KR20090112235A - Human chromosome microdeletion analysis chip and idiopathic infertility screening test - Google Patents

Abstract

The present invention relates to a step, a method and a chip for implementing the same in a high-throughput method to determine the presence or absence of AZFa, AZFb, AZFc region microdeletion of human Y chromosome long arm chromosome. Conventional methods for analyzing Y chromosome deletions, such as karyotyping, Southern blotting, and single polymerase chain reaction (PCR), have low-throughput, low specificity and accuracy. ), A long analysis time is required. Accordingly, the present invention provides multiplex PCR for STS markers and genes capable of comprehensive and specific analysis of AZFa, AZFb, and AZFc microdeletion sites in the chromosome interval 5-6 of human Y chromosome long arm. By incorporating high-specificity probes, we attempt to provide an accurate method for determining idiopathic infertility due to Yq chromosome microdeletion.

Description

Human Y chromosome microdeletion diagnosing chip and idiopathic infertility screening analysis therethrough}

The present invention relates to a chip for analyzing microdeletion of Y chromosomes, and more particularly, to determine the presence or absence of AZFa, AZFb, and AZFc region microdeletion of human Y chromosome long arm chromosome in high-throughput manner. It relates to a method and a chip.

The human Y chromosome is 60 Mb (mega base pairs) in length, which is smaller than the 165 Mb of the X chromosome and is about the same size as 21, the smallest of human chromosomes. Most of the Y chromosome, 40 Mb is composed of heterochromatin, the remaining 20 Mb is composed of euchromatin, and there is a pseudoautosomal region, PAR1 and PAR2, on both ends (see Fig. 1). . Sedative pigmentation consists of three areas. The X-transposable region is a 3.4 Mb long region translocated to Y on the X chromosome during evolution and has a high density of repeat sequences. The X-degenerated region has 16 X-associated genes present in a single copy. The ampliconic region contains over 10.2 Mb of large repeat sequences that show more than 99.9% homology, and the eight palindrome structures created by the repeat sequences provide intact genetic information even when mutations occur. Inverted conversion, which copies the genetic information of other palindromes, maintains the function of the gene, but also has the disadvantage of being vulnerable to deletion.

The relationship between the microdeletion of Yq11 and the spermatogenic failure in the long arm (q arm) of the Y chromosome is related to the chromosome Yq of azoospermia in Tiepolo L. et al. Subsequent studies have been reported since observing the microscopic observable deficiency at Yq and suggesting that there might be Azoospermia factor (AZF), a site involved in spermatogenesis (Tiepolo L. et al. Hum . Genet . (1976) 34: 119-124. Vollrath et al. Prepared a deletion map using sequence-tagged site (STS) markers that can be easily amplified by polymerase chain reaction (PCR) in people with abnormalities on the Y chromosome. Genes present have been associated with male infertility (Vollrath D et al. Science (1992) 258: 52-59).

There are four non-overlapping AZFa, AZFb, AZFc, and AZFd (proximal AZFc) sites in the Y chromosome armband. In 75% of patients with AZFa microdeletion, Sertoli cell-only syndrome (SCOS) without congenital living sperm cells is present, and in 25%, partial spermatogenic arrest is associated with severe oligozoospermia. , Microdeletion of AZFb and AZFc have been associated with atypical or rare spermosis, and AZFd microdeletion has been reported to range from sperm count to sperm count, ranging from normal sperm count to abnormal sperm morphology. Reijo R. et al. Nat . Genet . (1995) 10: 383-393). The frequency of microdeletion in AZF region was about 3-11% for AZFa, about 8-9% for AZFb, about 6-13% for AZFbc, and about 65-79% for AZFc. Simoni M. et al. Intl . J of Andrology (2004) 27: 240-249.

The microdeletion of the Y chromosome is closely related to testicular maturation, spermatogenesis, and spermatogenesis, which may cause male infertility, and is a major genetic factor of spermatogenesis. Yq chromosome microdeletion is de It is not inherited from the fertile father to son because it occurs as novo (Latin in the meaning of the new), but once the Yq microdeletion occurs in the sperm, it is inherited to all later male offspring. Diarrhea is also inherited when fertility is achieved through in vitro baby procedures with intracytoplasmic sperm injection (ICSI) (Aittomaki K. et al. Hum . Reproduction (2004) 19 (3): 472-476).

Ten to fifteen percent of fertile couples experience infertility, 40 to 50 percent of which are due to male causes, specifically varicose veins, vas deferens (complete or incomplete obstruction), and hormonal abnormalities. In addition to the causes of abnormal ejaculation, infection, and the like, anti-sperm antibodies on the surface of sperm, morphological malformations of sperm, and the like are examined, and primary tests are conducted. Among the causes of idiopathic male infertility that cannot be identified in the primary test, Yq11 microdeletion is emerging as an important factor.

Microdeletion of AZF (azoospermia factor) sites has been reported in 15-20% of idiopathic atherosclerosis and 7-10% of severe rare spermatosis (McElreavey et al. The Genetic Basis of Male Infertility (1999) 211 pp ).

Conventional methods for analyzing Y chromosome deletions, such as karyotyping, Southern blotting, single-stage STS markers or gene amplification in a single reaction, have low-throughput, low specificity. There have been drawbacks to require help, accuracy and long analysis time. Therefore, the present invention combines multiplex PCR and high-specificity probes for STS markers and genes capable of comprehensive and specific analysis of AZFa, AZFb, and AZFc microdeletion sites of the human Yq chromosome. By providing a high-throughput determination method for Yq chromosome microdeletion and a chip for implementing the same.

Analysis of Y chromosome deletion through 17 gene-based screening at Y5 and 6 of Y chromosome armband is preferable in terms of genotype-phenotype correlation, but DNAs such as enhancers and promoters other than gene exons Since there is a possibility that a microdeletion located at the regulatory region may be missing, the present invention intends to analyze microdeletion including STS markers as well as genes.

In addition, in order to prevent false positives of these sites due to partial microdeletion of AZFa and AZFb regions, by including multiple confirmatory STS markers that can confirm the analysis of genes and essential STS markers that are closely related to microdeletion of these sites. It aims to improve the accuracy of the results.

In order to achieve the object of the present invention, the analysis that can determine the high-throughput (microdeletion) occurring in the AZFa, AZFb and AZFc region of human Y chromosome eguromatin Provide a method.

In detail, the analysis method comprises the steps of: fabricating a chip in which 25 types of probes modified with an amino modifier at a 5'- or 3'-end are spotted;

Separating genomic DNA from a blood or semen sample by magnetic bead method;

Target DNA amplification products containing fluorescent dyes while amplifying 9 AZFa, 10 AZFb, and 9 AZFc sequence tagged-site markers and genes through multiplex PCR (polymerase chain reaction) Preparing a;

Inducing and washing a hybridization reaction between the multiplex PCR reaction product and a probe immobilized on a chip surface;

Simultaneously determining the presence or absence of AZFa, AZFb, AZFc microdeletion by scanning the chip with a laser of a wavelength specific to the fluorescent dye and measuring the fluorescence intensity according to the hybridization reaction result; And

It provides a human Y chromosome microdeletion analysis method comprising all of the above analysis step.

In the above assay method, the STS marker for the AZFa region is sY84, sY86, sY82, sY83, sY87, sY88 and includes DDX3Y as an AZFa microdeletion specific analytical gene;

STS markers for the AZFb region are sY127, sY134, sY129, sY105, sY114, sY143, sY152 and include RBMY as an AZFb microdeletion specific analytical gene;

STS markers for the AZFc region are sY254, sY255, sY153, sY158, sY586, sY554 and include DAZ as an AZFc microdeletion specific analytical gene; And

As an internal control, the present invention provides a high-throughput method for determining the presence or absence of human Y chromosome microdeletion including SRY and ZFY genes.

And, in order to achieve the object of the present invention preferably, at least one DNA selected from the group consisting of DNA (deoxyribonucleic acid) or PNA (peptide nucleic acid) oligonucleotide having a nucleotide sequence of SEQ ID NO: 1 to 50 or Provided are a chip for analyzing a human Y chromosome microdeletion including a PNA probe.

According to a preferred embodiment of the present invention, rapid high-throughput analysis is possible for 19 STS markers and five genes for microdeletion of the chromosome AZFa, AZFb, and AZFc regions of human Y chromosome long arm.

Yq chromosome microdeletion occurs as de novo, but once it is inherited in male offspring, the preferred practice of the present invention allows early diagnosis of idiopathic infertility and appropriate action. It can also be used as a screening test for germ cell cancer genetic factors due to Y chromosome microdeletion.

The present invention also provides a method for discriminating idiopathic infertility suitable for Koreans, including STS markers specific to Korean Y chromosome microdeletion, and a chip accordingly.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings and tables. Configurations shown in the drawings, tables and examples described herein are only one of the most preferred embodiments of the present invention and do not represent all of the technical idea of the present invention, various modifications that can be substituted for them at the time of the present application It should be understood that there may be equivalents and variations.

Also, within the scope of the present invention, various modifications may be made by those skilled in the art to which the present invention pertains, and such modifications are within the scope of the present invention.

< Example  1> Multiplex for analysis of Y chromosome microdeletion PCR  Primer design and synthesis involved in the reaction

Primers included in the multiplex PCR reactions performed earlier for chip reactions for microdeletion high-throughput analysis of Y chromosome armbands include genomic landmark sequences and STS. Entrez (http: //www.ncbi.nlm), which allows cross-searching between the dbSTS database of the US NCBI, which contains sequencing and mapping data for tagged sites, and the database of the National Center for Biotechnology Information (NCBI). .nih.gov / sites / gquery) was used to analyze the sequence of the relevant genes and STS markers detected.

Primers were selected for target regions to enable gene-specific or corresponding STS-specific amplification. Subsequently, primer primer (Primer premier version 5, Premier Biosoft International, Palo Alto, Calif., USA) and DNAiSmax (DNASIS MAX Version 2.7, MiraiBio Group, South San Francisco, CA, USA) programs were selected from the selected primer target sites. The primers were designed by application. The basic requirements for primer selection are as follows; Primer length (18-25 base pairs), melting temperature [Tm (℃), 58-63 ℃], GC content at 3 'end should not be high, and should not be complementary for more than 4 consecutive bases in primer sequence. The formation of the structure was prevented, and the formation of primer dimers was prevented by not allowing three or more identical bases to be positioned consecutively at the 3 'end. In addition, single specific polymorphism (SNP) was not included in the target site to ensure high specificity. In addition, it was verified that the primer sequence does not exhibit cross-reactive characteristics with other genes or STS markers constituting the chip of the present invention using various nucleotide sequence search tools such as the US NCBI sequencing database. . The details of the primers involved in the multiplex PCR reaction performed earlier for the chip reaction for microdeletion high-throughput analysis of the Y chromosome armband are described in Table 1. Primers used in the present invention were synthesized through US IDT (Integrated DNA Technologies, Inc., San Jose, CA, USA). According to a preferred embodiment of the present invention, a fluorescent dye labeled primer is used at the 5 'end or 3' end of the DNA sequence, or a general primer not labeled with the fluorescent dye is used, and the fluorescent dye is released during the multiplex PCR reaction. Both methods of labeling amplification products were identified by inducing labeled deoxyribonucleotide triphosphate to be inserted into the amplification products. Fluorescent dyes used in the present invention are 6-FAM (6-carboxyfluorescein), Cy5, Cy3, Cy5.5, JOE (6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein), Rhodamine Green TAMRA NHS (N-hydroxysuccinimide) Ester, Texas Red. The concentration of the primer was checked with an ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, Maine, USA) and resuspended in tertiary deionized sterile distilled water to a final range of 50-200 pmole / uL and aliquots. Frozen at -70 ° C.

Primer combinations involved in multiplex PCR reactions for microdeletion high-throughput analysis of Y chromosome armbands SEQ ID NO: STS marker or gene name, loci name, AZF region name Primer direction, base sequence (5'-3 ') Length 51 sY84, DYS273, AZFa Forward, AGAAGGGTCTGAAAGCAGGT 20 52 Reverse, AAGAAAGGGTCCCACTGAAT 20 53 sY86, DYS148, AZFa Forward, GTGACACACAGACTATGCTTC 21 54 Reverse, ACAGCAGCCTGAAATGGA 18 55 sY82, DYS272, AZFa Forward, ATCCTGCCCTTCTGAATCTC 20 56 Reverse, CAGTGTCCACTGATGGATGA 20 57 sY83, DYS11, AZFa Forward, CTTGAATCAAAGAAGGCCCT 20 58 Reverse, CAATTTGGTTTGGCTGACAT 20 59 sY87, DYS275, AZFa Forward, TCTGTTGCTTGAAAAGAGGG 20 60 Reverse, ACTGCAGGAAGAATCAGCTG 20 61 sY88, DYS276, AZFa Forward, TTGTAATCCAAATACATGGGC 21 62 Reverse, CACCCAGCCATTTGTTTTAC 20 63 DBY, DDX3Y, AZFa Forward, TCAGACAGTTTAGTTGGTTACTTCC 25 64 Reverse, AAGACACATCCTCAGCTAATCTTT 24 65 sY127, DYS218, AZFb Forward, GGCTCACAAACGAAAAGAAA 20 66 Reverse, CTGCAGGCAGTAATAAGGGA 20 67 sY134, DYS224, AZFb Forward, GTCTGCCTCACCATAAAACG 20 68 Reverse, ACCACTGCCAAAACTTTCAA 20 69 sY129, DYS220, AZFb Forward, AGCTTCAGGAGGTTCAAAAC 20 70 Reverse, GTCCCACTCCTATGGACTTCAC 22 71 sY105, DYS201, AZFb Forward, AAGGGCTTCTTCTCTTGCTT 20 72 Reverse, AGGGAGCTTAAACTCACCGT 20 73 sY114, DYS206, AZFb Forward, TGCACTCATGGAGACAACAG 20 74 Reverse, AACCAGGGTTTTCACTGAAA 20 75 sY143, DYS231, AZFb Forward, GCAGGATGAGAAGCAGGTAG 20 76 Reverse, CCGTGTGCTGGAGACTAATC 20 77 sY152, DYS236, AZFb Forward, AAGACAGTCTGCCATGTTTCA 21 78 Reverse, ACAGGAGGGTACTTAGCAGT 20 79 RBMY, multiple, AZFb Forward, ATGATGGCTACGGTGAGGC 19 80 Reverse, GGCGGATTCCTTTGGTCTTT 20 81 sY254, DAZ, AZFc Forward, GGGTGTTACCAGAAGGCAAA 20 82 Reverse, GAACCGTATCTACCAAAGCAGC 22 83 sY255, DAZ, AZFc Forward, GTTACAGGATTCGGCGTGAT 20 84 Reverse, CTCGTCATGTGCAGCCAC 18 85 sY153, DYS237, AZFc Forward, GCATCCTCATTTTATGTCCA 20 86 Reverse, CAACCCAAAAGCACTGAGTA 20 87 sY158, DYS241, AZFc Forward, CTCAGAAGTCCTCCTAATAGTTCC 24 88 Reverse, ACAGTGGTTTGTAGCGGGTA 20 89 sY586, G63907, AZFc Forward, GTGTGGCACATATGCCTATAAA 22 90 Reverse, TTGGTACATCCAGATGCAGAT 21 91 sY554, DAZ 1-4, AZFc Forward, GTTTCATGGGAAGTTGCTGGT 21 92 Reverse, CTTGGAGAAGTGAAATGCTTGTG 23 93 DAZ, DAZ 1-4, AZFc Forward, GACATCCACGTCATTAACAAACG 23 94 Reverse, AAGCTGCTTTGGTAGATACGGTT 23 95 sY14, SRY, not applicable Forward, GAATATTCCCGCTCTCCGGA 20 96 Reverse, GCTGGTGCTCCATTCTTGAG 20 97 sY18, ZFY, not applicable Forward, ATTTGGCTTGGAGTCAGTCA 20 98 Reverse, TATGAACCAATCTCACCCCC 20 99 SHGC-35340, ZFY, not applicable Forward, GTGTTTGCATTGCGACCAC 19 100 Reverse, TTTAAAGCCTGAGGCATCTGT 21

< Example  2> from clinical specimen Genomic DNA  detach

Genomic DNA was extracted from the peripheral blood sample by magnetic bead method. Peripheral blood in a volume of 0.01-0.02 mL was added to the 96-well or 384-well plate by number of samples and placed in the module. An 8-channel pipetting head is then fitted with a pipette of a pipette tip rack and then aspirates 500 uL of a buffer mix to dissolve the sample, so that each well of the lysis plate It was added to and reacted for 10 minutes. During the reaction, the reaction solution was mixed by pipetting up and down 15 times to promote the reaction. Thereafter, 70 uL of the reaction solution containing the magnetic beads was added, mixed with a pipette, and allowed to stand at room temperature for 1 minute, and then a magnetic separator was operated and reacted for an additional 2 minutes. The supernatant except the beads was eluted with a waste reservoir and the magnetic separator was shut down. Thereafter, 500 uL of lysis buffer containing no protease K was added and mixed with a pipette to uniformly spread the magnetic beads. After adding 50 uL of the purification buffer and mixing with a pipette, the mixture was allowed to stand at room temperature for 1 minute, and then a magnetic separator was operated and reacted for another 2 minutes. As above, the supernatant except the beads was eluted with a waste reservoir and the magnetic separator was stopped. Thereafter, 500 uL of washing buffer was added and the magnetic separator was operated twice to remove impurities. In order to recover the DNA as a final process, 100 uL of the elution buffer was loaded, and the pipette was repeatedly processed up and down 50 times for 5 minutes at room temperature, and the supernatant containing pure DNA was recovered and stored until the multiplex PCR reaction.

< Example  3> multiplex PCR Y chromosome microdeletion analysis chip through chip )of target  Gene and STS markers  Amplification

Prior to high-throughput chip analysis, target genes and STS markers were amplified by multiplex PCR reactions with genomic DNA isolated from the sample as a template. Thereafter, hybridization reactions with selective probes immobilized with the amplified reaction products on a chip can be used to determine the presence or absence of microdeletion of the Y chromosome long arm.

Multiplex PCR was performed to specifically amplify 9-10 genes / STS markers in a single reaction in the same PCR tube, including internal control for each AZF sub-region. . Internal controls included in the electrical multiplex PCR are sex determining region Y (SRY) and zinc finger protein (Y-linked). ZFY is a male-specific sequence that is not amplified in female samples. SRY demonstrates the presence of the testicular determining factor (TDF) sequence of the Y chromosome forearm, allowing the identification of XX males without ZFY. These internal controls are highly desirable as the control to be amplified regardless of Yq11 microdeletion. The compositional composition and PCR reaction conditions of the pre-made PCR mastermix of the multiplex PCR for each AZF sub-region described in this embodiment are shown in Tables 2, 3, and 4 below.

AZFa 9 Multiplex PCR Mastermix Composition and PCR Conditions  PCR reaction solution composition  Volume (uL)  PCR reaction conditions  Deionized Tertiary Sterilized Distilled Water  6  Initial denaturation  95 ° C, 15 minutes  1 time AZFa 9 primer premix; Table 1 SEQ ID NO (forward / reverse primer ) 1/2, 3/4, 5/6, 7/8, 9/10/10, 11/12, 13 / 14, 45/46, 47/48      4      Denaturation      95 ° C, 30 seconds         Episode 37  2X Multiplex PCR Premix  15  Annealing  57 ° C, 40 seconds  Template DNA (40 ng or more)  5  Extension  72 ° C., 40 seconds  Final volume  30   Extension  72 ° C., 7 minutes  1 time

AZFb 10 Multiplex PCR Mastermix Composition and PCR Conditions  PCR reaction solution composition  Volume (uL)  PCR reaction conditions  Deionized Tertiary Sterilized Distilled Water  4.6  Initial denaturation  95 ° C, 15 minutes  1 time AZFb 10 primer premix; Table 1 SEQ ID Number (forward / reverse primer) 15/16, 17/18, 19 / # 20, # 21 / # 22, # 23 / # 24, # 25 / # 26, # 27 / 28, 29/30, 45/46, 47/48      5.4      Denaturation      95 ° C, 30 seconds         Episode 37  2X Multiplex PCR Premix  15  Annealing  58 ° C, 30 seconds  Template DNA (40 ng or more)  5  Extension  72 ° C., 40 seconds  Final volume  30   Extension  72 ° C., 5 minutes  1 time

AZFc 9 Multiplex PCR Mastermix Composition and PCR Conditions  PCR reaction solution composition  Volume (uL)  PCR reaction conditions  Deionized Tertiary Sterilized Distilled Water  5.2  Initial denaturation  95 ° C, 15 minutes  1 time AZFc 9 primer premix; Table 1 SEQ ID NO (forward / reverse primer ) 31/32, 33/34, 35/36 , 37/38, 39/40, 41/42, 4 3 / 44, 45/46, 47/48      4.8      Denaturation      95 ° C, 30 seconds         Episode 37  2X Multiplex PCR Premix  15  Annealing  56 ℃, 40 seconds  Template DNA (40 ng or more)  5  Extension  72 ° C., 40 seconds  Final volume  30   Extension  72 ° C., 7 minutes  1 time

The final concentration of the PCR buffer in the 2X multiplex PCR premix was 50 mM KCl, 3.5 mM MgCl ₂ , 10 mM Tris-HCl, pH 8.2, 2.5 unit Taq polymerase, 300 uM dNTPs (Boehringer Mannheim, Mannheim, Germany). ), 10 mg / mL bovine serum albumin.

Sense primers of the Y chromosome microdeletion assay primers included in the multiplex PCR mastermix were synthesized by labeling Cy3 fluorescent dyes at their 5 'ends (Integrated DNA Technologies, Inc., Coralville, IA, USA). ).

In addition, the chip kit for Y chromosome microdeletion analysis, which is the object of the present invention, is prepared by adding Cy3 fluorescent dye-labeled deoxycytosine triphosphate (Cy3-dCTP) as a nucleotide constituting an amplification product during PCR. The sensitivity and specificity of the reaction could be improved. For the AZF sub-region multiplex PCR reaction using Cy3-dCTP, primer premix without fluorescent dye was used, and the final concentrations of the reactants constituting the PCR buffer were 50 mM KCl, 3.5 mM MgCl ₂ , and 10 mM Tris-HCl. , pH 8.2, 2.5 unit Taq polymerase, 300 uM dATP, 300 uM dGTP, 300 uM dTTP, 25 uM dCTP, 275 uM Cy3-dCTP (FluoroLink Cy3-dCTP, Amersham Pharmacia Biotech AB, Piscataway, NJ, USA) , 10 mg / mL bovine serum albumin.

< Example  4> Y chromosome microdeletion analysis chip ( chip Make up) Probe ( probe A) design and synthesis

Primer premier version 5, Premier Biosoft International, Palo Alto, to select specific probe target sites in the amplicon of the gene or STS markers by the internal control located on the Yp chromosome and the AZF sub-region of the Yq chromosome , CA, USA), DNASIS MAX Version 2.7, MiraiBio Group, South San Francisco, CA, USA, or OligoArray 2.0 (http://cbr-rbc.nrc-cnrc.gc.ca) The application of the program was designed and the details are shown in Table 5. Amino modifiers were labeled at the 5 'or 3' end of the DNA or PNA probe sequence of interest to synthesize the gene or STS marker-specific probe (MWG-Biotech AG, Ebersberg, Germany). When modifying the amino modifier at the 5 'end of the probe, 6 to 15 spacers were added to promote the reaction efficiency. No spacer was used when modifying the amino modifier at the 3 'end of the probe. The amine groups of the probe have the properties of primary amine groups and are attached to the aldehyde-activated or carboxyl-activated chip surface. The concentration of the probe was converted to absorbance values at 260 nm, and it was checked for impurities by Maldi-TOF (MALDI-TOF, Matrix Assisted Laser Desorption / Ionization Time-of-Flight), and high performance liquid chromatography (HPLC) After pure purification through liquid chromatography) was prepared so that the final concentration in sterile tertiary distilled water 100-300 pM.

Probe combinations that make up the Y chromosome microdeletion analysis chip Sequence listing number STS marker or gene name, loci name, AZF region name Probe orientation, nucleotide sequence (5'-3 ') Length One sY84, DYS273, AZFa Forward, ATGGGCTGGAGATTCAGTGGG 21 2 Forward, GGGCTGGAGATTCAGTGGGAC 21 3 sY86, DYS148, AZFa Forward, TCTCCTTGGGTTGTAGATGCTG 22 4 Forward, CCAGGGCTGGTTCCTCCT 18 5 sY82, DYS272, AZFa Reverse, GGAGATTCAGAAGGGCAGGATG 22 6 Reverse, GACACTGGAGATTCAGAAGGGCA 23 7 sY83, DYS11, AZFa Reverse, GATGTTTAGGGCTGCTGTTGCT 22 8 Reverse, CAGATGTTTAGGGCTGCTGTTG 22 9 sY87, DYS275, AZFa Reverse, TGAGGCCTGGGCAAAGACAAG 21 10 Forward, CTTGTCTTTGCCCAGGCCTCA 21 11 sY88, DYS276, AZFa Forward, GTAAAACAAATGGCTGGGTGC 21 12 Forward, GTAAAACAAATGGCTGGGTGCA 22 13 DBY, DDX3Y, AZFa Forward, TTAGCGATATTGACATGGGAGAA 23 14 Reverse, GTCAATATCGCTAAACTGCCAAGA 24 15 sY127, DYS218, AZFb Forward, TGGAATCTACCAAAGCCCACTGT 23 16 Reverse, GTGGGCTTTGGTAGATTCCAGT 22 17 sY134, DYS224, AZFb Forward, CATTCTACTTGAAGCGTTCTGTGAC 25 18 Reverse, CATTCTACTTGAAGCGTTCTGTGA 24 19 sY129, DYS220, AZFb Forward, GCAGCAACACGAATGTAGCTAGA 23 20 Reverse, TCTAGCTACATTCGTGTTGCTGC 23 21 sY105, DYS201, AZFb Forward, AATAAAGGGGAAACATTGAACCC 23 22 Forward, GGGAAACATTGAACCCCAGAGA 22 23 sY114, DYS206, AZFb Forward, TGGAGACAACAGTCTTGCCATAAG 24 24 Forward, TGGAGACAACAGTCTTGCCATAAGT 25 25 sY143, DYS231, AZFb Forward, GTCATATTTGGCTTCTGCTTGG 22 26 Reverse, GCTTTATCTAGCAACAGCCCAA 22 27 sY152, DYS236, AZFb Forward, AAGACAGTCTGCCATGTTTCAGC 23 28 Forward, CTGCCATGTTTCAGCTCTTTGAC 23 29 RBMY, multiple, AZFb Forward, GCCTCGGATGTCTTATGGTGGA 22 30 Reverse, TCCACCATAAGACATCCGAGGC 22 31 sY254, DAZ, AZFc Forward, AGGGCTGCTGGTCATTCGG 19 32 Reverse, CGAATGACCAGCAGCCCTTT 20 33 sY255, DAZ, AZFc Forward, CAGGATTCGGCGTGATTTGG 20 34 Reverse, CAAATCACGCCGAATCCTGTA 21 35 sY153, DYS237, AZFc Forward, GAACTCGCTTTATACTCAGTGCTTT 25 36 Reverse, AAGCACTGAGTATAAAGCGAGTTCT 25 37 sY158, DYS241, AZFc Forward, CTCACTATCTACCCGCTACAAACC 24 38 Reverse, CAGTGGTTTGTAGCGGGTAGATAGT 25 39 sY586, G63907, AZFc Forward, AAGCAATGGCTTCTCCAAATTATG 24 40 Reverse, AAAAGGCACTGTTTCTCCCATG 22 41 sY554, DAZ 1-4, AZFc Reverse, ATTCAAAACCAGCAACTTCCCA 22 42 Forward, GTTTCATGGGAAGTTGCTGGTTT 23 43 DAZ, DAZ 1-4, AZFc Reverse, TCACTGAACCGTATCTACCAAAGC 24 44 Forward, AAAATGTGCTAGGATTAGGGCAG 23 45 sY14, SRY, not applicable Reverse, ATGGGTCGCTTCACTCTATCCTG 23 46 Forward, TCCAGGATAGAGTGAAGCGACC 22 47 sY18, ZFY, not applicable Reverse, GGTGACTGACTCCAAGCCAAAT 22 48 Reverse, AAGTCCAGCATTTCTGCTTTGG 22 49 SHGC-35340, ZFY, not applicable Reverse, ATGGCGACTTAGAACAAATGGG 22 50 Reverse, AATATGGCGACTTAGAACAAATGG 24

< Example  5> Y chromosome microdeletion analysis chip ( chip Production

The Y chromosome microdeletion analysis chip according to the preferred embodiment of the present invention is six STS markers and one gene for AZFa region analysis, seven STS markers and one gene for AZFb region analysis, and AZFc region analysis. Six STS markers, one gene, and a high-specificity probe for the SRY and ZFY genes as immobilized controls were immobilized. A grid of high-specificity probes for Y chromosome microdeletion analysis consisting of 18 STS markers and 5 genes was designed, and 8 grids were included on one chip substrate, and an 8 well hybridization reaction chamber (8 A well hybridization reaction chamber was designed to simultaneously analyze 8 individual samples (see FIG. 2). After thawing at 70 ° C., the probe stock was stored at room temperature, and then diluted with deionized sterile tertiary distilled water to a final concentration of 10 nM and used as a working stock. Each probe with 50-fold dilution of working stock was subjected to a 1: 5-10 ratio (v / v) with 3X SSC spotting solution (500 mM NaCl, 3 mM sodium citrate, 1.5 MN, N, N-trimethylglycine, pH 6.8). Mixing was performed so that the concentration range of the probe dispensed into the final 96 well plate was 20-40 pmole / uL. The plate was mounted on a Microssys 5100 microarrayer (Cartesian Technologies, Ann Arbor, MI, USA) from which aldehyde-, thiocyanate-, amine-activated glass slides (CEL associates Inc., Houston, TX, USA) or epoxy- Two probes were spotted in order on the activated plastic chip surface. The average diameter of the spots is 80-150 micrometers and the distance between spots was maintained at 400-500 micrometers to minimize cross-talk effects between spots. Chip fabrication was carried out in a class 10,000 room maintaining 74% humidity. The probe spotted chips were baked at 120 ° C. for 1 hour, and then washed in 0.25% SDS (Sodium dodecyl sulfate) solution for 3 minutes and washed again with sterile tertiary distilled water. The chip was then reacted with a solution containing 0.2% sodium borohydride (NaBH 4) to block the probe. After washing twice with 3 distilled water, the water was removed and stored in a desiccator (dessicator) until the point of use.

< Example  6> Y chromosome microdeletion analysis chip ( chip ) Hybridization ( hybridization ) Response and result analysis

A multiplex PCR reaction for Y chromosome microdeletion high-throughput analysis according to one preferred embodiment of the present invention was performed for each AZF sub-region, followed by deionization in a total of 39 uL of 13 uL of each PCR reaction product. 39 μL of third sterile distilled water was added thereto, followed by thermal denaturation at 95 ° C. for 5 minutes, preservation on ice for 5 minutes, and spin down with a centrifuge. An 8 well hybridization chamber was placed on the chip surface and the well top was covered with a well cover. Thereafter, 60 uL of a hybridization reaction solution (3X SSC, 0.1% SDS, 0.2 mg / mL bovine serum albumin, pH 7) was preheated to the reaction mixture solution at 50 ° C. in the reaction mixture solution. After the addition and mixing, the reaction solution mixture was injected through the hole of the well cover, and care was taken not to generate bubbles. After fixing the chamber lid, the hybridization reaction was conducted at 50 ° C. for 30 minutes to induce specific nucleotide complementary binding between the probe on the chip surface and the multiplex PCR reaction product. The well cover of the chip surface where the hybridization reaction was completed was removed, the chip was immersed in the washing buffer 1 (0.1X SSC, 0.05% SDS) solution, washed with stirring at 2,000 rpm for 2 minutes, and repeated. After washing with washing solution 2 (2X SSC, 0.1% SDS) solution at 2,000 rpm for 2 minutes (washing) was repeated. Thereafter, the chips were dried by immersion in deionized tertiary sterile distilled water twice and centrifuged at 1,000 rpm. The chip was read using a ScanArray Lite (Packard Instrument Co., Meriden, CT, USA) scanner, and the mean fluorescence intensity and standard of the positive control spots using the analysis software (QuantArray 2.0). Signal-to-noise (S / R) ratio was obtained by comparing the error with the values around the spa and then scored as positive if this value was 5 or more (see FIG. 3).

Figure 1 is a schematic diagram showing the position in the chromosome of the analysis target gene and STS markers included in the Y chromosome microdeletion analysis chip of the present invention. The internal control SRY and ZFY genes are present at interval 1 of the Y chromosome forearm, the AZFa microdeletion is at interval 5 in the isochromosome of Y chromosome, and AZFb is at interval 5-6 linkage and AZFc is interval 6 It is present at the site. In addition, the genes for microdeletion analysis and STS markers included in the present invention are shown for each AZF sub-region.

Figure 2 is a schematic diagram of the Y chromosome microdeletion analysis chip manufactured according to the present invention, and shows a chip grid in which probes are arranged for each microdeletion site of AZFa, AZFb, and AZFc. An 8-well hybridization reaction chamber in which hybridization reaction between the labeled target DNA and the probes arranged on the chip is performed is shown schematically.

3, after fabrication of the chip in which the probe is immobilized according to the present invention, the target gene and STS markers are amplified by multiplex PCR reaction with genomic DNA isolated from the sample as a template, and hybridized with selective probes on the chip ( This is a flowchart showing the main steps of the chip analysis process for analyzing the presence or absence of Y chromosome microdeletion by performing a hybridization reaction.

Figure 4 shows the clinical results of the multiplex PCR for each AZF sub-region for the chip reaction for Y chromosome microdeletion analysis according to the present invention. The target DNA distribution of 7 clinical specimens can be confirmed through 9 multiplex PCR reactions including an internal control for microdeletion analysis of AZFa region. In addition, it was confirmed that target genes and STS markers were specifically amplified by 10 multiplex PCR reactions in AZFb region and 9 in AZFc region. M is the DNA molecular weight size marker, P is the positive control, and S number is the sample serial number.

5 shows a chip scan image (left picture) and a chip grid (right picture) showing the results of analyzing AZF target gene and STS markers for Y chromosome microdeletion high-throughput analysis according to the present invention. As a result of clinical cases of AZFb-c microdeletion, sY127 and sY134 STS markers and RBMY genes were deleted, and sY105 and sY152 were amplified while sY114 and sY143 markers were deleted. . In addition, AZFc microdeletion in which the sY254 and sY255 markers and the DAZ gene are deleted can be confirmed.

Figure 6 shows a chip scan image (left picture) and chip grid (right picture) showing the results of the analysis of the AZF target gene and STS markers for Y chromosome microdeletion high-throughput analysis according to the present invention, the AZFa microdeletion This is the result of clinical case analysis. The AZFa microdeletion can be confirmed by sY84 and sY86 STS markers and DDX3Y (DBY) genes, sY82 and sY88 are amplified and sY83 and sY87 breakpoint markers are deleted.

Figure 7 shows a chip scan image (left picture) and chip grid (right picture) showing the results of the analysis of the AZF target gene and STS markers for Y chromosome microdeletion high-throughput analysis according to the present invention, the AZFb microdeletion This is the result of clinical case analysis. AZFb microdeletion can be confirmed by the deletion of the sY127, sY129, sY134 markers and the RBMY gene, and the deletion of the sY114 and sY143 breakpoint markers as sY105 and sY152 are amplified.

8 shows a chip scan image (left picture) and a chip grid (right picture) showing the results of analyzing the AZF target gene and STS markers for Y chromosome microdeletion high-throughput analysis according to the present invention. This is the result of clinical case analysis. The AZFc microdeletion can be confirmed by the deletion of the sY254, sY255, sY153, and sY586 STS markers and the DAZ gene.

<110> PARK, MinKoo <120> Human Y chromosome microdeletion diagnosing chip and idiopathic          infertility screening analysis therethrough <160> 50 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sY84 probe <400> 1 atgggctgga gattcagtgg g 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sY84 probe <400> 2 gggctggaga ttcagtggga c 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sY86 probe <400> 3 tctccttggg ttgtagatgc tg 22 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> sY86 probe <400> 4 ccagggctgg ttcctcct 18 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sY82 probe <400> 5 ggagattcag aagggcagga tg 22 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sY82 probe <400> 6 gacactggag attcagaagg gca 23 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sY83 probe <400> 7 gatgtttagg gctgctgttg ct 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sY83 probe <400> 8 cagatgttta gggctgctgt tg 22 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sY87 probe <400> 9 tgaggcctgg gcaaagacaa g 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sY87 probe <400> 10 cttgtctttg cccaggcctc a 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sY88 probe <400> 11 gtaaaacaaa tggctgggtg c 21 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sY88 probe <400> 12 gtaaaacaaa tggctgggtg ca 22 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> DBY gene probe <400> 13 ttagcgatat tgacatggga gaa 23 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DBY gene probe <400> 14 gtcaatatcg ctaaactgcc aaga 24 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sY127 probe <400> 15 tggaatctac caaagcccac tgt 23 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sY127 probe <400> 16 gtgggctttg gtagattcca gt 22 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> sY134 probe <400> 17 cattctactt gaagcgttct gtgac 25 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> sY134 probe <400> 18 cattctactt gaagcgttct gtga 24 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sY129 probe <400> 19 gcagcaacac gaatgtagct aga 23 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sY129 probe <400> 20 tctagctaca ttcgtgttgc tgc 23 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sY105 probe <400> 21 aataaagggg aaacattgaa ccc 23 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sY105 probe <400> 22 gggaaacatt gaaccccaga ga 22 <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> sY114 probe <400> 23 tggagacaac agtcttgcca taag 24 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> sY114 probe <400> 24 tggagacaac agtcttgcca taagt 25 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sY143 probe <400> 25 gtcatatttg gcttctgctt gg 22 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sY143 probe <400> 26 gctttatcta gcaacagccc aa 22 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sY152 probe <400> 27 aagacagtct gccatgtttc agc 23 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sY152 probe <400> 28 ctgccatgtt tcagctcttt gac 23 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RBMY gene probe <400> 29 gcctcggatg tcttatggtg ga 22 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RBMY gene probe <400> 30 tccaccataa gacatccgag gc 22 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> sY254 probe <400> 31 agggctgctg gtcattcgg 19 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sY254 probe <400> 32 cgaatgacca gcagcccttt 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sY255 probe <400> 33 caggattcgg cgtgatttgg 20 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sY255 probe <400> 34 caaatcacgc cgaatcctgt a 21 <210> 35 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> sY153 probe <400> 35 gaactcgctt tatactcagt gcttt 25 <210> 36 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> sY153 probe <400> 36 aagcactgag tataaagcga gttct 25 <210> 37 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> sY158 probe <400> 37 ctcactatct acccgctaca aacc 24 <210> 38 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> sY158 probe <400> 38 cagtggtttg tagcgggtag atagt 25 <210> 39 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> sY586 probe <400> 39 aagcaatggc ttctccaaat tatg 24 <210> 40 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sY586 probe <400> 40 aaaaggcact gtttctccca tg 22 <210> 41 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sY554 probe <400> 41 attcaaaacc agcaacttcc ca 22 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sY554 probe <400> 42 gtttcatggg aagttgctgg ttt 23 <210> 43 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DAZ gene probe <400> 43 tcactgaacc gtatctacca aagc 24 <210> 44 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> DAZ gene probe <400> 44 aaaatgtgct aggattaggg cag 23 <210> 45 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sY14 probe <400> 45 atgggtcgct tcactctatc ctg 23 <210> 46 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sY14 probe <400> 46 tccaggatag agtgaagcga cc 22 <210> 47 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sY18 probe <400> 47 ggtgactgac tccaagccaa at 22 <210> 48 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sY18 probe <400> 48 aagtccagca tttctgcttt gg 22 <210> 49 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ZFY gene probe <400> 49 atggcgactt agaacaaatg gg 22 <210> 50 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ZFY gene probe <400> 50 aatatggcga cttagaacaa atgg 24  

Claims (10)

A high-throughput assay for microdeletion of human Y chromosomal eguromatin in the AZFa, AZFb and AZFc sites. The method of claim 1, further comprising: manufacturing a chip in which 25 types of probes modified with an amino modifier at a 5′- or 3′-end are spotted; Separating genomic DNA from a blood or semen sample by magnetic bead method; Target DNA amplification products containing fluorescent dyes while amplifying 9 AZFa, 10 AZFb, and 9 AZFc sequence tagged-site markers and genes through multiplex PCR (polymerase chain reaction) Preparing a; Inducing and washing a hybridization reaction between the multiplex PCR reaction product and a probe immobilized on a chip surface; Simultaneously determining the presence or absence of AZFa, AZFb, AZFc microdeletion by scanning the chip with a laser of a wavelength specific to the fluorescent dye and measuring the fluorescence intensity according to the hybridization reaction result; Human Y chromosome microdeletion analysis method comprising all of the above analysis step. The method according to claim 1 or 2, wherein the STS markers for the AZFa region are sY84, sY86, sY82, sY83, sY87, sY88 and include DDX3Y as an AZFa microdeletion specific assay gene; STS markers for the AZFb region are sY127, sY134, sY129, sY105, sY114, sY143, sY152 and include RBMY as an AZFb microdeletion specific analytical gene; STS markers for the AZFc region are sY254, sY255, sY153, sY158, sY586, sY554 and include DAZ as an AZFc microdeletion specific analytical gene; And Analytical method for determining the presence or absence of human Y chromosome microdeletion including the SRY, ZFY gene as a high-throughput as an internal control (high-throughput). According to claim 1 to 3, Human Y chromosome microdeletion analysis comprising at least one DNA probe (probe) selected from the group consisting of DNA oligonucleotide (deoxyribonucleic acid oligonucleotide) having a nucleotide sequence of SEQ ID NO: 1 to 50 DNA chip. 4. The human Y chromosome microdeletion analysis according to claim 1, further comprising one or more PNA probes selected from the group consisting of PNA oligonucleotides having a nucleotide sequence of SEQ ID NOs: 1 to 50. 5. PNA chip for. The method of claim 1, wherein the aldehyde-, thiisocyanate-, or carboxyl-activated glass slide is used, or an epoxy-activated plastic ( Human Y chromosome microdeletion analysis chip composed of plastic chips. The method of claim 1, wherein the idiopathic infertility is determined using a Y chromosome microdeletion analysis chip. 6. The method of claim 1, wherein the gene for germ cell cancer genetic causes due to Y chromosome microdeletion is analyzed using a Y chromosome microdeletion analysis chip. The method of claim 1, further comprising: specifically amplifying the sY84, sY86, sY82, sY83, sY87, sY88 STS markers and the DDX3Y gene for AZFa microdeletion analysis; Specifically amplifying the sY127, sY134, sY129, sY105, sY114, sY143, sY152 STS markers and RBMY genes for AZFb microdeletion analysis; Specifically amplifying the sY254, sY255, sY153, sY158, sY586, sY554 STS markers and the DAZ gene for AZFc microdeletion analysis; And Multiplex PCR kit for human Y chromosome microdeletion analysis comprising a primer and a reagent specifically amplifying the SRY, ZFY gene as an internal control in the multiplex PCR composition for each AZF sub-region. According to claim 1 to 3, STS markers included to determine the presence or absence of microdeletion of the AZFa, AZFb, AZFc region present in the human Y chromosome long sedative chromosome interval 5-6 is not only microdeletion itself but also the result A human Y chromosome microdeletion analysis method including a verifiable marker at the same time and analyzing the presence or absence of deletion of genes present in each AZF sub-region in addition to the anonymous STS marker.

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