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1 ISSN CN / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 573 PSCA SNP * SNP DNA PSCA SNP - PCR-RFLP PSCA rs A rs T SNP SNP PSCA R73 Analysis of SNP of PSCA Gene in Limited Gastric Cancer Cells Isolated by Laser Microdissection YANG Yi 1 1 GUO Yan 2 * ZHAO Xiao-Dong 1 LIU Bing-Ya 3 1 SHAO Zhi-Feng 2 1 Key Laboratory of Ministry of Education for Systems BiomedicineInstitute for Systems BiomedicineShanghai Jiao Tong UniversityShanghai China 2 State Key Laboratory of Oncogenes and Related GenesShanghai Cancer InstituteShanghai Jiao Tong University School of MedicineShanghai China 3 Shanghai Key Laboratory of Gastric NeoplasmsDepartment of SurgeryShanghai Institute of Digestive SurgeryRuijin HospitalShanghai Jiao Tong University School of MedicineShanghai China Abstract With the applications of genome-wide association study GWAS more and more predisposing gene were identified in gastric cancer The polymorphism detection of predisposing genes was applied in clinical diagnosis and research of gastric cancer Howeverthe detection was challenging No No 2010CB No * Tel Fax yanguo@ sjtu edu cn Received January Accepted March Supported by the National Natural Science Foundation of China No National Basic Research Program of China 973 ProgramNo 2010CB and State Key Laboratory of Oncogenes and Related Genes No * Corresponding author Tel Fax yanguo@ sjtu edu cn

LIU Bing-Ya 3 1 SHAO Zhi-Feng 2 1 Key Laboratory of Ministry of Education for Systems BiomedicineInstitute for Systems BiomedicineShanghai Jiao Tong UniversityShanghai 200240China 2 State Key

2 with limited samplessuch as limited gastric mucosal cells for early diagnosis With laser microdissection and GenomePlex library whole genome amplification methodssingle nucleotide polymorphism SNP of the prostate stem cell antigen PSCA in limited pure gastric cancer cells were studied Using polymerase chain reaction-restriction fragment length polymorphism PCR-RFLP and sequencing method rs and rs the predisposing mutations of PSCA genein microdissected gastric cancer tissuewere identified The results showed that the sensitivity and reliability of predisposing mutation detection were greatly increased after pure gastric cancer cells isolation Multi-gene SNP detection methods for limited sample developed can be applied to the genetic analysis of other types of limited sample Whole genome amplification products can also be used for high-throughput DNA microarray and next-generation sequencing analysis Key words gastric cancer limited sample whole genome amplification single nucleotide polymorphism SNP prostate stem cell antigen PSCA DOP-PCR 1617 rolling circle amplification 1819 hyper-branched genome-wide association studygwas strand displacement amplification 2021 single nucleotide polymorphismsnp GenomePlex library prostate stem cell antigenpsca rs A rs T PSCA rs rs CDH1 9 CD14 myeloperoxidase thymidylate array-cgh Illumina synthase WGA4 PCR GenElute PCR Clean-up Kit Sigma- Aldrich PfuUltraⅡ Fusion HS DNA Polymerase 13 Agilent Technologies QIAamp DNA Mini Kit QIAquick PCR QIAGEN polyethylene NaphthalatePEN 1 mm Carl 1415 PCR Zeiss MicroImaging PALM laser microdissection

rs2976392 and rs2976392the predisposing mutations of PSCA genein microdissected gastric cancer tissuewere identified The results showed that the sensitivity and reliability of predisposing mutation

3 6 PSCA SNP 569 system Carl Zeiss MicroImaging μm PEN 1 mm 100% 2 min PSCA rs min rs % 1 min 18 2 mg 30 s 70% 90% 100% QIAamp DNA Mini Kit DNA 2 ~ 3 s ng / μl 1 PCR-RFLP PSCA rs bp PCR 1 3 Pvu Ⅱ 5'- 32 μl 10 CTG GCCATCTGTCCGCAGCT-3' 2 μl K 1 μl 9 μl 5'-CAGATGGAGGAGGATGCTGGA-3'PCR 10 μl 117 bp 4 rs PCR g 10 min pfuultra DNA PCR DNA 95 3 min s 65 30s 50 1 h 99 4 min s min PCR 2 μl μl min min min 1 μl PCR 2 min 500 ng 50 μl PCR 10 NEB Ⅱ5 μl 10U PVUⅡ 75 5 min 37 1 h min PCR 7 5 μl % PAGE μl Titanium Taq 51 μl PCR-RFLP PSCA rs min 1-2 bp s 65 5 min 25 A T 1 HpyCH4Ⅳ 5'-GAA - 20 GenElute PCR GGGCAAGCAGCACAGCCTAC-3' PCR Nanodrop 120 bp 1 5% 1 4 DNA 95 3 min s s Taq A 4 μl 10 mmol / L datp 0 4 μl 5 U / μl rtaq 1 μl μl 70 1 μl 30 min A PCR 500 ng T μl 10 NEB 5 μl 10U PCR-RFLP PSCA DNA NanoDrop A A 260 / A G A 1 QIAquick PCR PCR 50 μl ACCCGCTGGTGTTGACTGT-3' 5'- rs PCR pfuultra s min PCR QIAquick PCR HpyCH4Ⅳ PCR 50

3 min 94 30 s 65 30s 50 1 h 99 4 min 72 40 s30 72 5 min PCR 2 μl 1 95 1 μl 16 20 min 24 20 min 37 20 min 1 μl PCR 2 min 500 ng 50 μl PCR 10 NEB Ⅱ5 μl 10U PVUⅡ 75 5 min 37 1 h 65 20 min PCR 7 5 μl 10

4 μl 37 1 h min 12% PAGE 1 6 PSCA bp NCBI rs SNP PCR DNA 9 SNP A PSCA PCR PvuⅡ G Fig PvuⅡ A G NanoDrop A A 260 / A 280 DNA SNP ng / μl PEN SNP 5 μl 1 5% Fig bp 2 PEN Fig PCR-RFLP PSCA SNP PSCA rs SNP 99 bp 18 bp 2 Fig 4 Fig 2 1 5% Agarose gel electrophoresis of whole genome amplification products from several gastric cancer cells Several gastric cancer cells were isolated by laser microdissectionthen genomic DNA was amplified with GenomePlex library method M DL2000 DNA marker 1 Whole genome amplification products from chosen gastric cancer cells The products showed typical smear 2 Empty PEN membrane as negative control There were no visible products Fig 4 rs patterns analysis with 12% PAGE gel electrophoresis after PvuⅡ digestion PSCA gene contained with rs sites was PCR amplified Forward primer sequence was 5'-CTGGCCATCTGTCCGCAGCT-3' and reverse primer sequence was 5'-CAGATGGAGGAGGA TGCTGGA-3' The length of PCR product was 117 bp PCR product was digested with PvuⅡ enzymes M 20 bp DNA ladder marker 1 Genotype of the PCR products from DNA extracted from gastric cancer tissuewhich showed that there existed A / G genotype 2 Genotype of the PCR products from WGA products of several gastric cancer cells which showed that there existed A / G genotype 2 3 rs T 22 PCR HpyCH4 Ⅳ

from several gastric cancer cells Several gastric cancer cells were isolated by laser microdissectionthen genomic DNA was amplified with GenomePlex library method M DL2000 DNA marker 1 Whole genome

5 6 PSCA SNP 571 C 100 bp 20 bp 2 Fig 5 SNP SNP 2 HpyCH4Ⅳ T C DNA SNP SNP PSCA rs rs Fig 5 rs patterns analysis with 12% PAGE gel electrophoresis after HpyCH4Ⅳ digestion PSCA gene contained with rs sites was PCR amplified Forward primer sequence was 5'-GAAACCCGCTGGTGTTGACTGT- 3'and reverse primer sequence was 5'-GGGCAAGCAGC ACAGCCTAC-3' The length of PCR product was 120 bp PCR product was digested with HpyCH4Ⅳ enzymes M 20 bp DNA ladder marker 1 Genotype of the PCR products from DNA extracted from gastric cancer tissuewhich showed that there existed T / C genotype 2 Genotype of the PCR products from WGA products of several gastric cancer cells which showed that there existed T / C genotype PSCA SNP rs A rs A 1 SNP SNP PSCA SNP rs T SNP rs A 9 1 A 2 5 PSCA PCR rs DNA PSCA rs G Fig 6A 1 A Fig 6B PSCA rs G 1 A PSCA A 1 SNP 3 SNP

bp PCR product was digested with HpyCH4Ⅳ enzymes M 20 bp DNA ladder marker 1 Genotype of the PCR products from DNA extracted from gastric cancer tissuewhich showed that there existed T / C genotype 2

6 Fig 1 Isolation of several gastric cancer cells with laser microdissection Frozen gastric cancer was sectionedspread on slides with PEN membrane and stained with hematoxylin A Isolated gastric cancer cells were marked B Marked gastric cancer cells were laser microdissectedpressure capsulated and collected Fig 3 A typical random cloning and sequencing of whole genome amplification products from several gastric cancer cells The green parts of the figure were the cloning vector sequences The yellow parts were the fixed sequences of the adaptor The purple parts were the random sequences of the adaptors The middle parts of the sequences can completely match to chromosome 19 Fig 6 rs patterns analysis of DNA from gastric cancer tissue by cloning and sequencing PSCA gene contained with rs sites was PCR amplified PCR product was cloned and Sanger sequenced A The gray part was the primer sequences for PCR and red frame was the mutation introduced into the primer The red part was G allele of the PSCA gene rs locus B The gray part was the primer sequences for PCR and red frame was the mutation introduced into the primer The red part was A allele of the PSCA gene rs locusanother red one the was another SNP

from several gastric cancer cells The green parts of the figure were the cloning vector sequences The yellow parts were the fixed sequences of the adaptor The purple parts were the random sequences

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stage and bone metastasis in prostate cancer J Oncogene 200019 10 1288-1296 4 Ross SSpencer S DHolcomb Iet al Prostate stem cell antigen as therapy target tissue expression and in vivo efficacy of an

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TABLE OF CONTENTS Page

TABLE OF CONTENTS Page Acknowledgements... VI Declaration... VII List of abbreviations... VIII 1. INTRODUCTION... 1 2. BASIC PRINCIPLES... 3 2.1. Hereditary Colorectal Cancer... 3 2.1.1. Differential diagnosis...