title: Quantitative haplotyping of PCR products by nonsynchronous pyrosequencing with di-base addition.
abstract: Molecular haplotyping is becoming increasingly important for studying the disease association of a specific allele because of its ability of providing more information than any single nucleotide polymorphism (SNP). Computational analysis and experimental techniques are usually performed for haplotypic determination. However, established methods are not suitable for analyzing haplotypes of massive natural DNA samples. Here we present a simple molecular approach to analyze haplotypes of conventional polymerase chain reaction (PCR) products quantitatively in a single sequencing run. In this approach, specific types and proportions of haplotypes in both individual and pooled samples could be determined by solving equations constructed from nonsynchronous pyrosequencing with di-base addition. Two SNPs (rs11176013 and rs11564148) in the gene for leucine-rich repeat kinase 2 (LRRK2) related to Parkinson's disease were selected as experimental sites. A series of DNA samples, including these two heterozygous loci, were investigated. This approach could accurately identify multiple DNA samples indicating that the approach is likely to be applied for haplotyping of unrestricted conventional PCR products from natural samples, and be especially applicable for analyzing short sequences in clinical diagnosis. Graphical Abstract One DNA sample consisting of 4 different DNA templates with different proportion are sequenced by nonsynchronous pyrosequencing with di-base addition. The number of incorporated nucleotides produced by a single sequencing reaction equals to the total of incorporated nucleotides. Four independent equations are constructed from the pyrograms of nonsynchronous pyrosequencing data. Molecular haplotypes of two adjacent SNPs can be quantitatively identified by solving these equations.
authors.affiliation: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, Jiangsu, 210096, China.
authors.forename: Rongfang
authors.initials: R
authors.lastname: Pan
authors.affiliation: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, Jiangsu, 210096, China. xiaopf@seu.edu.cn.
authors.forename: Pengfeng
authors.initials: P
authors.lastname: Xiao
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000235:genetics; D006239:Haplotypes / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 / Q000235:genetics; D008962:Models, Theoretical; D010300:Parkinson Disease / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004247:DNA; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
keywords: Di-base; Equations; Haplotype; Nonsynchronous pyrosequencing; Quantitative analysis