New method for isolation and sequence determination of 5'-terminal regions of bacteriophage φX174 in vitro mRNAs

Abstract

We have determined the nucleotide sequences of the 5'-terminal oligonucleotides, produced by RNase T_1 digestion of bacteriophage φX174 mRNAs synthesized in vitro. The major sequences are: pppCpGp(Ap), pppApUpCpGp(Cp), pppAp(Ap)_2UpCp(Up)_2Gp(Gp), and pppAp(Ap)_3UpCp(Up)_2Gp(Gp). The sequences of several minor 5'-terminal oligonucleotides were also determined. For this research we have devised a simple isolation procedure, for the 5'-terminal oligonucleotides, based upon hydroxylapatite chromatography and two-dimensional thin-layer separation. This method allows for the rapid and quantitative recovery of all oligonucleotides containing 5'-triphosphate end groups and should be generally useful for sequence on 5' termini of mRNAs.

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