Methods and compositions for multiplex pcr amplifications
OwnerTzvi TZUBERY, Shira Glasner +5inventors
InventorsTzvi TZUBERY, Shira Glasner, Tal Salomon, Boaz Arieli, Aryeh Gassel, Maoz Tal, Raphael Gassel
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Abstract
The present invention concerns in general PCR reaction mixtures comprising a mixture of hot-start primers and non-hot-start primers for a given target sequence, including multiplex PCR reaction mixtures; methods utilizing same for detection of one or more target polynucleotide sequences; and kits comprising same.
Claims25
A PCR reaction mixture comprising:
- a) a first set of primers for amplifying a first target polynucleotide sequence;
- b) a second set of primers for amplifying a second target polynucleotide sequence;
- and
- c) an activating enzyme,
- wherein the primers that hybridize to at least one end of said first target polynucleotide sequence are a mixture of hot-start primers and non-hot-start primers, wherein the reaction product of amplifying said first target polynucleotide sequence using said non-hot-start primers is a substrate for amplification using said hot-start primers;
- and
- wherein said hot-start primers contain an inactivating chemical modification that is reversed by the action of said activating enzyme, wherein said hot-start primers become a substrate for said activating enzyme when said hot-start primers are hybridized to a complementary sequence at elevated temperatures.
A method of detecting a first target polynucleotide sequence and a second target polynucleotide sequence in a test sample, wherein the nucleic acids of said test sample have been derived from a clinical specimen and have not been extensively purified, the method comprising the steps of:
- (a) amplifying nucleic acids from said test sample using a PCR reaction mixture;
- and
- (b) detecting the amplified PCR product, wherein the PCR reaction mixture comprises:
- i) a first set of primers, comprising at least one forward primer and at least one reverse primer, that is capable of amplifying said first target polynucleotide sequence;
- and ii) a second set of primers, comprising at least one forward primer and at least one reverse primer, that is capable of amplifying said second target polynucleotide sequence, wherein at least one of said forward primer and said reverse primer of said first set of primers is provided as a mixture of hot-start primers and non-hot-start primers.
Dependencies are inferred from the claim text.