HPV. Development and Optimization of Oligonucleotide Microarray for Detection and Sub-typing of Human Papillomavirus

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1 3 3 Vol3 No Life Science Research June HPV *,, 2,, 3, : HPV(Human papillomavirus) HPV (6 6 8 ), (Tm) ~60 mer,, HPV,, HPV,, : (HPV); ; ; :R372 :A : (2009) Development and Optimization of Oligonucleotide Microarray for Detection and Sub-typing of Human Papillomavirus WEI Min MA Wen-li * SUN Zhao-hui 2 ZHANG Jin-fang LI Ling ZHENG Wen-ling 3 Institute of Genetic Enginerring Southern Medical University Guangzhou 5055 Guangdong China 2Clinical Laboratory General Hospital of Guangzhou Command Guangzhou 5055 Guangdong China 3 Southern China Genomics Research Center Guangzhou Guangdong China Abstract: An oligonucleotide microarray assay for detection and sub-typing of human papillomavirus HPV was developed and optimized Biological softwares Arraydesigner 20 and BLAST program were applied to analyze the whole genome of four different HPV types 6 6 and 8 to design ~ 60 mer oligonucleotide probes with high specificity and similar melting temperature Tm The cultured HPV samples were labeled with fluorescence dyes Cy3 using a method of restriction display PCR RD-PCR The labeled samples were hybridized with the oligo microarray HPV DNA samples were hybridized specifically with multiple spots correspondingly to show positive signals and the corresponding HPV subtypes were recognized as well while no signals were detected of all the negative and blank controls With optimization of glass slide treatment and fluorescence labeling method the hybrid specificity and fluorescence signal intensity of oligo microarray were heightened Key words human papillomavirus oligonucleotide microarray RD-PCR detection Life Science Research ~25 : : : D WSTJJ : 978- Tel weimin78@26com * 96- Tel Fax wenli668@gmailcom

2 3 :HPV 2 HPV / [] HPV HPV HPV HPV [2] HPV HPV6 HPV HPV6 HPV8 39% HPV De Villier EM 2 2 HPV 3 5 [3,] HPV Taq T DNA TaKaRa 5 -GTTTGGCTGGTCTCCATC-3 SIP 5 -CCAGCCAAACCCA-3 SIR 5 - HPV GGTTTGGCTGGTGTG-3 ABI 3900 Cy3 U 5 -Cy3- UGTTTGGCTGGTCTCCATC-3 Cy3 9 HPV mer Trilink 60~ pmd-8 T Sau3A 70 mer 2 GenBank HPV6 HPV HPV6 HPV8 DNA Array Designer 20 BLAST HPV HPV Tm HPV 0 Table HPV specific, positive and negative control oligonucleotide probes of microarray Target Probe No Region Sequence 5-3 HPV6 HPV HPV6 HPV8 Positive probes Negative probes Negative probes E2 E6 L E6 L E2 CCCAACCACCCGTGGAGGCTAATGGACATATATT AATTTCTGCACCCACTATAACGTCACACCCT GGCACTGGGCCTCCTCAAAGGCACCACATAAAC ATGCCATTGTAACTGTAACATATCATAGTGAG ATGCCTATAAGAACCTAAAGGTTGTGTGGCGAG ACAACTTTCCCTTTGCAGCGTGTGCCTGTTGCT AACAAGTTTGCATTACCTGATTCATCCCTGTTTG ACCCCACTACACAGCGTTTAGTATGG TGCTGAACCATTTGACCCTATCCCTGACCCTGTC CAACATTCTGTTACACAGTCTTATCTTACCTCCA GCAATGTTTCAGGACCCACAGGAGCGACCCAGA AAGTTACCACAGTTATGCACAGAGCTG ACATTAGGAAAACGAAAAGCTACACCCACCACC TCATCTACCTCTACAACTGCTAAACGCA CCTGCCACACCACTAAGTTGTTGCACAGAGACT CAGTGGACAGTGCTCCAATCCTCACTGCATT AGTGGCTAACCCTGAGTTTCTTACACGTCCATC CTCTTTAATTACATATGACAACCCGGC GCCTACCAACAAGTGTCAGTGGCTAACCCTGAG TTTCTTACACGTCCATCCTCTTTAATTACA GTTTGGCTGGTGTGGATCGTTTGGCTGGTGTGGA TCGTTGGCTGGTGTGGATCGTTTGG GTATTAATTATTGCTAGCTGATCATACCACGTTA GTCGTTAAGCATGCATGCAGCTAGGA GTAACGTTAAGAGACTACCATTGCACATGCCCT AAGAACAGGTACAATAGAGTAGGTACA Tm /

3 (poly-l-lysine) 3 % - GOPS 95% 30 min 500 r / min 5 32 Poly-L-lysine PCR Product Purification Kit 70 ml poly-l-lysine 70 ml PBS 600 ml Sau3A PCR 500 r / min h 33 poly-l-lysine 95 5 min 95 02% 30 s s s s 72 PDITC 0% 898% min min 2 h 500 r / min % SDS 95 5 min GOPS / poly-l-lysine GOPS-PLL % SDS 0 SSC+% SDS 0 SSC 50% DMSO g / L Pixsys poly-l-lysine Agilent-2565 B GOPS-PLL 5 5 Cy3 9 mer signal-to- GenePix 00A GenPix Pro 60 noise ratio SNR DMSO 2 g / L A HPV6 HPV8 DNA DNA 50% dimlthyl Sau3A sulfoxide DMSO Pixsys ~ 000 Cartesian 250~500 GOPS-PLL B C RD- PCR BIO-RAD RD-PCR 65 mj [5] 6 22 DNA μg Sau3A μl 37 h GATC SIP SIR Sau3A T DNA Cy3 PCR 3S 5 μl DNA 2 50% Formamide 0 SSC 2 2 SSC + Array-Pro poly-l-lysine GOPS-PLL DNA 3

4 3 :HPV 23 HPV HPV DNA 23 HPV 0 GOPS-PLL HPV RD- PCR 5 HPV M 2 M M A B HPV6, 8 DNA (A) HPV6,8 DNA Sau3A ; (B) HPV6 DNA Cy3- ; (C) HPV8 DNA Cy3- Fig Agarose gel electrophoresis of digestion and amplification of HPV6 HPV8 plasmids DNA A Agarose gel electrophoresis of restricted fragments of HPV6 HPV8 plasmids DNA digested by Sau3A B Agarose gel electrophoresis of amplified products of HPV6 plasmids DNA with Cy3-labeled universal primers C Agarose gel electrophoresis of amplified products of HPV8 plasmids DNA with Cy3-labeled universal primers M DL 2000 HPV6 plasmid DNA 2 HPV8 plasmid DNA C M M HPV RD- PCR RD-PCR Fig2 Agarose gel electrophoresis of amplified HPV fragments of RD-PCR and RD-gradient PCR M D000 Lane Amplified fragments of RD-PCR Lane 2 Amplified fragments of RD-gradient PCR Fig Scanning results of optimized microarray hybridization A Sample of HPV6 B Sample of HPV C Sample of HPV6 D Sample of HPV8 E Sample of human DNA A B A ~5 H6 ~0 Positive controls A6 ~0 H ~5 Blank controls D~5 G6~0 Negative controls B~0 HPV6 Result of immobilization ratio experiment of probes C~0 D6~0 HPV probes E~0 F6~0 HPV6 probes F~5 G~5 HPV8 probes 3 Fig3 two different slides A poly-l-lysine coated slide B GOPS-PLL slide

5 HPV L Gp5 / 6 β HPV E6 E7 PCR Cy5 Cy3 Klaassen HPV [5] DNA HPV DNA 20 mer 53 PCR Hybrid Capture β DNA HPV HPV 5 HPV Systems HPV FDA HC 20 ~25 mer HPV PCR [6] HPV PCR General Primermediated PCR GP-PCR HPV HPV HPV PCR HPV HPV [7,8] HPV PCR Type-Specific PCR TS-PCR 60 mer PCR HPV Restriction Display PCR RD-PCR HPV RFLP RD Dot blotting microtiter enzyme immunoassays reverse hybridization line probe assays [9~] [6] 60 mer DNA DNA [ 2,3] HPV Biomedlab Company Seoul Korea [] 22 HPV HPV

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