35] The use of random-sequence oligonucleotides for determining consensus sequences

Abstract

Publisher Summary This chapter discusses the utilization of random-sequence oligonucleotides to determine consensus sequences. It describes the development of an alternative method that is useful in defining the sequence requirements of a genetic element. A collection of recombinant deoxyribonucleic acid (DNA) molecules is made in which random or highly degenerate DNA replaces a genetic element of interest. A selection or screen is made to isolate from this collection those sequences that confer an equivalent function. A comparison is made of DNA sequences that satisfy a particular selection results in a consensus that defines the genetic element. Thus, unlike conventional mutagenesis, which uncovers the nonfunctional derivatives of a wild-type sequence, the method presented in the chapter uses the bias of selective pressure to select functional sequences from random DNA. In principle, the method is applicable for any genetic element that confers a phenotype that is subject to a selection or screen. The first step in determining the consensus sequence for such a genetic element is to construct an appropriate vector. The crucial features of the vector are as follows: (1) it lacks the genetic element of interest, (2) it contains all other sequences necessary to pass the selection, and (3) it contains unique restriction endonuclease cleavage sites that can be used to clone DNA segments at the position of the deleted genetic element. Oligomers were synthesized and kindly provided by Alexander Nussbaum using the phosphite triester method on an Applied Biosystems DNA Synthesizer Model 380A. The three variations of this method are described in the chapter and have important implications concerning the design of further experiments on other genetic elements.