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System and method for high resolution analysis of nucleic acids to detect sequence variations

  • US 9,139,882 B2
  • Filed: 11/22/2010
  • Issued: 09/22/2015
  • Est. Priority Date: 03/28/2007
  • Status: Active Grant
First Claim
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1. A method for nucleic acid sequence amplification, comprising:

  • identifying a target nucleic acid sequence from one or more segments of DNA, said target nucleic acid sequence having primers with melting temperatures that are within at least 15°

    C. of the melting temperature of the target nucleic acid sequence;

    obtaining a pair of oligonucleotide primers with an annealing curve (TA) that overlaps with a denaturation curve (TD) of the target nucleic acid sequence, in such a manner as to minimize the temperature range between the higher of the melting temperature of a pair of oligonucleotide primers and the melting temperature of the target nucleic acid sequence, wherein the temperature range does not exceed 15°

    C.; and

    amplifying the target nucleic acid sequence by thermocycling the target nucleic acid sequence and the pair of oligonucleotide primers within a 15°

    C. temperature range,wherein thermocycling comprises;

    i. denaturing the target nucleic acid sequence; and

    ii. annealing of the pair of oligonucleotide primers; and

    iii. extension of the target nucleic acid sequence by the pair of oligonucleotide primers,wherein an upper thermal cycle temperature in the thermocycling is selected to minimize non target denaturation and maximize target denaturation.

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