Study On The Analysis Of Haplotypes By Nons Ynchronous Pyrosequencing

Molecular haplotype,referring to specific combinations of single nucleotide polymorphisms(SNPs),can reflect more biological information than a single SNP.As a major branch of study in human genomics,analysis of haplotypes plays an important role in many fields of researches,such as molecular association studies and linkage analyses.To solve the problem that established experimental analyses cannot determine the haplotype qualitatively and quantitatively simultaneously,besides,to improve the efficiency,accuracy and reliability of experimental methods for haplotypic studies,pyrosequencing with particular ability of quantitative analysis was introduced in our study.In this paper,we developed a novel method for analyzing haplotypes of conventional polymerase chain reaction(PCR)products,both types and contents of haplotypes in individual or pooled samples could be obtained in a single sequencing process.It may provide a fast and reliable experimental technique for haplotypic analysis of conventional PCR products.The main contents of the present study are as follows:(1)The methodological principle of haplotypic determination by nonsynchronous pyrosequencing.Based on pyrosequencing,an analytical method was proposed to calculate the content of particular haplotype in individual or pooled samples from sequencing peak intensities.The theory was:contents of four haplotypes which mostly existing in regions containing two adjacent independent diallele loci were regarded as four unknowns,values of these unknowns could be obtained by solving four independent equations,and then the haplotype and its content were determined.Asynchronous synthesis sequencing was introduced to extend the nucleotide of the same position in different DNA templates in different sequencing reactions to construct independent equations for solving unknowns.With theoretical analysis,we found that sequencing with di-base addition could make the four different haplotypic templates obtain regular asynchronous synthesis information;according to the principle that the sequencing signal intensity of a single DNA template is proportional to the number of synthesized nucleotides and the amount of DNA template,the construction of independent equations was based on the fact that sequencing signal intensity of pooled samples containing four different haplotypic templates was equal to the additivity of sequencing intensity from these different templates.Finally,the thesis targeted two SNPs(rs11176013(A/G)and rs11564148(A/T))relating to Parkinson's disease(PD)as study objects and confirmed methodological possibility of haplotypic analysis with nonsynchronous pyrosequencing theoretically.(2)Analysis of the feasibility of haplotypic determination by nonsynchronous pyrosequencing.The oligonucleotide sequences obtained by artificial synthesis were used as single-stranded DNA templates for experimental implementation and the feasibility of the methodological principle was analyzed.The experimental results showed that coefficients of equations obtained from a series of sequencing peak intensities of different initial amounts of DNA templates with di-base addition maintained at a constant value after the standardized treatment.This meant actual coefficients of equations were not affected by amounts of templates,moreover,this constant value was similar to the theoretical numbers of extended nucleotides from haplotypic templates.With the selected amount of template(0.5 pmol),haplotypes of homozygous templates and mixed templates prepared with different contents and components were analyzed.Results showed that types and contents of haplotypes in homozygous and mixed templates were similar to the theoretic,confirming that it was feasible and reliable for analyzing haplotypes of DNA templates with this proposed method.In addition,for homopolymeric fragments,experimental results showed that any template containing homopolymeric regions less than seven nucleotides under the amount of less than 1 pmol only needed one extra di-base addition to ensure that nucleotides were completely incorporated.(3)Analysis of PCR products using haplotypic determination by nonsynchronous pyrosequencing.First,genomic DNA was extracted from blood samples to design primers to obtain PCR products containing targeted SNPs and then plasmids representing particular haplotypes from the PCR products were constructed.Second,with the constructed particular plasmids,mixed plasmids were prepared with different contents and components.These mixed plasmids were used as known samples,simulating the individual(or pooled)blood samples for analysis of haplotypes.Optimizations of experimental conditions were also conducted and this novel method was further confirmed applicable to PCR products.Finally,genomic DNA of the actual blood samples was taken as unknown samples and the contents of particular haplotypes were analyzed.The analyzed results were verified by TA cloning and overall assessment was made for this proposed method with these experimental results.The results showed that haplotypic determination of PCR products from unknown samples with the proposed method was successful.This method may provide a reliable,simultaneously qualitative and quantitative method for haplotypic determination and alternative platform for linkage analysis of disease and haplotypes.