Multiplex Pyrosequencing.
We describe here the development of a new and simple single-tube multiplex Pyrosequencing assay. Genomic DNA or cDNA was employed to PCR amplify region(s) using biotinylated and normal primer(s). Subsequent to capture of PCR products on streptavidin-coated beads, single-stranded DNA separation and hybridization of multiple sequencing primers, Pyrosequencing was performed. The obtained pyrogram resulted in a unique pattern in which the intensity of the signal determined the number of incorporated nucleotide(s). Here, we demonstrate the use of this multiplex Pyrosequencing for single nucleotide polymorphisms genotyping and microbial typing.
Pourmand
Nader
N
Stanford Genome Technology Center, Stanford University, 855 California Avenue, Palo Alto, CA 94304, USA.
Elahi
Elahe
E
Davis
Ronald W
RW
Ronaghi
Mostafa
M
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid
D004279:DNA, Viral; D004247:DNA