Method, Kit and System for Enhanced Nested Pcr
AssigneeThomsen Bioscience
InventorsGert Bolander Jensen, Lars Thomsen, Oene Robert Veltman
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Abstract
The present invention relates to a method, a kit and a system for sensitive detection of a target nucleic acid molecule. The target nucleic acid molecule may e.g. be specific for a certain microorganism, e.g. a pathogenic microorganism such as B. anthracis. The methods and kits relate to so-called nested PCR and especially improvements, which renders the nested PCR technique better suited for automated analysis.
Claims30
A method of amplifying, and optionally also detecting, a target nucleic acid sequence, the method comprising the steps of:
- a) providing a sample that may or may not comprise a target nucleic acid sequence,
- b) providing a pair of outer primers and a pair of inner primer, a nucleic acid polymerase and standard reagents for PCR, the melting temperature (Tm) of the pair of outer primers being at least 2° C. higher than the Tm of the pair of inner primers,
- c) contacting the sample with the pair of outer primer and the pair of inner primers, and standard reagents for PCR, thus obtaining the reaction mixture,
- d) cycling, at least two times, the temperature of the reaction mixture between a first denaturation temperature, a first annealing temperature and a first extension temperature, the first annealing temperature being approximately the same as or lower than the lowest Tm of the outer primer pair and higher than the highest Tm of the inner primer pair, and
- e) cycling, at least two times, the temperature of the reaction mixture between a second denaturation temperature, a second annealing temperature and a second extension temperature, the second annealing temperature being similar to or lower than the lowest Tm of the inner primer pair.
A kit comprising a pair of outer primers and a pair of inner primer, the melting temperature (Tm) of the pair of outer primers being higher than the Tm of the pair of inner primers.
An analysis system for detection of a microorganism, the analysis system comprising a pair of outer primers and a pair of inner primer, the melting temperature (Tm) of the pair of outer primers being higher than the Tm of the pair of inner primers.
Dependencies are inferred from the claim text.