Preparation Of HBV Specific Nucleotide Fragments For HBV Genotyping Microarray

lntroductionHepatitis B is a liver disease caused by HBV(hepatitis B virus).It is now widely accepted that there are eight HBV genotypes named A to H.Most investigations showed that the geographic distribution,clinical manifestation,method of treatment, curative effect and prognosis of chronic hepatitis B are related closely with the infected HBV genotypes.Therefore,HBV genotyping has become an important aspect in the research of hepatitis B gene diagnosis.HBV genotyping by gene microarray technology has the merits of high sensitivity,using least sample and simple procedures etc. However,several drawbacks also exist at present.:1.The currently available HBV genotyping microarrays can test only one genotype on one chip and can not test eight genotypes on the same chip;2.Templates are critical in construct gene microarray and generally they are derived from the patients' serum.Different geographic distribution of different HBV genotypes made the access of serum of all eight genotypes very difficult and expensive for any area;3.Many traditional microarray methods are still tedious and the binding of sample DNA with probe is unstable and easily defluxed in washing, influencing the hybridization signal.These weak points have significantly hindered the development of HBV genotyping microarray technique.By careful comparison and analysis of the genomic sequences of all HBV genotypes,we find that it is possible to differentiate the 8 genotypes by different combination of bases 228,244,288,324,360,403 in the opening reading frame of S gene consensus sequence in HBV DNA.These specific sequences are short,easy to prepare,and applicable for detecting all eight HBV genotypes on one chip.SOEing(gene splicing by overlap extension) is a simple method to introduce specific sequences of other genotypes by introducing point mutations at the 6 sites as above by SOEing method.Using these specific sequences as the templates of the HBV genotyping microarray,we solved the problem of obtaining all the 8 HBV genotype samples.Our lab designed an Air-heterotherm PCR amplify system and in-situ extension gene chip-box to combine PCR and hybridization into one step.Hybridization and extension proceed in liquid-solid phase while asymmetrical PCR goes in the liquid phase.Thus HBV sample is genotyped and gene microarray procedures shortened. Because of the characteristics of primer designing,this method could be used to check single base mutation.After extension the probe and the sample DNA binding is more reliable,stable,and the signal stronger.Our preliminary results confirmed our anticipation and laid the basis for constructing an All-subtypes HBV genotyping microarray and its clinical application.Materials and MethodsMaterialsgenotype C HBV sample was stock of our labJM109,pMD-18T,rTaq,pyrobest,EcoRI,NcoI,Vent_R—(exo^-) DNA polymeraseEZ-10 Spin Column Plasmid Mini-preps kit,EZ Spin Column DNA Gel Extraction Kit(Shanghai biotechnology)Biometra Personal PCR system,GermanyBiochip sample spotter Micro GrindⅡ600,Biorobtics Ltd,EnglandConfocal laser scanner(Gene TAC^TM LSⅣ,Genomic Solutions Inc.USA) Air-heterotherm PCR amplify system and in-situ extension gene chip-box(self-made by our laboratory)Methods1.Compare and analyze with bioinformatics software the genomic sequences of eight(A-H) genotypes of HBV collected from NCBI database and screening for base points of genotyping.2.Design primers and probes according to the chosen sites and analyze their specificity.3.Prepare by SOEing and sequencing specific nucleotide fragments containing genotyping base sites.4.Using VentR-(exo-) DNA polymerase without 3'-5' exonuclease activity to confirm the validity of prepared fragments by conventional PCR. 5.Preparation of genechip:Probes are spotted on slices by biochip sample spotter according to pre-designed microarray.Chips are then hydrated,caked and installed to. the gene chip.box.6.Confirmation of validity of gene chip with the prepared fragment as template.Results1.Six specific base points for HBV genotyping are selected by bioinformatic analysis.2.8 specific fragments containing genotyping base point are successfully prepared with SOEing method.3.The validity of the 6 genotyping base sites in our prepared 8(A-H) specific fragments have been confirmed by conventional PCR.4.Biochip test using our specific nucleotide fragment as template gave result as anticipated.Conclusion1.SOEing is a simple method for introducing site specific point mutation.It could be used to prepare template DNA sequences for HBV genotyping by microarray.2.The accuracy and precision of the prepared specific nucleotide fragments have been proved by sequencing,conventional PCR and our novel nucleotide probe in-situ extension gene chip test.3.It is proved preliminarily that the novel In-situ extension gene chip-box can be used for HBV genotyping.