Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1994-01-19
1995-11-28
Parr, Margaret
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
536 243, 536 242, C12P 1934
Patent
active
054707242
ABSTRACT:
Methods for amplifying DNA sequences of interest are disclosed. The methods can be performed using only one primer and are also useful in cloning protocols and for sequencing large DNAs. The methods comprise cleaving a sample DNA using an agent, such as a restriction endonuclease, that produces discrete DNA fragments; ligating the fragments to "adapter" polynucleotides having a ligatable end and first and second self-complementary sequences separated by a spacer sequence, thereby forming ligated duplexes; denaturing the ligated duplexes to form templates; annealing molecules of an oligonucleotide primer to the templates, the primers being homologous to a primer target site associated with the sequence of interest; extending the primers using a DNA polymerizing agent to form duplex products; and denaturing the duplex products. Subsequent multiple cycles of annealing primers, extending the primers, and denaturing duplex products are usually performed so as to achieve the desired degree of amplification. Sequencing of large DNAs is performed using multiple rounds of DNA amplification, each round employing a primer homologous with a primer target site in the sequence of interest previously amplified. Cloning is facilitated by including a replication origin and selectable marker in the adapters.
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Houtteman Scott William
Parr Margaret
State of Oregon Acting by and through the Oregon State Board of
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