A method of simultaneously analyzing RNA and DNA in a sample, the method comprising the step (a) contacting the sample with a reverse primer from a first primer pair directed to a target RNA region to effect reverse transcription of RNA into cDNA with a reverse transcriptase; (b) subsequently contacting the sample with (i) a forward primer from the first primer pair directed to a second cDNA region, (ii) a forward and a reverse primers from a second primer pair targeted to a DNA region, and (ii) a DNA polymerase to simultaneously amplify the target cDNA and target DNA region; and (c) analyzing the amplified target cDNA region and/or amplified target DNA region. Also encompassed are uses of the method to analyze gene expression and mutations, kits comprising primers, enzymes, buffers.

A method of simultaneously analyzing RNA and DNA in a sample, the method comprising the step (a) contacting the sample with a reverse primer from a first primer pair directed to a target RNA region to effect reverse transcription of RNA into cDNA with a reverse transcriptase; (b) subsequently contacting the sample with (i) a forward primer from the first primer pair directed to a second cDNA region, (ii) a forward and a reverse primers from a second primer pair targeted to a DNA region, and (ii) a DNA polymerase to simultaneously amplify the target cDNA and target DNA region; and (c) analyzing the amplified target cDNA region and/or amplified target DNA region. Also encompassed are uses of the method to analyze gene expression and mutations, kits comprising primers, enzymes, buffers.

The present disclosure provides methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a mixed sample of DNA comprising DNA from both the mother of the fetus and from the fetus, and optionally from genotypic data from the mother and father. The ploidy state is determined by using a joint distribution model to create a plurality of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.

The present disclosure provides methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a mixed sample of DNA comprising DNA from both the mother of the fetus and from the fetus, and optionally from genotypic data from the mother and father. The ploidy state is determined by using a joint distribution model to create a plurality of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.

A method and a kit for detecting Mycobacterium tuberculosis are provided. The method includes a step of performing a nested qPCR assay to a specimen. The nested qPCR assay includes a first round of amplification using external primers and a second round of amplification using internal primers and a probe. The external primers have sequences of SEQ ID NOs. 1 and 2, and the internal primers and the probe have sequences of SEQ ID NOs. 3 to 5.

A method and a kit for detecting Mycobacterium tuberculosis are provided. The method includes a step of performing a nested qPCR assay to a specimen. The nested qPCR assay includes a first round of amplification using external primers and a second round of amplification using internal primers and a probe. The external primers have sequences of SEQ ID NOs. 1 and 2, and the internal primers and the probe have sequences of SEQ ID NOs. 3 to 5.

Embodiments of present disclosure are directed to methods for amplifying nucleic acid, comprising two steps: a first step of preparing a reaction mixture comprising the target nucleic acid and a second step of processing the reaction mixture in a thermocycler. During a first phase of the processing step, the thermocycler may be configured to heat the reaction mixture to a first temperature and cool the reaction mixture to a second temperature repeatedly for a first plurality of cycles. During the first phase, fluorescence probes do not anneal to template strands and do not emit fluorescence signals. During a second phase of the processing step, the thermocycler may heat the reaction mixture to a third temperature and cool the reaction mixture to a fourth temperature repeatedly for a second plurality of cycles. During the second phase, fluorescence probes anneal to the template strands and are degraded by DNA polymerase to emit fluorescence signals for detection and/or quantification of the target nucleic acid. Methods for amplifying nucleic acid in accordance with the disclosure may be employed for nucleic acid amplification and detection in clinical and research settings.

Embodiments of present disclosure are directed to methods for amplifying nucleic acid, comprising two steps: a first step of preparing a reaction mixture comprising the target nucleic acid and a second step of processing the reaction mixture in a thermocycler. During a first phase of the processing step, the thermocycler may be configured to heat the reaction mixture to a first temperature and cool the reaction mixture to a second temperature repeatedly for a first plurality of cycles. During the first phase, fluorescence probes do not anneal to template strands and do not emit fluorescence signals. During a second phase of the processing step, the thermocycler may heat the reaction mixture to a third temperature and cool the reaction mixture to a fourth temperature repeatedly for a second plurality of cycles. During the second phase, fluorescence probes anneal to the template strands and are degraded by DNA polymerase to emit fluorescence signals for detection and/or quantification of the target nucleic acid. Methods for amplifying nucleic acid in accordance with the disclosure may be employed for nucleic acid amplification and detection in clinical and research settings.

The technology described herein is directed to methods of determining oligonucleotide sequences, e.g. by enriching target sequences prior to sequencing the sequences.

Described herein are methods and assays for detection of recombination and/or rearrangement events in a cell. In some embodiments, the methods and/or assays relate to Linear Amplification Mediated (LAM)-PCR. In some embodiments, the recombination event is a V(D)J recombination event.