Direct solid-phase sequence analysis of the human p53 gene by use of multiplex polymerase chain reaction and alpha-thiotriphosphate nucleotides.
Among the candidate cancer-prognostic genes is the p53 tumor suppressor gene, which, when mutated, plays an important role in the development of many types of cancers. To facilitate robust large-scale DNA analysis of microdissected tumor biopsies, we describe a multiplex/nested PCR approach for a simultaneous outer amplification of exons 4-9 of the human p53 gene with parallel amplification of the HLA-DQB1 locus, involving a total of 14 primers. This approach reduces the required number of cells for analysis and avoids any variation in the amplifications of the individual p53 exons during the common outer amplification step. The HLA sequencing allows sample identification because the DQB1 locus is highly polymorphic and is thereby patient-specific. The p53 and HLA amplicons are analyzed by solid-phase sequencing in a semiautomated format. To improve the DNA sequence quality, we used 2'-deoxyribonucleoside 5'-O-1-thiotriphosphates in the sequencing reactions.
Berg
C
C
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Hedrum
A
A
Holmberg
A
A
Pontén
F
F
Uhlén
M
M
Lundeberg
J
J
D001322:Autoanalysis; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D005091:Exons; D016158:Genes, p53; D006683:HLA-DQ Antigens / Q000235:genetics; D059866:HLA-DQ beta-Chains; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*; D013873:Thionucleotides
D006683:HLA-DQ Antigens; D059866:HLA-DQ beta-Chains; C069051:HLA-DQB1 antigen; D013873:Thionucleotides; D004259:DNA-Directed DNA Polymerase