DNA extension and analysis with rolling primers

Details DNA extension and analysis with rolling primers DNA extension and analysis with rolling primers

1997-08-22

1999-10-05

Jones, W. Gary

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

536 231, 536 243, 935 76, 935 77, 935 78, C12Q 168, C07H 2102, C07H 2104, C12N 1500

Patent

active

059622288

ABSTRACT:
A novel "primer walking" method for DNA sequencing is provided that comprises repeated cycles nucleotide identification by selective extension and primer advancement along a template by template mutation. An important feature of the invention is providing a set of primers, referred to herein as "rolling primers" that contain complexity-reducing nucleotides for reducing the number of primers required for annealing to every possible primer binding site on a sequencing template. Another important feature of the invention is the systematic replacement of at least one of the four nucleotides in the target polynucleotide with its cognate complexity-reducing nucleotide or complement thereof. Sequencing is initiated by annealing rolling primers differing only in their terminal nucleotides to a primer binding site of a sequencing template so that only the rolling primer whose terminal nucleotide forms a perfect complement with the template leads to the formation of an extension product. After amplifying the double stranded extension product to form an amplicon, the terminal nucleotide, and hence its complement in the template, is identified by the identity of the amplicon. The primer binding site of the template of the successfully amplified polynucleotide is then mutated by, for example, oligonucleotide-directed mutagenesis so that a subsequent rolling primer may be selected from the set that forms a perfectly matched duplex with the mutated template at a site which is shifted towards the direction of extension by one nucleotide relative to the binding site of the previous rolling primer. The steps of selective extension, amplification and identification are then repeated. In this manner, the primers "roll" along the polynucleotide during the sequencing process, moving a base at a time along the template with each cycle.

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