66] Detection of a single base substitution between human leukocyte IFN-αA and -α2 genes with octadecyl deoxyoligonucleotide probes

Abstract

Publisher Summary This chapter describes the hybridization conditions to detect single base changes in plasmids coding for interferon (IFN)-αA and IFN-α2. Chemically synthesized deoxyoligonucleotides complementary to unique DNA segments have been used to detect cloned DNA sequences. Because deoxyoligonucleotide-DNA duplexes with a single base mismatch have a significantly lower thermal stability than those without any mismatch, they can be efficiently applied for the detection of site-specific mutations. There is a single base substitution of A for G between human leukocyte IFN-αA and IFN-α2 genes, which results in an amino acid substitution of lysine (αA) for arginine (α2) at the amino acid #23 of the interferon protein. Strong hybridization is observed between the oligonucleotide probe αA and pLeIFA25 or between the probe αA and pBR325-α2, whereas the hybridization between the probe αA and pBR325-α2 or between the probe α2 and pLeIFA25, where there is a single base pair mismatch, is very weak when hybridization was performed at 37° or 42°.