Isolation and Transcription of 3' Terminal Regions of Unique DNA Molecules

Abstract

ONE of the major difficulties in the determination of the primary structure of DNA is the isolation of small specific polydeoxyribonucleotide fragments which can be analysed directly or transcribed into RNA. So far, this difficulty has been overcome only in the case of regions that can be selectively protected from endonucleolytic attack by binding to highly purified specific proteins or to ribosomes. We have developed a method which allows the selective isolation and subsequent transcription of fragments derived from the 3′ termini of any unique DNA molecule. The procedures were established by using the DNA from bacteriophage λ; this provided an indication of the validity of the technique for relatively high molecular weight DNA. The method involves the limited digestion of the DNA with spleen acid deoxyribonuclease to produce fragments of chosen length, the selective extension of the fragments derived from the 3′ termini of the original DNA by addition of dATP residues with terminal deoxynucleotidyl transferase, and the separation of the extended fragments from the bulk of the DNA by chromatography on oligo (dT) cellulose (Fig. 1). The RNA transcripts prepared from these fragments exhibited an absolute specificity for the 3′ terminal regions of A DNA in a variety of hybridisation experiments. Two-dimensional fingerprint analysis of ribonuclease T₁ digests of the transcription products gave a simple and characteristic oligonucleotide pattern which could be used for sequence determination. This method can also be adapted to the isolation of specific fragments from internal regions of DNA molecules.