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1 HEREDITAS (Beijing) , 30(12): ISSN 技术与方法 DOI: /SP.J MLPA- Y 周大文 1, 管翌华 2, 许淼 1, 颜景斌 1, 黄英 1, 张敬之 1, 任兆瑞 1.,, ; 2., : 为了探讨 MLPA- 微阵列技术用于检测性染色体异常的可行性和精确性, 针对 Y 染色体上的 3 个基因 TSPY(p11.2) PRY(q11) 和 RBMY(q11.2) 设计 MLPA 探针, 应用 MLPA- 微阵列技术对 15 例已知染色体核型的样品进行检测, 将检测结果与各样品核型分析和 PCR 的检测结果进行对照和比较结果表明, MLPA- 微阵列技术对上述各基因位点的检测结果与样品染色体核型基本吻合, 特别是对二例核型分析没有获得染色体结构异常信息的样品, MLPA- 微阵列技术检测出 Y 染色体微小的缺失或指示某些未知染色体片段的信息, 并与 PCR 检测结果完全相符表明文章报道的 MLPA- 微阵列技术能够检测核型分析无法分辨的微小变化和异常, 显示 MLPA- 微阵列技术在染色体异常分析中具有很高的检测效率和准确性, 相对于染色体核型分析具有明显的优势, 在临床染色体病诊断中具有较大的应用前景 : MLPA; 微阵列 ; 染色体异常 1 Preliminary application of MLPA-based array in detecting Y chromosome abnormalities ZHOU Da-Wen 1, GUAN Yi-Hua 2, XU Miao 1, YAN Jing-Bin 1, HUANG Ying 1, ZHANG Jing-Zhi 1, REN Zhao-Rui 1 1. Shanghai Institute of Medical Genetics, School of Medicine, Shanghai Jiao Tong University, Shanghai , China; 2. Center Laboratory, Shanghai Children s Hospital, Shanghai Jiao Tong University, Shanghai , China Abstract: To explore the feasibility and accuracy of MLPA-based array (Array-MLPA) in detecting sex chromosome abnormalities, MLPA probes were designed to target against three gene loci, TSPY (p11.2), PRY (q11), and RBMY (q11.2) in human Y chromosome. Array-MLPA approach was applied to test abnormalities of Y chromosome in 15 patient samples with known karyotypes. The data were compared with karyotyping and PCR analyses. The results showed that the copy number of each site detected by Array-MLPA was basically consistent with karyotyping analysis. Moreover, small deletions of chromosomes that were not found by routine karyotyping analysis were identified by the approach described, which fully agreed with PCR analysis, indicating that Array-MLPA was able to detect small abnormalities of chromosomes that cannot be found by karyotyping analysis. Compared to the routine karyotyping method, Array-MLPA has the advantages of high 收稿日期 : ; 修回日期 : 基金项目 : (863 )( 2007AA02Z400), ( 2006BAI05A07, 2006BAI05A08), ( 2008ZD004), ( SHDC ), ( B204) [Supported by the grants from the National High Technology Research and Development Program of China (No. 2007AA 02Z400), the National Key Technology Research and Development Program of China (No. 2006BAI05A07, 2006BAI05A08), Joint Key Project of Shanghai Health Bureau (No. 2008ZD004)], Joint Key Project of New Frontier Technology in Shanghai Municipal Hospitals (No. SHDC ), and the State and Shanghai Key Discipline (No.B204)]. 作者简介 : (1982 ),,,, zdwmail2006@yahoo.com.cn 通讯作者 : (1945 ),,,, zhrren@sjtu.edu.cn; (1959 ),,,, jzhang38@hotmail.com

2 1630 HEREDITAS (Beijing) efficiency and reliability in chromosomal analysis, which has great potential in clinical application of diagnosis of chromosome abnormalities. Keyword: MLPA; array; chromosome abnormality,,,, (Fluorescence in situ hybridization, FISH), 20 kb~5 Mb,, FISH (Multiplex ligation-dependent probe amplification, MLPA) Schouten [1] 2002, 1 bp DNA, PCR FISH Southern blotting, MLPA- (Array-MLPA) MLPA (Array), MLPA, [2] MLPA- 200 DMD dystrophin, DNA [3] MLPA-,, 研究对象 15 1, DNA Array-MLPA 表 1 检测样品及染色体核型 Table 1 Samples and the karyotypes Sample S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S 主要试剂 Karyotype 46, XX 46, XX 46, XX /, mar / / Yq-? DNA /, MLPA (MLPA buffer buffer PCR buffer dilution buffer PCR ) (1 blocker 1 SSPE 3 SSPE 5 SSPE) Pamgene 主要仪器和分析软件 PCR Eppendorf, Pamgene-FD10 Olympus, MLPA- Pamgene, 4, [2, 3] BioNavigator Pamgene 外周血 DNA 的提取 2~3 ml(acd ), - DNA

3 12 : MLPA- Y 1631 DNA 260 nm 280 nm A 260 A 280, A 260 /A 280 DNA MLPA 探针 Y 3 TSPY(p11.2) PRY(q11) RBMY(q11.2) Schouten [1], (Tag sequence) (Stuffer sequence),, [3], (Universal primers), : PamF: CCAGTGAATTGTAATACGAC; PamR: CTCACTAAAGGGAACAAAAG, (Target sequence), GenBank 5 4 nmol/l MLPA 反应 MLPA Schouten [1], 5 μl 100 ng/μl DNA 98 5 min,, 1.5 μl 1.5 μl MLPA buffer 95 5 min h, 22 μl min 98 5min, 20 μl PCR, 10 μmol/l PAM-F( Cy5) PAM-R 1 μl, PCR 95 5 min, s s 72 C 1 min, 33, min, 芯片杂交检测 Pamgene-FD10 10 μl Cy5 MLPA 5 μl 5 9 μl H 2 O 1 μl blocker 25 μl, 95 5 min min, 1 buffer, 100 ms, 300 ms, 1000 ms, 3000 ms BioNavigator 数据分析 CCD 4 (16-bit TIFF ) BioNavigator, (Local background) 4, ( 1 ) PCR 检测 MLPA- Y 3 3 ( 2), MLPA- PCR 25 μl, 1 μldna, dntp 2 μl, 10 EXbuffer 2.5 μl, 10 μmol/l 1 μl, EXTaq 0.2 μl(1 U), : 95 5 min; (95 1 min, min, s) 32 ; min 5 μl PCR 1% MLPA- MLPA- 0.3 ( 30%), 0.3 MLPA- 1 Y, MLPA- 表 2 PCR 引物序列及扩增长度 Table 2 PCR primers and amplification length Gene TSPY PRY RBMY Primer sequence(5 3 ) F: TTACCTCCGTACCATCTACC R: GAAGTCAGCCTCCAACTAAG F: CACTGGTTGCTCAGGAAGAT R: AGAGGCTAGGATTGCAACT F: CCGTTATCCTCTTCAGTCAC R: CATCTAGAGGCCATGCATAC Amplification length (kb)

1632 HEREDITAS (Beijing) 2008 30 图 1 部分样品的 MLPA- 微阵列芯片检测情况 S12 3 S13 S14 S15 TSPY S14 RBMY 3 Fig.

The signal intensities in TSPY loci of S13, S14, and S15 and RBMY locus of S14 were positive. The results of other sites of the three samples were negative., ( ) Y ( ), ( ) ( )( 3) MLPA-, S13( /, mar?

4 1632 HEREDITAS (Beijing) 图 1 部分样品的 MLPA- 微阵列芯片检测情况 S12 3 S13 S14 S15 TSPY S14 RBMY 3 Fig. 1 Results of part samples detected by Array-MLPA The results showed that all the three loci of S12 gave negative signals. The signal intensities in TSPY loci of S13, S14, and S15 and RBMY locus of S14 were positive. The results of other sites of the three samples were negative., ( ) Y ( ), ( ) ( )( 3) MLPA-, S13( /, mar?) TSPY MLPA-, S14( /) PRY MLPA- 2.2 PCR Y 3 (TSPY RBMY PRY) PCR ( 2), MLPA-, PCR MLPA-, S13 TSPY PCR, S14 PRY, MLPA- PCR 3 3 MLPA 2002 [4, 5],,,, MLPA- DNA MLPA,, MLPA- 表 3 所有样品的 Array-MLPA 和 PCR 检测结果的比较 Table 3 Comparison of all samples detected by both Array-MLPA and PCR Array-MLPA PCR Sample TSPY PRY RBMY TSPY PRY RBMY S S S S S S6 S7 S8 S9 S10 S11 S12 S S S15 + +

5 12 : MLPA- Y 1633 图 2 部分样品的 PCR 检测结果 M: 1kb DNA (NEB); B: ; N: ; P: Fig. 2 PCR results of part samples M: 1 kb Marker (NEB); B: Blank; N: Negative control; P: Positive control.,, 30 min [2, 3], 4, [6, 7], [3] MLPA-,,, MLPA-,,,, 5 Mb, MLPA-, MLPA-, S14 Y (45, XO/), MLPA- Y PRY, PCR PRY, MLPA- ( mar ), MLPA- S13 MLPA- Y TSPY, (mar) Y, FISH MLPA- PCR,,, MLPA- [8~10],,,, MLPA-,

6 1634 HEREDITAS (Beijing) ,, MLPA-,, MLPA,, MLPA-, 参考文献 (References): [1] Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequencesby multiplex ligation-dependent probe amplification. Nucleic Acids Res, 2002, 30(12): e57. [DOI] [2] Berry IR, Delaney CA, Taylor GR. Detecting ligated fragments on oligonucleotide microarrays: optimizing chip design, array multiplex ligation-dependent probe amplification modification, and hybridization parameters. Methods Mol Biol, 2007, 381: [DOI] [3] Zeng FY, Ren ZR, Huang SZ, Kalf M, Mommersteeg M, Smit M, White S, Jin CL, Xu M, Zhou DW, Yan JB, Cheng MJ, van Beuningen R, Huang SZ, den Dunnen J, Zeng YT, Wu Y. Array-MLPA: Comprehensive detection of deletions and duplications and its application to DMD patients. Hum Mutat, 2007, 29(1): [DOI] [4] Roelfsema JH, White SJ, Ariyürek Y, Bartholdi D, Niedrist D, Papadia F, Bacino CA, den Dunnen JT, van Ommen GJ, Breuning MH, Hennekam RC, Peters DJ. Genetic heterogeneity in Rubinstein-Taybi syndrome: Mutations in both the CBP and EP300 genes cause disease. Am J Hum Genet, 2005, 76(4): [DOI] [5] Vink GR, White SJ, Gabelic S, Hogendoorn PC, Breuning MH, Bakker E. Mutation screening of EXT1 and EXT2 by direct sequence analysis and MLPA in patients with multiple osteochondromas: Splice site mutations and exonic deletions account for more than half of the mutations. Eur J Hum Genet, 2005, 13(4): [DOI] [6] van Beuningen R, van Damme H, Boender P, Bastiaensen N, Chan A, Kievits T. Fast and specific hybridization using flow-through microarrays on porous metal oxide. Clin Chem, 2001, 47(10): [DOI] [7] Harteveld CL, Voskamp A, Phylipsen M, Akkermans N, den Dunnen JT, White SJ, Giordano PC. Nine unknown rearrangements in 16p13.3 and 11p15.4 causing alpha- and beta-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification. J Med Genet, 2005, 42(12): [8] Evans MI, Henry GP, Miller WA, Bui TH, Snidjers RJ, Wapner RJ, Miny P, Johnson MP, Peakman D, Johnson A, Nicolaides K, Holzgreve W, Ebrahim SA, Babu R, Jackson L. International, collaborative assessment of prenatal karyotypes: Expected limitations if only chromosome-specific probes and fluorescent in-situ hybridization are used. Hum Reprod, 1999, 14(5): [DOI] [9] Lewin P, Kleinfinger P, Bazin A, Mossafa H, Szpiro-Tapia S. Defining the efficiency of fluorescence in situ hybridization on uncultured amniocytes on a retrospective cohort of prenatal diagnoses. Prenat Diagn, 2000, 20(1): 1 6.[ DOI] [10] Thein AT, Abdel-Fattah SA, Kyle PM, Soothill PW. An assessment of the use of interphase FISH with chromosome specific probes as an alternative to cytogenetics in prenatal diagnosis. Prenat Diagn, 2000, 20(4): [DOI]

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr PCR 龙平, 崔占虎, 李虔全, 徐建平, 张春红, 周立社 * *, 李旻辉 ( 包头医学院, 内蒙古包头 014060) DNA 30 28 DNA trnl-trnf PCR PCR 136 bp 323 bp 10% PCR PCR Astragalus membranaceus Fisch. Bge. var. mongho- licus Bge. Hsiao A. membranaceus