A multi-enzyme model for pyrosequencing

Abstract

Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid real-time sequence determination. This technique employs four enzymatic reactions in a single tube to monitor DNA synthesis. Nucleotides are added iteratively to the reaction and in case of incorporation, pyrophosphate (PPi) is released. PPi triggers a series of reactions resulting in production of light, which is proportional to the amount of DNA and number of incorporated nucleotides. Generated light is detected and recorded by a detector system in the form of a peak signal, which reflects the activity of all four enzymes in the reaction. We have developed simulations to model the kinetics of the enzymes. These simulations provide a full model for the Pyrosequencing four-enzyme system, based on which the peak height and shape can be predicted depending on the concentrations of enzymes and substrates. Simulation results are shown to be compatible with experimental data. Based on these simulations, the rate-limiting steps in the chain can be determined, and K(M) and kcat of all four enzymes in Pyrosequencing can be calculated.

Quantification of CpG methylation at the 5′‐region of XIST by pyrosequencing from human serum

“…The biotinylated PCR product (25 mL) was purified using streptavidin-Sepharose beads (Amersham Biosciences, Uppsala, Sweden). The single strand PCR product acted as a template in a pyrosequencing reaction [18]. For sequencing, we used the pyrosequencing primer as 5 0 -GTTATTTTAGATTTGT-3 0 (gi:37704377, 931-946).…”

Determining the global DNA methylation status of rat according to the identifier repetitive elements

“…Pyrosequencing analyzes ssDNA templates by synthesizing complementary strands [17], and thus can be used for the measurement of DNA methylation after bisulfite treatment [18]. Our method for measuring the CpG methylation of ID elements may represent the global DNA methylation in the rat genome and can be used for other applications.…”

Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)–PCR

“…The pyrosequencing technique has become more widely used during the last several years as reaction chemistry [3,4] and understanding of reaction kinetics [5,6] have improved, and as higher throughput hardware/software (www.pyrosequencing.com) and chemistries [7,8] have been developed. Published applications have included microbial genotyping [9][10][11][12] mutation detection [13], sequencing of heteroplasmic DNA [14], SNP genotyping [15,16], SNP allele frequency determination [17,18] SNP-based allelic loss mapping [19], gene copy number change [20] and CpG methylation analysis [21][22][23], among others.…”