The present invention provides methods, compositions and kits for enriching and determining nucleotide sequences of a plurality of target loci from a sample comprising nucleic acids. The methods comprise one or more cycles of primer extension followed by PCR amplification of target sequences using nested target-specific primers.

Disclosed is a method for high-throughput detection of genome-wide modifications in a nucleic acid genome obtained from a cell or tissue caused by the activity of a designer nuclease comprising the following steps: a) Extraction of the genomic DNA from cells that were exposed to a designer nuclease under conditions which allow the designer nuclease to introduce a DNA double-strand break (DSB) in the genomic DNA of the cell, b) fragmentation of the nucleic acid to obtain random fragments, c) performing an end repair in order to obtain blunt ends, d) ligation with a linker comprising a sequence complementary to a so called “linker primer”, e) performing a first nucleic acid amplification reaction with a “linker primer” and a so called “ON-target primer”, whereby one primer is located upstream and one primer is located downstream of the on-target site, wherein at least one decoy primer is present in the reaction mixture, f) performing a second nucleic acid amplification reaction whereby so called “nested primers” are added to the reaction mixture, whereby one primer is complementary to the on-target locus and one primer complementary to the linker sequence, g) performing a further nucleic acid amplification reaction whereby at least one code containing primers are added to the reaction mixture, h) sequencing of the nested and barcoded amplification product, and i) aligning the sequenced products with suitable bioinformatic means to a reference sequence to identify a chromosomal location that contains a genomic modification based on at least one DNA double strand break.

Disclosed is a method for high-throughput detection of genome-wide modifications in a nucleic acid genome obtained from a cell or tissue caused by the activity of a designer nuclease comprising the following steps: a) Extraction of the genomic DNA from cells that were exposed to a designer nuclease under conditions which allow the designer nuclease to introduce a DNA double-strand break (DSB) in the genomic DNA of the cell, b) fragmentation of the nucleic acid to obtain random fragments, c) performing an end repair in order to obtain blunt ends, d) ligation with a linker comprising a sequence complementary to a so called “linker primer”, e) performing a first nucleic acid amplification reaction with a “linker primer” and a so called “ON-target primer”, whereby one primer is located upstream and one primer is located downstream of the on-target site, wherein at least one decoy primer is present in the reaction mixture, f) performing a second nucleic acid amplification reaction whereby so called “nested primers” are added to the reaction mixture, whereby one primer is complementary to the on-target locus and one primer complementary to the linker sequence, g) performing a further nucleic acid amplification reaction whereby at least one code containing primers are added to the reaction mixture, h) sequencing of the nested and barcoded amplification product, and i) aligning the sequenced products with suitable bioinformatic means to a reference sequence to identify a chromosomal location that contains a genomic modification based on at least one DNA double strand break.

The present invention relates to a composition for a sequential polymerase chain reaction and a gene amplification method using the same. The composition for a sequential polymerase chain reaction comprises a template gene, a DNA polymerase, dNTP, a first forward primer, a first reverse primer, a second reverse primer and a third reverse primer so that a sequential polymerase chain reaction can be induced in one container. Unlike conventional nested PCR, a reactant-adding step is omitted in the sequential polymerase chain reaction according to the present invention, and thus the inflow of contaminants from the outside is low, the yield of polymerase chain reaction (PCR) of genes can be increased, and highly-reproducible polymerase chain reaction results can be obtained even if the presence of genes is very low.

The present invention relates to a composition for a sequential polymerase chain reaction and a gene amplification method using the same. The composition for a sequential polymerase chain reaction comprises a template gene, a DNA polymerase, dNTP, a first forward primer, a first reverse primer, a second reverse primer and a third reverse primer so that a sequential polymerase chain reaction can be induced in one container. Unlike conventional nested PCR, a reactant-adding step is omitted in the sequential polymerase chain reaction according to the present invention, and thus the inflow of contaminants from the outside is low, the yield of polymerase chain reaction (PCR) of genes can be increased, and highly-reproducible polymerase chain reaction results can be obtained even if the presence of genes is very low.

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.

qRT-PCR primers capable of detecting EML4-ALK gene variant based on circulating tumor cells at a more sensitive detection limit than a conventional method. Also disclosed is a lung cancer patient screening method which is capable of detecting EML4-ALK gene variant using circulating tumor cells even in a lung cancer patient on whom ALK-FISH testing has been difficult to perform, due to difficulty in collecting a solid lung cancer tissue sample, and is able to determine whether an anticancer drug targeting the EML4-ALK gene variant may be applied to the lung cancer patient.

The subject invention pertains to the rapid amplification and real-time monitoring of nucleic acid sequences at a constant temperature. Specifically, fluorescent or electroactive-labeled loop probes and primers are designed to be specific to identified regions of a target nucleic acid sequences and subsequently amplify and permit the identification of the presence of the target sequence. In this method, a DNA polymerase with no 5′ to 3′ exonuclease activity is added to extend the primers and probes, while a nicking endonuclease is added to specifically identify the amplified nucleic acid product and cleave the label from the loop probe-extended DNA duplex.

The subject invention pertains to the rapid amplification and real-time monitoring of nucleic acid sequences at a constant temperature. Specifically, fluorescent or electroactive-labeled loop probes and primers are designed to be specific to identified regions of a target nucleic acid sequences and subsequently amplify and permit the identification of the presence of the target sequence. In this method, a DNA polymerase with no 5′ to 3′ exonuclease activity is added to extend the primers and probes, while a nicking endonuclease is added to specifically identify the amplified nucleic acid product and cleave the label from the loop probe-extended DNA duplex.