A novel method for detecting polymerase chain reaction product utilizing the 5 prime yields 3 prime exonuclease activity of Thermus aquaticus DNA polymerase

Conference · Mon Mar 11 00:00:00 EST 1991 · FASEB Journal (Federation of American Societies for Experimental Biology); (United States)

OSTI ID:5378700

Holland, P M; Watson, R; Abramson, R D; Gelfand, D H ^[1]

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1. Cetus Corp., Emeryville, CA (United States)

The polymerase chain reaction (PCR) method of DNA amplification is a powerful and sensitive technique which has been greatly simplified by the use of the thermostable enzyme Thermus aquaticus (Taq) DNA polymerase. In addition to its polymerase activity, Taq DNA polymerase also has a 5{prime}{yields}3{prime} exonuclease activity. Utilizing this activity, the authors have developed a PCR detection method which generates signal simultaneously with target sequence amplification. No additional probing, blotting or hybridization assays of amplified product are necessary. A 5{prime}{yields}3{prime} exonuclease cleaves 5{prime} terminal nucleotides of nicked double stranded DNA. The authors have generated a substrate suitable for exonuclease activity in a PCR assay by the addition of a labeled oligonucleotide probe designed to hybridize within the target sequence. During amplification, the 5{prime}{yields}3{prime} exonuclease activity of Taq DNA polymerase degrades the probe into smaller fragments which can be differentiated from undegraded probe. The presence of probe does not influence PCR product formation. Hydrolysis occurs only when probe is bound specifically to template. The presence of high complexity DNA in the PCR mixture does not compromise the specificity of probe interaction. In a PCR assay, the amount of detectable label may be modified by altering cycle number, target copy number or probe concentration. The size of labeled fragments may also be modified by varying base composition of the probe. This detection method is advantageous over present day procedures in that it is neither labor intensive nor requires significant skills. The minimal sample handling could reduce the risk of sample contamination, thereby increasing accuracy.

• OSTI ID: 5378700
• Report Number(s): CONF-9104107-; CODEN: FAJOE
• Journal Information: FASEB Journal (Federation of American Societies for Experimental Biology); (United States), Vol. 5:4; Conference: 75. annual meeting of the Federation of American Societies for Experimental Biology (FASEB), Atlanta, GA (United States), 21-25 Apr 1991; ISSN 0892-6638
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DNA POLYMERASES
BIOASSAY
ENZYME ACTIVITY
NUCLEASES
BACTERIA
DNA REPAIR
GENE AMPLIFICATION
SUBSTRATES
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
ENZYMES
ESTERASES
HYDROLASES
MICROORGANISMS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PROTEINS
REPAIR
TRANSFERASES
550200* - Biochemistry