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).

A nucleic acid amplification method includes a step of heating a first region of a container housing a droplet containing a target nucleic acid and a sample necessary for amplification of the target nucleic acid to a denaturation temperature of the target nucleic acid and heating a second region different from the first region to a synthesis temperature of the target nucleic acid, and an amplification step of repeating a cycle through a denaturation stage at which the droplet housed in the container is moved to and retained in the first region and a synthesis stage at which the droplet is moved to and retained in the second region at a plurality of times. At the amplification step, periods of part of cycles of the plurality of cycles are made shorter than periods of the other cycles.

A nucleic acid amplification method includes a step of heating a first region of a container housing a droplet containing a target nucleic acid and a sample necessary for amplification of the target nucleic acid to a denaturation temperature of the target nucleic acid and heating a second region different from the first region to a synthesis temperature of the target nucleic acid, and an amplification step of repeating a cycle through a denaturation stage at which the droplet housed in the container is moved to and retained in the first region and a synthesis stage at which the droplet is moved to and retained in the second region at a plurality of times. At the amplification step, periods of part of cycles of the plurality of cycles are made shorter than periods of the other cycles.

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 is directed to methods and compositions for generating a pool of oligonucleotides. The invention finds use in preparing a population or subpopulations of oligonucleotides in solution. The pool of oligonucleotides finds use in a variety of nucleic acid detection and/or amplification assays.

Methods and containers are provided for identifying a species, illustratively a bacterial species. Illustrative methods comprise amplifying various genes in the nucleic acid from the bacterial species in a single reaction mixture using pairs of outer first-stage primers designed to hybridize to generally conserved regions of the respective genes to generate a plurality of first-stage amplicons, dividing the reaction mixture into a plurality of second-stage reactions, each using a unique pair of second-stage primers, each pair of second-stage primers specific for a target bacterial species or subset of bacterial species, detecting which of the second-stage reactions amplified, and identifying the bacterial species based on second-stage amplification. Methods for determining antibiotic resistance are also provided, such methods also using first-stage primers for amplifying genes known to affect antibiotic resistance a plurality of the second-stage reactions wherein each pair of second-stage primers specific for a specific gene for conferring antibiotic resistance.

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.

Disclosed are DNA polymerases having increased reverse transcriptase efficiency relative to a corresponding, unmodified polymerase. The polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the DNA polymerases.

Assays and methods for detecting resistance to beta-lactam antibiotics including detection of multiple β-lactamase family specific gene targets by polymerase chain reaction or microarray. One or more kits including primers and/or probes for identification of β-lactamase genes selected from the group consisting of one or more of the following: MOX-like, FOX-like, ACC-like, ACT/MIR-like, CMY-2-like, DHA-like, CTX-M-14-like, CTX-M-15-like, VIM-like, NDM-like, IMP-like, KPC-like, and OXA-48-like, OXA-51-like, OXA-143-like, OXA-58-like, OXA-23-like, OXA-24/40-like, TEM-like, and SHV-like. A kit may also include one or more primers and/or probes for the identification a non-beta lactamase gene family which confers antibiotic resistance, such as the MCR-1 gene.