Mutation detection by pyrosequencing: sequencing of exons 5-8 of the p53 tumor suppressor gene.
The ability to sequence a large number of DNA samples rapidly and accurately for detection of all possible mutations is a critical goal for the future application of DNA sequencing in routine medical diagnostics. Pyrosequencing() is a non-electrophoretic real-time DNA sequencing method that uses the luciferase-luciferin light release as the detection signal for nucleotide incorporation into target DNA. For pyrosequencing of the human p53 gene, a nested multiplex PCR method for amplification of exons 5-8 was prepared. In order to investigate the use of pyrosequencing in mutation detection, DNA samples from skin-cancer patients were used. Two forms of nucleotide dispensation strategy were used, cyclic and programmed. Bi-directional pyrosequencing was performed and the overlapping sequence data produced were assembled to determine the sequence of the gene. Reliable sequencing data were obtained with both dispensation strategies, but some advantages were obtained using the programmed nucleotide dispensation approach, such as longer and faster reads, and fewer out-of-phase problems. The accuracy of pyrosequencing for detection of p53 mutations and allele distribution was demonstrated.
KTH, Royal Institute of Technology, Department of Biotechnology, Teknikringen 34, SE-100 44, Stockholm, Sweden. carlos@biochem.kth.se
C A
CA
Garcia
A
A
Ahmadian
B
B
Gharizadeh
J
J
Lundeberg
M
M
Ronaghi
P
P
Nyrén
D001483:Base Sequence; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded / Q000235:genetics; D005091:Exons / Q000235:genetics; D016147:Genes, Tumor Suppressor / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D012878:Skin Neoplasms / Q000235:genetics; D013698:Templates, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded; D016159:Tumor Suppressor Protein p53