Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1993-05-06
1995-11-28
Parr, Margaret
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, 4351723, C12N 1570, C12Q 168
Patent
active
054707226
ABSTRACT:
A method that permits the rapid amplification of unknown DNA that flanks a known site, such that one can walk into an uncharacterized region of DNA. In this method, human genomic DNA is restriction enzyme digested and then ligated to a 5' phosphorylated-oligonucleotide so that the 5' end of each strand of genomic DNA is extended and phosphorylated. The phosphorylated-oligonucleotide is constructed to render 5' end extensions that are complementary to the known sequence. Following denaturation and re-annealing under dilute conditions that promote intrastrand annealing and under high stringency, only those DNA strands containing the known sequence will form a stem-loop structure with a recessed and phosphorylated 5' end, rendering a substrate for a subsequent heat-stable ligation reaction to another oligonucleotide. This second oligonucleotide is complementary to the sequence immediately adjacent to the phosphorylated-oligonucleotide high stringency annealing site. The heat-stable ligation reaction appends a known sequence to the DNA segments containing the two known contiguous DNA sequences used for oligonucleotide annealing. This heat-stable ligation of known sequence permits the subsequent highly specific amplification of the unknown flanking DNA.
REFERENCES:
patent: 4661450 (1987-04-01), Kemp et al.
Jones and Winistorfer, Nucleic Acids Res., 20:595-600 (1992).
Jones and Winistorfer, PCR Methods Applic., 2:197-203 (1993).
Campbell Eggerton
Parr Margaret
Pepper Frederick W.
University of Iowa Research Foundation
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