[29] Hybridization with synthetic oligonucleotides

This chapter describes procedures for the use of synthetic oligonucleotides for Southern blot experiments and gene bank screening. It also describes the effects of various mismatches on the efficiency of hybridization. In the study discussed in the chapter, d(A-G-C-A-C-C-T-T-T-C-T-T-A-G-C), complementary to bases 24-39 of the yeast iso-1-cytochrome c messenger ribonucleic acid (mRNA), was synthesized in a laboratory by the phosphotriester method. Deoxyribonucleic acid (DNA) fragments were transferred to nitrocellulose paper by the blotting procedure of Southern or the plaque transfer procedure of Benton and Davis. All hybridizations were carried out in 2 × SSC with 0.2% of polyvinylpyrrolidone, Ficoll, and bovine albumin in sealed plastic bags. Hybridization to exonuclease III-digested λ DNA was observed, and primer extension and DNA sequencing work showed that hybridization with the correct site had occurred. However, it was not possible to determine the number of mismatched base pairs that were present. The effects of the type and position of mismatch on oligonucleotide hybridization were also tested.