11] Long synthetic oligonucleotide probes for gene analysis

Abstract

This chapter describes the procedures for using long synthetic oligonucleotide probes to identify individual genes within a family of closely related sequences. Different methods, such as: (1) genomic DNA isolation, (2) oligonucleotide synthesis and purification, (3) southern blot analysis, and (3) hybridization, are described. High molecular weight DNA was isolated from the peripheral blood leukocytes (PBLs), Epstein-Barr virus (EBV)-transformed B cell lines, and tissues according to published methods, with minor modifications. Synthetic oligonucleotide probes were synthesized on a Coder 300 by the phosphoramidite method using the “trityl off” program. 14 probes were cleaved from the columns and dried under vacuum according to manufacturer's directions. Methods were used for southern blot analysis: standard southern blotting, and in-gel southern blotting. Hybridization with synthetic oligonucleotide was carried out as previously described in “Seal-a-meal” bags. Various results regarding the following experiments are also discussed in this chapter.