Detection of mutations in PCR products from clinical samples by surface plasmon resonance.
Two different strategies for scanning and screening of mutations in polymerase chain reaction (PCR) products by hybridization analysis are described, employing real-time biospecific interaction analysis (BIA) for detection. Real-time BIA was used to detect differences in hybridization responses between PCR products and different 17-mer oligonucleotide probes. For the analysis using a biosensor instrument, two different experimental formats were investigated based on immobilization of either biotinylated PCR products or oligonucleotide probes onto a sensor chip. Applied on the human tumour suppressor p53 gene, differences in hybridization levels for full-match and mismatch situations employing both formats allowed the detection of point mutations in exon 6 PCR products, derived from a breast tumour biopsy sample. In addition, a mutant sample sequence could be detected in a 50/50 background of wild type exon 6 sequence. The suitability of the different formats for obtaining a regenerable system and a high throughput of samples is discussed.
Nilsson
P
P
Department of Biochemistry and Biotechnology, KTH-Royal Institute of Technology, Stockholm, Sweden.
Persson
B
B
Larsson
A
A
Uhlén
M
M
Nygren
P A
PA
D001426:Bacterial Proteins / Q000737:chemistry; D015374:Biosensing Techniques; D001710:Biotin / Q000737:chemistry; D001943:Breast Neoplasms / Q000235:genetics; D002624:Chemistry, Clinical / Q000295:instrumentation / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D016158:Genes, p53; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019809:Streptavidin; D013499:Surface Properties; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D001426:Bacterial Proteins; D016159:Tumor Suppressor Protein p53; D001710:Biotin; D019809:Streptavidin