ISSN 1007-7626 CN 11-3870 / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology 2012 6 28 6 567 ~ 573 PSCA SNP 1 1 2 * 1 3 1 2 1 200240 2 200032 3 200025 SNP DNA PSCA SNP - PCR-RFLP PSCA rs2976392 A rs2294008 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 200240China 2 State Key Laboratory of Oncogenes and Related GenesShanghai Cancer InstituteShanghai Jiao Tong University School of MedicineShanghai 200032China 3 Shanghai Key Laboratory of Gastric NeoplasmsDepartment of SurgeryShanghai Institute of Digestive SurgeryRuijin HospitalShanghai Jiao Tong University School of MedicineShanghai 200025China 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 2012-01-17 2012-03-25 No 30900271 973 No 2010CB529205 No 91-10-09 * Tel 021-34206632 Fax 021-34206059 E-mail yanguo@ sjtu edu cn Received January 172012 Accepted March 252012 Supported by the National Natural Science Foundation of China No 30900271 National Basic Research Program of China 973 ProgramNo 2010CB529205 and State Key Laboratory of Oncogenes and Related Genes No 91-10-09 * Corresponding author Tel 86-21-34206632 Fax 021-34206059 E-mail yanguo@ sjtu edu cn
568 28 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 rs2976392 and rs2976392the 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 4 2 1 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 2223 2-4 rs2976392 A rs2294008 T PSCA rs2976392 rs2294008 2 5 6-8 CDH1 9 CD14 myeloperoxidase thymidylate array-cgh Illumina synthase 1 10-12 1 1 54 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
6 PSCA SNP 569 system Carl Zeiss MicroImaging 1 2 10 μm PEN 1 mm 100% 2 min PSCA rs2976392 4 min rs3394008 1% 1 min 18 2 mg 30 s 70% 90% 100% QIAamp DNA Mini Kit DNA 2 ~ 3 s 40 1 82 36 5 ng / μl 1 PCR-RFLP PSCA rs2976392 1-3 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 rs2976392 PCR 20 800 g 10 min pfuultra DNA PCR DNA 95 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 1 5 12% PAGE μl Titanium Taq 51 μl PCR-RFLP PSCA rs2294008 95 3 min 1-2 bp 1 94 30 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 94 30 s 63 30 s Taq A 4 μl 10 mmol / L datp 0 4 μl 5 U / μl rtaq 1 μl 10 3 6 μl 70 1 μl 30 min A PCR 500 ng T 22 50 μl 10 NEB 5 μl 10U 1 5 - PCR-RFLP PSCA DNA NanoDrop A 260 0 73 A 260 / A 280 1 G A 1 QIAquick PCR PCR 50 μl ACCCGCTGGTGTTGACTGT-3' 5'- rs2294008 PCR pfuultra 72 40 s 30 72 5 min PCR QIAquick PCR HpyCH4Ⅳ PCR 50
570 28 μl 37 1 h 65 20 min 12% PAGE 1 6 PSCA 5 033 bp NCBI rs2976392 13 SNP 3 8 10 11 PCR 1 5 17 DNA 9 SNP 2 2 2 1 A PSCA PCR PvuⅡ G Fig 1 2 2 2 PvuⅡ A G NanoDrop A 260 1 684A 260 / A 280 DNA SNP 1 8584 2 ng / μl PEN SNP 5 μl 1 5% Fig 2 1 100 bp 2 PEN Fig 3 2 4 PCR-RFLP PSCA SNP PSCA rs2976392 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 rs2976392 patterns analysis with 12% PAGE gel electrophoresis after PvuⅡ digestion PSCA gene contained with rs2976392 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 rs2294008 T 22 PCR HpyCH4 Ⅳ
6 PSCA SNP 571 C 100 bp 20 bp 2 Fig 5 SNP SNP 2 HpyCH4Ⅳ T C DNA SNP SNP PSCA rs2976392 rs2294008 Fig 5 rs2294008 patterns analysis with 12% PAGE gel electrophoresis after HpyCH4Ⅳ digestion PSCA gene contained with rs2294008 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 rs2976392 A rs2976392 A 1 SNP SNP PSCA SNP rs2294008 T SNP rs2976392 2 1 A 9 1 A 2 5 PSCA PCR rs2976392 DNA PSCA rs2976392 9 8 G Fig 6A 1 A Fig 6B PSCA rs2976392 2 1 G 1 A PSCA A 1 SNP 3 SNP
572 28 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 rs2976396 patterns analysis of DNA from gastric cancer tissue by cloning and sequencing PSCA gene contained with rs2976396 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 rs2976392 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 rs2976392 locusanother red one the was another SNP
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