Detection of mutations in RET proto-oncogene codon 634 through double tandem hybridization.
We developed a procedure to detect the 7 point mutations at Cys634 of the proto-oncogene RET, which is responsible for medullary thyroid carcinoma (MTC). Genomic DNA was prepared from blood samples obtained from normal and MTC-affected individuals belonging to a family with a history of the disease. The RET genotype for each individual was first established by performing restriction and sequencing analyses. Single-stranded target DNA was prepared by asymmetric polymerase chain reaction (PCR) amplification of a 93-bp fragment containing Cys634. The target was annealed with pairs of prelabeled stacking oligonucleotides designed to create appropriate 7-nucleotide gaps, which served as the sites of subsequent hybridization with glass-immobilized 7-mer probes. The target-stacking oligonucleotide duplexes were hybridized with DNA chips containing a set of eight 7-mer probes designed to detect the wild-type sequence and the seven point mutations described. We tested two sets of immobilized probes containing internal or 5'-terminal codon-634 single-base variations. Both groups of probes were able to discriminatively identify the mutations. The hybridization patterns indicated that the disease in this family was due to the C634Y mutation, in accord with the original sequence analysis. The hybridization-based mutation assignment was additionally supported by determination of the control homozygous and heterozygous hybridization patterns produced with synthetic targets having the normal or codon 634 mutant sequences. The effects of mismatch type and nearest-neighbor sequences on the occurrence of false-positive (mismatched) hybridizations are discussed.
Maldonado-Rodríguez
Rogelio
R
Escuela Nacional de Ciencias Biológicas, IPN, México, 11340, DF, México.
Espinosa-Lara
Mercedes
M
Barrera-León
Oscar
O
Colin-Tovar
Carmen
C
González-Yebra
Beatriz
B
Salcedo-Vargas
Mauricio
M
Santiago-Hernández
J Carlos
JC
Méndez-Tenorio
Alfonso
A
Beattie
Kenneth L
KL
D000595:Amino Acid Sequence; D001483:Base Sequence; D018276:Carcinoma, Medullary / Q000097:blood / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D006579:Heterozygote; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D013964:Thyroid Neoplasms / Q000097:blood / Q000235:genetics
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; D003545:Cysteine