of area, AZFb areas, the classical label site in AZFc areas, the detection primer in respective labels site
Survey path.
In each areas of AZF, EAA/EMQN recommends the classical site for having for the micro-deleted detection of respective regions, such as AZFa areas
SY84 and the sY134 in sY86, AZFb area and the sY255 and sY254 in sY127, AZFc area.In AZFa areas, AZFb areas and AZFc
Qu Zhong, most common absent region are AZFc areas, account for more than 79% always lacked.In addition to above-mentioned classical deletion segment, EAA/EMQN
Also the expansion detection site and detection method for each area are recommended, these expand detection site except being deposited in classical site primer
Outside the effect verified in the case of doubting, while it may also confirm that the micro-deleted scope of Y chromosome.Wherein in the expansion in AZFc areas
In detection site, sY1291 is mostly important, and has potential value of clinical studies（Krausz et al;2013）.The site
One positioned at AZFc areas is referred to as in the subprovince of " gr/gr ", and early stage was once used for the research that Y chromosome genealogical tree missing develops
（Repping et al;2003）, and point out as with family genetic polymorphism " gr/gr " missing may because eliminate it is several
The AZFc areas hereditary information of half and then the generation for influenceing sperm, are an important male sterility risks and assumptions.
But in follow-up research, had been found that using the EAA/EMQN sY1291 recommended detection method in the presence of certain
False positive, in the sample that AZFc areas missing occurs, sY1291 is presented more than 99% as missing, and in normal sample
AZFc areas also may occur in which missing, i.e., certain Polymorphic Population is showed in normal sample（Krausz et al;2009）.
Therefore, analyzed, it is necessary to a kind of more structurally sound sY1291 positions in order to preferably micro-deleted to male AZF
Point inspection policies and method.
The content of the invention
It is an object of the invention to the weak point for the sY1291 detection methods recommended for EAA/EMQN and
The actual current demand that sY1291 missings are examined, there is provided a kind of high specificity and AZFc areas classics deletion segment missing concordance rate
High people's Y chromosome label site sY1291 detection method, and application.
People's Y chromosome label site sY1291 provided by the invention detection method, is related to sY1291 sites PCR primer
The strategy of amplification region（Inspection policies）, new specific PCR amplimer is designed, to people's Y chromosome label site sY1291
Detected；Concretely comprise the following steps：
（1）Determine the amplification region of sY1291 sites PCR primer.Its strategy is to avoid European andrology association/European molecule
The micro-deleted molecule diagnostic operation guide of Y chromosome of genetic experiment Quality Control coorporative network (EAA/EMQN) combined publication（2013 editions）In
The amplification region of the sY1291 sites PCR primer of recommendation, recommend the large fragment duplicate block of detection zone proximity in EAA/EMQN
Design new specific PCR amplimer in domain.Therefore the detection zone that the present invention selects includes：I areas（ChrY:25,504,754-
25,505,032）, II areas（ChrY:25,207,966-25,208,244）, III areas（ChrY:26,841,711-26,841,
989）, IV areas（ChrY:27,120,413-27,120,691）.I areas are located at EAA/EMQN with II areas and recommend sY1291 site primers
The upstream in region, III areas are located at the downstream in EAA/EMQN recommendation sY1291 site primers region with IV areas；Wherein, the 5 ' ends in I areas
SY1291 site primers region 3 ' is recommended to hold 38bp away from EAA/EMQN（See accompanying drawing 1）.
（2）Pcr amplification primer thing is designed, nucleotides sequence is classified as shown in SEQ ID No.1 and SEQ ID No.2, PCR amplifications
Primer covers the I areas in people Yq11.223-Yq11.23 regions（ChrY:25,504,754-25,505,032）, II areas（ChrY:
25,207,966-25,208,244）, III areas（ChrY:26,841,711-26,841,989）, IV areas（ChrY:27,120,
413-27,120,691）；
Using pcr amplification primer thing provided by the present invention（Nucleotide sequence shown in SEQ ID No.1 and SEQ ID No.2）Expanded
Increase people Yq11.223-Yq11.23 regions I areas（ChrY:25,504,754-25,505,032）, II areas（ChrY:25,207,966-
25,208,244）, III areas（ChrY:26,841,711-26,841,989）With IV areas（ChrY:27,120,413-27,120,
691）Sequence homology>99%.
（3）Using pcr amplification primer thing, people's Y chromosome label site sY1291 is detected, is divided into 3 kinds of situations：
（a）Performing PCR amplification is entered using archaeal dna polymerase, pcr amplification product is examined using 1.0%-2.0% agarose gel electrophoresis
Survey, no PCR amplifications target product is sample sY1291 site deletions, and it is that sY1291 is not lacked there are PCR amplification target product persons；
（b）Fluorescence is carried out to the end of nucleotide sequence 5 ' shown in the end of nucleotide sequence 5 ' shown in SEQ ID No.1 or SEQ ID No.2
Element mark（By taking FAM as an example）, performing PCR amplification is entered using archaeal dna polymerase, PCR primer uses 3500Dx or same type gene sequencing
Instrument carries out Capillary Electrophoresis fragment analysis, and it is that sY1291 sites do not lack PCR amplification target product fluorescence peak persons occur, is not occurred
PCR amplification target product fluorescence peak persons are sY1291 site deletions；
（c）Fluorescence labeling probe is designed, its nucleotide sequence is carried out as shown in SEQ ID No.3 using real time fluorescent PCR method
Detection, there is target fragment PCR amplification curves person and do not lacked for sY1291 sites, do not occur target fragment PCR amplification curve persons
For sY1291 site deletions.
Wherein, situation（a）With situation（b）Reaction system in, pcr amplification primer thing SEQ ID No. 1 and SEQ ID No.
2 working concentration is 50nmol/L to 300nmol/L；Situation（c）Reaction system in, pcr amplification primer thing SEQ ID No.1 with
SEQ ID No. 2 working concentration is 300nmol/L to 500nmol/L, and probe SEQ ID No.3 working concentration is
150nmol/L to 250nmol/L.
The sY1291 missing detection methods of the present invention, available for the micro-deleted detections of people AZF.
The beneficial effects of the invention are as follows：
SY1291 detection methods provided by the present invention, effectively expand the detection range to AZFc areas absent region.The present invention
In, redesign new pcr amplification primer thing at the sY1291 detection zones upstream 38bp that EAA/EMQN recommends（SEQ ID
Nucleotide sequence shown in NO.1 and SEQ ID NO.2）.The I areas of new pcr amplification primer analyte detection（ChrY:25,504,754-25,
505,032）SY1291 detection zones 3 ' are recommended to hold 38bp away from EAA/EMQN in 5 ' ends.In addition to I areas, new pcr amplification primer thing is simultaneously
It has detected II areas（ChrY:25,207,966-25,208,244）, III areas（ChrY:26,841,711-26,841,989）With IV
Area（ChrY:27,120,413-27,120,691）.The total covering Yq11.223-Yq11.23 in four regions has more than 1912kb
Effect expands the detection range to AZFc absent regions.And with the classical deletion segment in Y chromosome AZFc areas（SY255 with
sY254）Missing concordance rate reaches 100%, overcomes EAA/EMQN and recommends sY1291 loci detection methods and the classical missing in AZFc areas
Site（SY255 and sY254）The problem of testing result is inconsistent, the missing false positive issue in normal male crowd.
Detection method provided by the invention, high specificity is high with AZFc areas classics deletion segment missing concordance rate, can be effective
The range of loss in determination AZFc areas is assisted, while these detections are quick, easy, can expand popularization in the micro-deleted field of Y chromosome
Using.
Brief description of the drawings
Fig. 1：People's Y chromosome label site provided by the present invention sY1291 inspection policies.Accompanying drawing show label site
The structure diagram in people Yq11.223-Yq11.23 regions where sY1291.Wherein, black vertical line show EAA/EMQN recommendations
SY1291 site primers region.The perpendicular rectangle frame pcr amplification primer thing in sY1291 sites provided by the present invention of white is detected
Region, wherein I areas are located at recommends at sY1291 detection zones upstream 38bp away from EAA/EMQN.Y residing for each region contaminates shown in figure
Colour solid physical location information is referring to human genome database Hg19（http://genome.ucsc.edu/）.
Fig. 2：The present invention combines PCR agarose electrophoresis detection sY1291（Embodiment 1）.In accompanying drawing, M swimming lanes produce for PCR
Thing molecular weight markers, 1-6 are 6 different male's samples.From left to right：For EAA/EMQN recommend by totally 6 swimming lanes by first group of 1-6
Method detects 6 sample AZFc areas classics deletion segments（SY255 and sY254, sY255 amplified fragments are 123bp, and sY254 is
380bp）, No. 3 with No. 4 samples be AZFc areas missing sterility male's samples, No. 1, No. 2, No. 5 with No. 64 samples be normal
Male's sample；Second group of 1-6 swimming lane is same 6 samples, recommends sY1291 loci detection methods using EAA/EMQN（Expand piece
Section is 527bp）, No. 1 does not lack with No. 2 samples, and No. 5 are missing false positive with No. 6 samples；3rd group is that the inventive method detects
SY1291 sites（Amplified fragments are 279bp）, 6 pattern detection results are consistent with AZFc areas classics deletion segment testing result.
Fig. 3 combines real-time fluorescence PCR method for the present invention and detects sY1291, while detects EAA/EMQN and recommend reference gene
（Sex determining gene, SRY）（Embodiment 3）, as a result show that SRY has corresponding amplification curve to sY1291 sites, be male
SY1291 sites do not lack sample.
Fig. 4 combines real-time fluorescence PCR method for the present invention and detects sY1291, while detects EAA/EMQN and recommend reference gene
（Sex determining gene, SRY）（Embodiment 3）, as a result show that sry gene amplification curve normally detects, do not detect sY1291 sites
Amplified signal, it is male's sY1291 site deletion samples.
Embodiment
Content for a better understanding of the present invention, is described further with reference to specific embodiment.It should be understood that following tool
Body embodiment is merely to illustrate effectiveness of the invention, rather than limitation of the present invention.In addition, the people's gene using commercialization
Organize the biological material of DNA extraction kit or other chemical reagent from people（Such as people's whole blood, throat swab, saliva, hair follicle or muscle groups
Knit）Middle extraction human gene group DNA is used for subsequent detection, is not comprised in the claim of the present invention, but be used as people's gene
The old process or previous steps of group DNA detection, this human gene group DNA's extraction process should not also turn into the application of the present invention
Limitation.Therefore, follow-up each embodiment is started from after human gene group DNA extracts and complete.
Each embodiment uses SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 nucleotide sequence and its modification
Feature is shown in Table 1, difference of the 5 ' ends of nucleotide sequence according to follow-up PCR primer detection method wherein shown in SEQ ID NO.2, can
Add fluorescein（Such as FAM）Modification（For Capillary Electrophoresis fragment analysis）Or it is not added with fluorescein modification（Embodiment 1 and embodiment
3）.
The pcr amplification primer thing in sY1291 sites of the present invention of table 1 and probe
Note：SEQ ID No.2 5 ' ends can not mark fluorescent element（Agarose electrophoresis）Or mark fluorescent is plain (Capillary Electrophoresis).
Embodiment 1：The present invention combines PCR primer agarose electrophoresis and carries out sY1291 site primers
Using PCR primer shown in SEQ ID No.1 and SEQ ID No.2（Wherein SEQ ID No.2 mark without fluorescein）, press
PCR reaction conditions expand to male gene group DNA shown in PCR reaction systems shown in table 2 and table 3, and pcr amplification product uses
2.0% agarose carries out electrophoresis.
No. 1-6 totally 6 male's samples according to AZFc area's classics deletion segment (sY255 and sY254) testing results and table
Type is divided into two classes, No. 3 with the male sterility sample that No. 4 samples are AZFc areas classics deletion segment, No. 1, No. 2, No. 5 with No. 6
Sample is the normal normal male sample of seminal parameters.AZFc areas classics deletion segment (sY255 and sY254) detection method uses
The detection method that EAA/EMQN recommends.Above-mentioned 6 sample ZFc areas classics deletion segment (sY255 and sY254) testing result,
EAA/EMQN recommends sY1291 loci detection methods testing result and the inventive method detection sY1291 sites result to see accompanying drawing 2
It is shown.
The present invention of table 2 combines the PCR reaction systems in agarose electrophoresis detection sY1291 sites
。
The present invention of table 3 combines the PCR reaction conditions in agarose electrophoresis detection sY1291 sites
。
As a result visible, AZFc areas classics site is present in male's sample of missing, method detection provided by the present invention
SY1291 sites also lack, but EAA/EMQN recommends the sample that sY1291 loci detection methods lack in AZFc classics deletion segment
In missing may be present, both can behave as missing in the normal normal male sample of seminal parameters can also appear as not lacking, i.e.,
EAA/EMQN recommends the testing result in sY1291 sites false positive to be present.
Embodiment 2：The present invention combines the analysis of PCR primer Capillary Electrophoresis fluorescence light segments and carries out sY1291 site primers
Using PCR primer shown in SEQ ID No.1 and SEQ ID No.2（Wherein SEQ ID No.2 use FAM fluorescein marks
Note）, the PCR reaction conditions shown in PCR reaction systems and table 5 as shown in table 4,1169 male's DNA samples are entered with performing PCR amplification,
PCR primer carries out Capillary Electrophoresis using 3500Dx genetic analyzers.
1169 male's samples include 232, the normal male's sample of seminal parameters, sterility male 937.All samples
This is using the kit ratified through Chinese food Drug Administration for the micro-deleted detections of AZF（Human chromosome AZF areas are micro-
Lacking nucleic acid reagent box, (PCR- fluorescence probe methods, 20153400764) state's tool note is accurate to carry out AZFc areas classics site（SY255 with
sY254）Detection, while using EAA/EMQN recommend sY1291 sites detected.Statistical result is shown in Table 6 and table 7.
The present invention of table 4 combines the PCR reaction systems in capillary electrophoresis detection sY1291 sites
。
The present invention of table 5 combines the PCR reaction conditions in agarose electrophoresis detection sY1291 sites
。
From the point of view of the uniformity with the uniformity of phenotype and with AZFc areas classics deletion segment testing result, EAA/EMQN
The positive predictive value for recommending sY1291 detection methods to lack AZFc areas is 64.92% (198/305), to the moon of AZFc areas missing
Property predicted value is 100.00% [(212+652)/864], and false positive recall rate is 11.02% [(20+87)/(232+739)].
The EAA/EMQN of table 6 recommends sY1291 loci detection methods to 1169 male's pattern detection results
Note：SY255 and sY254 missings are detected using state's accurate 20153400764 kits of tool note.
It is of the invention from the point of view of the uniformity with the uniformity of phenotype and with AZFc areas classics deletion segment testing result
The positive predictive value that sY1291 detection methods lack to AZFc areas is 100.00% (198/198), pre- to the feminine gender of AZFc areas missing
Measured value is 100.00% [(232+739)/971], and false positive recall rate is 0.00% [(0+0)/(232+739)].Obviously, compare
The sY1291 detection methods that EAA/EMQN recommends, sY1291 detection methods provided by the present invention are remarkably improved to be lacked for AZFc
The positive predictive value of mistake（Improved by 64.92% to 100.00%）, and significantly reduce the false positive rate of sY1291 missings（By
11.02% is down to 0.00%）.
The sY1291 loci detection methods of the present invention of table 7 are to 1169 male's pattern detection results
Note：SY255 and sY254 missings are detected using state's accurate 20153400764 kits of tool note.
Embodiment 3：The present invention combines real-time fluorescence sonde method and carries out sY1291 site primers
Using PCR primer shown in SEQ ID No.1 and SEQ ID No.2（Mark fluorescent is not plain by SEQ ID No.2）、SEQ ID
Fluorescence probe shown in NO.3, PCR reaction conditions carry out real-time fluorescence detection shown in PCR reaction systems and table 9 as shown in table 8.
Reference gene in the micro-deleted detections of AZF of EAA/EMQN recommendations is detected in the system simultaneously（Sex determining gene, SRY）.Inside
In the presence of joining gene by fluorescence signal, there is sY1291 sites amplification curve person and do not lack sample for sY1291 sites（Fig. 3）, do not go out
Existing sY1291 sites amplification curve person is sY1291 site deletion samples（Fig. 4）.
The present invention of table 8 combines the PCR reaction systems in real-time fluorescence detection sY1291 sites
。
The present invention of table 9 combines the PCR reaction conditions in real-time fluorescence detection sY1291 sites
。
The invention provides it is a kind of for people Y chromosome AZFc areas label site sY129, different from EAA/EMQN recommend
Method, new inspection policies, and provide detectable EAA/EMQN and recommend totally 4 regions, conjunction before and after sY1291 detection ranges
Pcr amplification primer thing of the meter more than 1912kb.This inspection policies combines different follow-up PCR primer analysis means, can be formed not
Same sY1291 loci detection methods, PCR combinations agarose electrophoresis, PCR combination Capillary Electrophoresis as shown in the Examples are glimmering
Light fragments analysis method, real-time fluorescence PCR method etc..SY1291 detection methods and Chinese CFDA is recommended to ratify to be used for EAA/EMQN
AZFc areas classics site（SY255 and sY254）The kit of missing detection is control, is shown by large sample detection, the present invention
There is provided sY1291 site primers strategy compares EAA/EMQN with method and recommends sY1291 loci detection methods, has and AZFc areas
The higher positive uniformity in classical absent region, positive predictive value, negative predictive value and extremely low false positive rate, and contribute to
AZFc range of loss defines.
In this description, the present invention is described with reference to its specific embodiment.But it is clear that it can still make
Various modifications and alterations such as use high score without departing from the spirit and scope of the present invention on follow-up PCR primer analysis means
Distinguish that solubility curve, or the primer to being provided or probe are marked using different types of fluorescein, or recommend in EAA/EMQN
In sY1291 upstreams limited range (0-200bp) new PCR primer or probe are designed using the design fundamentals of PCR primer
Deng.Therefore, specification is recognized is illustrative and be not restrictive for the purpose of the present invention.
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Claims (6)
1. a kind of people's Y chromosome label site sY1291 detection method, it is characterised in that concretely comprise the following steps：
（1）The amplification region of sY1291 sites PCR primer is determined, its strategy is to avoid European andrology association/European molecule
The micro-deleted molecule diagnostic operation guide of Y chromosome of genetic experiment Quality Control coorporative network (EAA/EMQN) combined publication（2013 editions）In
The amplification region of the sY1291 sites PCR primer of recommendation, recommend the large fragment duplicate block of detection zone proximity in EAA/EMQN
Design new specific PCR amplimer in domain；The detection zone I, which is located at EAA/EMQN, to be recommended on sY1291 site primers region
Swim at 38bp；Therefore the detection zone of selection includes：I areas：ChrY:25,504,754-25,505,032, II areas：ChrY:25,
207,966-25,208,244, III areas：ChrY:26,841,711-26,841,989, IV areas：ChrY:27,120,413-27,
120,691；I areas are located at the upstream in EAA/EMQN recommendation sY1291 site primers region with II areas, and III areas and IV areas are located at EAA/
EMQN recommends the downstream in sY1291 site primers region；Recommend sY1291 site primers region 3 ' away from EAA/EMQN in the 5 ' ends in I areas
Hold 38bp；
（2）Pcr amplification primer thing is designed, nucleotides sequence is classified as shown in SEQ ID No.1 and SEQ ID No.2, the pcr amplification primer thing
Cover I areas, II areas, III areas, the IV areas in Yq11.223-Yq11.23 regions；
（3）Using pcr amplification primer thing, people's Y chromosome label site sY1291 is detected, is divided into 3 kinds of situations：
（a）Performing PCR amplification is entered using archaeal dna polymerase, pcr amplification product is examined using 1.0%-2.0% agarose gel electrophoresis
Survey, no PCR amplifications target product is sample sY1291 site deletions, and it is that sY1291 is not lacked there are PCR amplification target product persons；
（b）Fluorescence is carried out to the end of nucleotide sequence 5 ' shown in the end of nucleotide sequence 5 ' shown in SEQ ID No.1 or SEQ ID No.2
Element mark, performing PCR amplification is entered using archaeal dna polymerase, PCR primer carries out capillary using 3500Dx or same type gene sequencer
Electrophoresis fragment analysis, it is that sY1291 sites do not lack PCR amplification target product fluorescence peak persons occur, does not occur PCR amplification targets
Product fluorescence peak person is sY1291 site deletions；
（c）Fluorescence labeling probe is designed, its nucleotide sequence is carried out as shown in SEQ ID No.3 using real time fluorescent PCR method
Detection, there is target fragment PCR amplification curves person and do not lacked for sY1291 sites, do not occur target fragment PCR amplification curve persons
For sY1291 site deletions.
2. people's Y chromosome label site sY1291 according to claim 1 detection method, it is characterised in that use
Pcr amplification primer thing：SEQ ID No.1 and SEQ ID No.2 expand people Yq11.223-Yq11.23 regions I areas, II areas, III
The sequence homology in area and IV areas>99%.
3. people's Y chromosome label site sY1291 according to claim 1 detection method, it is characterised in that step
（1）In situation（a）With situation（b）Reaction system in, pcr amplification primer thing SEQ ID No. 1 and SEQ ID No. 2 work
It is 50nmol/L to 300nmol/L to make concentration；Situation（c）Reaction system in, pcr amplification primer thing SEQ ID No.1 and SEQ
ID No. 2 working concentration is 300nmol/L to 500nmol/L, and probe SEQ ID No.3 working concentration is 150nmol/L
To 250nmol/L.
4. the pcr amplification primer thing of the detection for people's Y chromosome label site sY1291, it is characterised in that nucleotides sequence is classified as
Shown in SEQ ID No.1 and SEQ ID No.2.
5. it is used for the pcr amplification primer thing of people's Y chromosome label site sY1291 detection, its feature as claimed in claim 4
It is, in addition to the probe for people's Y chromosome label site sY1291 detections, its nucleotides sequence are classified as SEQ ID No.3 institutes
Show.
6. the detection method of people's Y chromosome label site sY1291 as described in one of claim 1-3, micro-deleted in people AZF
Application in detection.
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