Compositions, kits, and methods for performing rapid polymerase chain reaction (PCR) to amplify a target nucleic acid in a biological sample are disclosed. The methods include the use of at least one hybridization stabilizer and/or the adjustment of the thermocycling profiles between initiation and propagation phases of the amplification process. Also disclosed are methods of detecting the target nucleic acid following amplification thereof, as well as reaction mixtures that may be utilized in said methods.

The present invention relates to the field of ribonucleotide analysis. More specifically, the present invention provides compositions and methods for detection for nucleic acids using probe pair litigation. In particular embodiments, the compositions and methods of the present invention utilize a probe set comprising (1) a first multi-partite probe comprising a 5′ phosphorylated donor probe and a first bridge probe, wherein the 5′ phosphorylated donor probe specifically hybridizes to a target nucleic acid; and (ii) a second multi-partite probe comprising a 3′ acceptor probe and a second bridge probe, wherein the 3′ acceptor probe specifically hybridizes to the target nucleic acid adjacent to the 5′ donor probe and the second bridge probe is 5′ phosphorylated.

The present invention relates to the field of ribonucleotide analysis. More specifically, the present invention provides compositions and methods for detection for nucleic acids using probe pair litigation. In particular embodiments, the compositions and methods of the present invention utilize a probe set comprising (1) a first multi-partite probe comprising a 5′ phosphorylated donor probe and a first bridge probe, wherein the 5′ phosphorylated donor probe specifically hybridizes to a target nucleic acid; and (ii) a second multi-partite probe comprising a 3′ acceptor probe and a second bridge probe, wherein the 3′ acceptor probe specifically hybridizes to the target nucleic acid adjacent to the 5′ donor probe and the second bridge probe is 5′ phosphorylated.

The present disclosure provides methods and kits for performing high multiplex PCR using molecular barcodes. The methods disclosed herein separately extend a set of primers (BC primers) that each comprise a target-specific sequence, a molecular barcode and a universal sequence, and amplify the resulting extension products using another set of primers (LA primers) that each comprise another target-specific sequence and a universal sequence. The methods may further comprise amplification using universal primers (preferably comprising an adapter).

The present disclosure provides methods and kits for performing high multiplex PCR using molecular barcodes. The methods disclosed herein separately extend a set of primers (BC primers) that each comprise a target-specific sequence, a molecular barcode and a universal sequence, and amplify the resulting extension products using another set of primers (LA primers) that each comprise another target-specific sequence and a universal sequence. The methods may further comprise amplification using universal primers (preferably comprising an adapter).

The present invention relates to a primer for PCR obtained by, directly or through inosine as a linker, linking a complementary nucleotide sequence or a complementary nucleotide sequence including a mis-matched nucleotide sequence to the 5′-terminal of a forward or reverse primer; and to a PCR method including a step of mixing a nucleic acid template in a PCR composition including the primer and then performing PCR on the mixture. The primer for PCR of the present invention includes a complementary nucleotide sequence or a mis-matched nucleotide sequence in a complementary nucleotide sequence, which is linked to the 5′-terminal thereof directly or via a linker, thereby lowering the sensitivity increase due to the increase in amplification products and reducing non-specifically occurring reactions in PCR.

The present invention relates to a primer for PCR obtained by, directly or through inosine as a linker, linking a complementary nucleotide sequence or a complementary nucleotide sequence including a mis-matched nucleotide sequence to the 5′-terminal of a forward or reverse primer; and to a PCR method including a step of mixing a nucleic acid template in a PCR composition including the primer and then performing PCR on the mixture. The primer for PCR of the present invention includes a complementary nucleotide sequence or a mis-matched nucleotide sequence in a complementary nucleotide sequence, which is linked to the 5′-terminal thereof directly or via a linker, thereby lowering the sensitivity increase due to the increase in amplification products and reducing non-specifically occurring reactions in PCR.

The present invention provides compositions, kits and methods for the selective hybridization and capture of a specific target nucleic acid. The specific target nucleic acid may be present in a heterogeneous mixture of nucleic acids. Selective hybridization and capture provides a target nucleic acid that is substantially free of non-target and/or contaminating nucleic acids. Target nucleic acids that have been selectively hybridized and captured using the current invention are then used in subsequent analysis, wherein the presence of non-target and/or contaminating nucleic acids that interfere with said subsequent analysis have been substantially reduced or eliminated, thereby providing improved analysis results. The invention offers the further advantage of requiring less stringent purification and/or sterility efforts than conventionally needed in order to ensure that enzymes and other reagents used in subsequent analysis, or present in the environment in which an assay is performed, are free of bacterial or other contaminating nucleic acids.

The present invention provides compositions, kits and methods for the selective hybridization and capture of a specific target nucleic acid. The specific target nucleic acid may be present in a heterogeneous mixture of nucleic acids. Selective hybridization and capture provides a target nucleic acid that is substantially free of non-target and/or contaminating nucleic acids. Target nucleic acids that have been selectively hybridized and captured using the current invention are then used in subsequent analysis, wherein the presence of non-target and/or contaminating nucleic acids that interfere with said subsequent analysis have been substantially reduced or eliminated, thereby providing improved analysis results. The invention offers the further advantage of requiring less stringent purification and/or sterility efforts than conventionally needed in order to ensure that enzymes and other reagents used in subsequent analysis, or present in the environment in which an assay is performed, are free of bacterial or other contaminating nucleic acids.

The present invention provides methods for immobilizing target nucleic acids on a solid support utilizing combinatorial capture probe pairs. These pairs contain first and second capture oligonucleotides that each comprise a target binding region, a capture region and a stem region positioned between the target binding and capture regions. The target binding regions comprise nucleic acid sequences that allow them to hybridize to adjacent regions on the target nucleic acid. The stem regions have nucleic acid sequences that are complementary to each other and the capture regions each comprise a sequence that when positioned adjacent to one another produce a combined nucleic acid sequence that is complementary to a portion of an oligonucleotide bound to a solid support. When the first and second capture oligonucleotides are annealed to the target nucleic acid, the stem regions are brought together allowing them to hybridize, which in turn brings the capture regions together to produce a combined nucleic acid sequence. This combined nucleic acid sequence is then able to hybridize to the oligonucleotide bound to the solid support thereby immobilizing the target nucleic acid.