DIRECT SEQUENCING OF LARGE RECOMBINANT MOLECULES

Award Information
Agency:
Department of Health and Human Services
Branch
n/a
Amount:
$50,000.00
Award Year:
1990
Program:
SBIR
Phase:
Phase I
Contract:
n/a
Agency Tracking Number:
13844
Solicitation Year:
n/a
Solicitation Topic Code:
n/a
Solicitation Number:
n/a
Small Business Information
Collaborative Research Inc
2 Oak Pk, Bedford, MA, 01730
Hubzone Owned:
N
Socially and Economically Disadvantaged:
N
Woman Owned:
N
Duns:
n/a
Principal Investigator:
Douglas R Smith
(617) 275-0004
Business Contact:
() -
Research Institution:
n/a
Abstract
THIS PROJECT ADDRESSES THE NEED FOR EFFICIENT METHODS TO RAPIDLY DETERMINE THE DNA SEQUENCE OF YEAST ARTIFICIAL CHROMOSOME (YAC) OR COSMID CLONES WITHOUT SUBCLONING SMALL FRAGMENTS. THE METHOD COMBINES MULTIPLEX DNA SEQUENCING WITH LIGASE-MEDIATED POLYMERASE CHAIN REACTION (PCR) AMPLIFICATION OF SPECIFIC RESTRICTION FRAGMENTS. PURIFIED YAC OR COSMID DNA IS DIGESTED WITH TWO DIFFERENT RESTRICTION ENZYMES TO GENERATE FRAGMENTS WITH ONE BLUNT ENDAND ONE AMBIGUOUS 4-BASE 5'-PROTRUDING END. SYNTHETIC OLIGONUCLEOTIDES CONTAINING MULTIPLEX TAG SEQUENCES ARE SPECIFICALLY LIGATED TO THESE ENDS, AND PRIMERS HOMOLOGOUS TO THE TAGS ARE USED TO AMPLIFY THE FRAGMENTS BY PCR. PRODUCTS OF THE PCR REACTIONS ARE POOLED, AND CHEMICAL SEQUENCING REACTIONS ARE PERFORMED. THE SAMPLES ARE FRACTIONATED ON SEQUENCING GELS, ELECTROBLOTTED, AND INDIVIDUAL SEQUENCE LADDERS VISUALIZED ON AUTORADIOGRAMS AFTER SEQUENTIAL HYBRIDIZATION USING MULTIPLEX PROBES. WITH APPROPRIATE SELECTION OF RESTRICTIONENZYMES, THERE IS A HIGH PROBABILITY THAT ANY PARTICULAR 4-BASE END WILL OCCUR ONLY ONCE IN THE STARTING CLONE. EXTENSIVE OVERLAPS CAN BE GENERATED BY USING SEVERAL SETS OFINTERRELATED DOUBLE RESTRICTION DIGESTS. PHASE I STUDIES WILL FOCUS PRIMARILY ON TECHNICAL ASPECTS OF THE LIGATION AND PCR REACTIONS. A SET OF MULTIPLEX SEQUENCING REACTIONS WILL BE PERFORMED, AND THE APPLICABILITY OF THE METHOD WILL BE EVALUATED.

* information listed above is at the time of submission.

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