Diagnosing X-linked Ichthyosis by Monoplast Single-round Duplex PCR

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1 13 4 Vol13 No Life Science Research Aug PCR X- *,,,,,,,, : X- (X-linked ichthyosis, XLI), XLI, XLI (preimplantation genetic diagnosis, PGD) PCR amelogenin (Amel), 958% 909%, 15%; Amel-Y 919% 927%, 14% 0; ADO 62% PCR XLI, XLI PGD : PCR; X- (XLI); ; amelogenin ; (PGD) : R3943 :A : (2009) Diagnosing X-linked Ichthyosis by Monoplast Single-round Duplex PCR ZHONG Chang-gao LI Lu-yun LU Chang-fu GONG Fei LIAO Hong-qing ZHANG Zhan GAO Bai-di ZHANG Shuo-ping LU Guang-xiu * Institute of Reproduction and Stem Cell Engineering Central South University Reproductive & Genetic Hospital CITIC-XIANGYA Changsha Hunan China Abstract X-linked ichthyosis XLI is a sort of sex linked recessive inherited diseases It is one of the most common congenital metabolic diseases of the mankind Diagnosing XLI on monoplast level using monoplast PCR that can provide a basis for preimplantation genetic diagnosis PGD of XLI The gene and the amelogenin Amel gene were amplified by single-round duplex PCR with single lymphocytes from the patient the normal female and single blastomere from the normal person The amplification success rates were 958% and 909% on gene in the groups of single lymphocytes and single blastomere from the normal person respectively The false positive rate was 15% on gene in the groups of single lymphocytes from the patient The amplification success rates were 919% and 927% on the gene loci of and Amel-Y respectively The contamination rates were 14% and 0 on the above 2 gene loci respectively The allele dropout rate ADO was 62% on the Amel loci To some extent the protocol of monoblast single-round duplex PCR for diagnosing XLI is accurate specific and reliable It will help us further develop the PGD of XLI : : : JJY3120 : * Tel lgxdirector@sinacom

2 4 : PCR X- 333 Key words monoblast single-round duplex PCR X-linked ichthyosis XLI gene amelogenin gene preimplantation genetic diagnosis PGD Life Science Research ~336 X XLI HEPES 1 IVC-HTF HEPES 02 μl 25 μl 200 μl 02 μl [1] XLI steroid sulfatase 113 PCR X Xp kb 10 85% ~90% -F 5 - GCTTCAGCTGTGCTGTCCTT-3 -R 5 -CCCAAGCCTTCAGGTGATAA-3 PCR 223 bp [5] [2] PGD amelogenin Amel-F 5 -ATCAGAG 1989 Handyside CTTAAACTGGGAAGCT-3 Amel-R 5 -CCTGGG PGD CTCTGTAAAGAATAG-3 PCR X [3] PGD 105 bp Y 111 bp 12 [4] 121 PGD PCR 20 μl 65 X 10 min -70 PCR 122 PCR X Sigma 1 PCR Buffer 03 mmol / L dntp 0625 U HotStarTaq Ivitrogen PCR IVC-HTF HEPES min 30 s 96 2 min In Vitro Care International s s s s 60 [4] 02 μl 25 μl 25 s s min 200 μl 4 02 μl 123 PCR μl 200 mmol/l Tris Gly [6] Tricine ph 473 Sigma 25 μl 02 μmol / L 008 μmol/l Amel 2 mmol/l MgCl 2 01% 1 μl 1 loading buffer 10% V acrylamide gel electrophoresis PAGE 2 h Tyrode 1 min IVC-HTF

3 PCR PCR 68 PCR bp Amel 15% 1 / 68 Amel X Y 105 bp X % 63 / bp Y 3 X Y ADO 105 bp Amel 2 Amel ADO 62% 4/ bp 111 bp 37 1 Amel X allele dropout 27% 1 / 37 1 ADO Table 1 1 Amplification results of single lymphocytes and blastomeres Cell types No Noof blank control Amplified gene Amplification success rate False positive rate Single lymphocyte of patient of deletion type Single lymphocyte of normal female Blastomere of normal person Amel-Y Amel-Y 15% 1 / % 63 / % 63 / % 46 / % 44 / % 40 / % 40 / % 24 / 44 15% 1 / 68 27% 1 / / / / 23 bp M PCR M: DNA ; 1~4: PCR ; 5~9: PCR ; 10~14: PCR ; 15 16: gdna ; 17~20: HEPES Fig1 The amplification results of monoplast with single-round duplex PCR M puc Mix Marker 8 molecular weight marker Lanes 1~4 the PCR products of single lymphocyte from the patient Lanes 5~9 the PCR products of single lymphocyte from the normal female Lanes 10~14 the PCR products of single blastomere from the normal person gdna positive control of the male and the female respectively Lane 17~20 blank control with washing solution of single lymphocyte from the patient single lymphocyte from normal female single blastomere from the normal person and blank control with HEPES culture medium respectively 22 PCR PCR % 46 / 48 Amel X % 44 / 48

4 4 : PCR X PCR PCR 23 PCR PCR PCR DNA PCR 909% 40/44 Amel X % 40 / Amel Y % 24 / HotstarTaq 10 Touchdown PCR 3 59 DNA XLI % 149 / 160 PCR XLI PCR 1 PCR 50% 50% 50% PCR PCR XLI 85%~90% XLI 3 PGD PGD XLI X- XLI PGD SRY Amel SRY PGD SRY Amel PGD Amel X / Y 1 X PCR Y PCR PCR bp [5,8] bp 111 bp PCR Genebank Amel X Y PCR PCR 105 bp 111 bp [5] 106 bp 112 bp Amel SRY primer extension ADO preamplification PEP Y PCR Zhang Amel X Wells [4,7] PCR 10 [9,10] PEP 78% ~90% 96 ADO 65 Amel PGD PCR 938% 61/65 ADO 62% 4/65

5 [11] 5%~20% % 909% Amel-Y 919% 147/ % [10] 90% PCR DNA 68 Yan-Lin LI Qi PAN Qian et al 1 15% South Univ Med Sci DNA 73 1 Amel- X 14% 1 / 73 hybridization[j] Nucleic Acids Res 1999 Amel-Y PCR XLI XLI PGD (References): Xue-jun YANG Sen Progress of molecular pathogenesis on X-linked ichthyosis[j] F Med Sci Sect Dermatol Venereol [3] GOOSSENS V HARTON G MOUTOU C et al ESHRE PGD Consortium data collection VIII cycles from January to December 2005 with pregnancy follow-up to October 2006[J] Hum Reprod [4] ZHANG L CUI X F SCHMITT K et al Whole genome amplification from a single cell Implications for genetic analysis[j] Proc Nat Acad Sci USA [5] [J] MA Single cell PCR for sex determination in preimplantation genetic diagnosis[j] J Cent [6] De RYCKE M GEORGIOU I SERMON K et al PGD for autosomal dominant polycystic kidney disease type 1[J] Mol Hum Reprod [7] WELLS D SHERLOCK J K HANDYSIDE A H et al Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic [8] NAKAHORI Y HAMANO K IWAYA M et al Sex identification by polymerase chain reaction using X-Y homologous primer[j] Am J Med Genet [9] RAY P F HANDSIDE A H Increasing the denaturation temperature during the first cycles of amplification reduces allele drop out from single cells for preimplantation diagnosis [J] Mol Hum Reprod [10] PCR [J] ZHONG Chang- [1] [J] gao LI Lu-yun LU Chang-fu et al Diagnosing achondroplasia LU Guo-fang ZHANG Ying Progress by single cell nested-pcr [J] Chin J Med Genet of pathogenesis on genetic ichthyosis[j] Chinese Journal of Bir-th Health and Heredity [11] HARPER J C WELLS D Recent advances and future [2] developments in PGD[J] Prenat Diagn [J] XIAO Feng-li ZHANG 1199 CA BIOSIS (BA) Medical Index AJ NCBI CABI B 2007 Journal of Genetics and Genomics 2008 SCI-E ISSN CN /R M ISSN CN /R M wwwchinagenecn 5, : 1 : : ybxue@geneticsaccn : swli@geneticsaccn