Single-base mutational analysis of cancer and genetic diseases using membrane bound modified oligonucleotides.
A convenient format for the detection of PCR amplified sequences is the hybridization of the PCR products to oligonucleotide probes which are immobilized on a solid phase. We describe a new method for site-specific attachment of such probe oligonucleotides to nylon membranes. The method is based on the formation of an amide bond between carboxyl groups present on the membranes and amino-linkers situated on the 5' end of the oligonucleotides. The covalent attachment is via a carbodiimide mediated condensation. The single, 5' end attachment of the oligonucleotides to the membrane surface leaves the probe free to interact with complementary sequences, thus increasing the hybridization efficiency relative to methods where heat or ultraviolet light is used for non-specific fixation. Using biotinylated PCR products in hybridization reactions along with a non-radioactive chemiluminescent detection system, high efficiency hybridization is obtained as well as a very good signal to noise ratio. The method has been applied successfully to the detection of RAS point mutations, cystic fibrosis deletion and point mutations and others. The sensitivity, simplicity and reproducibility of this method make it an ideal tool for the diagnosis of infectious and genetic diseases, as well as analysis of mutations in neoplasias, HLA typing and other areas.
Zhang
Y
Y
Department of Human Genetics, Cetus Corp., Emeryville, CA 94608.
Coyne
M Y
MY
Will
S G
SG
Levenson
C H
CH
Kawasaki
E S
ES
D001483:Base Sequence; D002460:Cell Line; D003550:Cystic Fibrosis / Q000235:genetics; D004247:DNA; D004252:DNA Mutational Analysis; D011905:Genes, ras; D030342:Genetic Diseases, Inborn / Q000235:genetics; D006801:Humans; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction; D013499:Surface Properties
D008567:Membranes, Artificial; D015345:Oligonucleotide Probes; D004247:DNA