US 12,391,985 B2
Method of determining PIK3CA mutational status in a sample
Evrykleia Lianidou, Athens (GR); and Athina Markou, Athens (GR)
Assigned to PHARMASSIST LTD, Athens (GR)
Filed by PHARMASSIST LTD, Athens (GR)
Filed on Mar. 21, 2022, as Appl. No. 17/699,761.
Application 17/699,761 is a continuation in part of application No. 15/501,457, granted, now 11,279,979, previously published as PCT/GR2015/000036, filed on Jul. 28, 2015.
Claims priority of provisional application 62/034,231, filed on Aug. 7, 2014.
Prior Publication US 2022/0298565 A1, Sep. 22, 2022
Int. Cl. C12Q 1/6858 (2018.01); C12Q 1/6816 (2018.01); C12Q 1/6853 (2018.01); C12Q 1/6886 (2018.01)
CPC C12Q 1/6858 (2013.01) [C12Q 1/6816 (2013.01); C12Q 1/6853 (2013.01); C12Q 1/6886 (2013.01); C12Q 2600/106 (2013.01); C12Q 2600/118 (2013.01); C12Q 2600/156 (2013.01); C12Q 2600/158 (2013.01)] 14 Claims
 
1. A method for analyzing presence of a PIK3CA mutant allele DNA in a DNA sample, said method comprising the steps of performing an asymmetric and allele specific Polymerase Chain Reaction (PCR) of said mutant allele DNA in the DNA sample, and performing a melting analysis of DNA produced in the PCR, wherein said PCR is carried out by the use of:
a mutant allele specific primer complementary to a 3′ (three prime) end of a first strand of the mutant allele DNA to be amplified, said mutant allele specific primer comprises a mutation site and a mismatch to corresponding wild type DNA,
an unlabeled blocking probe, comprising the sequence of SEQ ID NO: 15, that is an oligonucleotide complementary to a wild type sequence of a first strand of wild type DNA corresponding to a first strand of the mutant allele DNA at a corresponding position in which a mutation to be detected is present, said unlabeled blocking probe comprising an additional mismatch to said first strand of the mutant allele DNA besides the mutation site, and which probe is blocked from acting as a primer for DNA synthesis in the PCR reaction, and
a common primer that is complementary to a 3′ end of a second strand of the mutant allele DNA to be amplified by the PCR, and
wherein said melting analysis is carried out by the use of:
the unlabeled blocking probe, which also functions as a melting probe, and
a detectable component for measuring a melting temperature of double-stranded DNA components at least including a first double-stranded component comprising the melting probe bound to an amplified mutant allele DNA strand or a second double-stranded component comprising the melting probe bound to a wild type allele DNA strand, wherein the melting temperature differs between the first double-stranded component and the second double-stranded component.