The present invention provides a method for analysis of target nucleic acids which are present in low amounts. In particular, the method comprises the following steps: i. providing a sample wherein target nucleic acids are present in a low amount, ii. generating a reduced representation library of said target nucleic acids by a method comprising: fragmenting said target nucleic acids; ligating adaptors to said fragments; and selecting a subset of said adaptor-ligated fragments, iii. massively parallel sequencing said reduced representation library, and iv. identifying variants in said target nucleic acids by analyzing results obtained by said sequencing.

The present invention provides a method for analysis of target nucleic acids which are present in low amounts. In particular, the method comprises the following steps: i. providing a sample wherein target nucleic acids are present in a low amount, ii. generating a reduced representation library of said target nucleic acids by a method comprising: fragmenting said target nucleic acids; ligating adaptors to said fragments; and selecting a subset of said adaptor-ligated fragments, iii. massively parallel sequencing said reduced representation library, and iv. identifying variants in said target nucleic acids by analyzing results obtained by said sequencing.

Methods for detecting length polymorphisms in a DNA sample are provided. The methods include steps of amplifying a target region of the DNA in the sample, wherein the DNA sample is diluted prior to the amplifying step to provide a plurality of amplified samples in which 0 or 1 target DNA strand is amplified in each amplified sample and wherein at least one amplified sample includes 1 target DNA strand; depositing the plurality of amplified samples onto a surface; imaging the plurality of amplified samples deposited on the surface using atomic force microscopy (AFM) to determine an amplicon length distribution; comparing the amplicon length distribution to a corresponding amplicon length distribution obtained from a reference DNA sample that does not contain a length polymorphism in the target region; and detecting a length polymorphism in the target region of the DNA sample when the amplicon length distribution is distinct from the corresponding amplicon length distribution.

Methods for detecting length polymorphisms in a DNA sample are provided. The methods include steps of amplifying a target region of the DNA in the sample, wherein the DNA sample is diluted prior to the amplifying step to provide a plurality of amplified samples in which 0 or 1 target DNA strand is amplified in each amplified sample and wherein at least one amplified sample includes 1 target DNA strand; depositing the plurality of amplified samples onto a surface; imaging the plurality of amplified samples deposited on the surface using atomic force microscopy (AFM) to determine an amplicon length distribution; comparing the amplicon length distribution to a corresponding amplicon length distribution obtained from a reference DNA sample that does not contain a length polymorphism in the target region; and detecting a length polymorphism in the target region of the DNA sample when the amplicon length distribution is distinct from the corresponding amplicon length distribution.

Polynucleotide sequencing methods include incubating unlabeled nucleotides with a cluster of template polynucleotide strands having the same sequence when the identity of the previously added labeled nucleotide is being detected. The detection step provides time for the addition of the unlabeled nucleotides to be incorporated into the copy strands in which the previously added labeled nucleotide did not get incorporated. Thus, at the end of the detection step, all or most of the copy strands will be in phase and ready to incorporate the appropriate labeled nucleotide in the subsequence incorporate step.

A diagnosis kit is disclosed. The diagnosis kit according to an embodiment of the present invention includes a concentration channel into which a sample containing a methylated DNA is introduced, a concentration chamber connected to the concentration channel, wherein the methylated DNA is concentrated by an ion concentration polarization (ICP) phenomenon and moves to the concentration chamber, a sensing chamber connected to the concentration chamber to allow the methylated DNA inside the concentration chamber to move, allow the methylated DNA moved from inside of the concentration chamber to be hybridized, and allow a methylated DNA binding protein to be bound to the hybridized methylated DNA, and a sensor configured to acquire a first electrochemical signal inside the sensing chamber when the methylated DNA is hybridized and acquire a second electrochemical signal inside the sensing chamber when the methylated DNA binding protein is bound.

A diagnosis kit is disclosed. The diagnosis kit according to an embodiment of the present invention includes a concentration channel into which a sample containing a methylated DNA is introduced, a concentration chamber connected to the concentration channel, wherein the methylated DNA is concentrated by an ion concentration polarization (ICP) phenomenon and moves to the concentration chamber, a sensing chamber connected to the concentration chamber to allow the methylated DNA inside the concentration chamber to move, allow the methylated DNA moved from inside of the concentration chamber to be hybridized, and allow a methylated DNA binding protein to be bound to the hybridized methylated DNA, and a sensor configured to acquire a first electrochemical signal inside the sensing chamber when the methylated DNA is hybridized and acquire a second electrochemical signal inside the sensing chamber when the methylated DNA binding protein is bound.

Disclosed herein are methods of detecting sepsis in a patient in need thereof, the method comprising: receiving a patient sample, a pathogenic primer mix, a fluorophore label probe, and a dPCR buffer in one or more wells of a microfluidic device and detecting fluorescent light signal from the droplets to determine sepsis in the patient. The microfluidic device comprises a droplet generation channel in fluid communication with the one or more wells, adapted to generate droplets comprising the patient sample, one or more pathogenic primers, a fluorophore label probe, and dPCR buffer. Furthermore, the microfluidic device comprises a chamber in fluid communication with the droplet generation channel for collecting the droplets generated by the droplet generation channel and performing a PCR reaction in the droplets.

Disclosed herein are methods of detecting sepsis in a patient in need thereof, the method comprising: receiving a patient sample, a pathogenic primer mix, a fluorophore label probe, and a dPCR buffer in one or more wells of a microfluidic device and detecting fluorescent light signal from the droplets to determine sepsis in the patient. The microfluidic device comprises a droplet generation channel in fluid communication with the one or more wells, adapted to generate droplets comprising the patient sample, one or more pathogenic primers, a fluorophore label probe, and dPCR buffer. Furthermore, the microfluidic device comprises a chamber in fluid communication with the droplet generation channel for collecting the droplets generated by the droplet generation channel and performing a PCR reaction in the droplets.

Methods are provided that, for example, include (a) combining ssDNA containing a modified nucleotide (e.g., a ssDNA with a modified nucleotide proximate to its 5′ end) with a DNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate a first ssDNA fragment having a 3′OH and a second ssDNA fragment having the modified nucleotide); wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio (m/m); and (b) cleaving at least 95% of the ssDNA at the modified nucleotide. In some embodiments, a method may comprise (a) combining (i) a ssDNA comprising a modified nucleotide (e.g., proximate to its 5′ end) with (ii) a DNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate (after cleavage) a first ssDNA fragment having a 3′OH and a second ssDNA fragment comprising the modified nucleotide) wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio and cleaving at least 95% of the ssDNA at the modified nucleotide. In some embodiments, methods provided herein may include (a) combining (i) a ssDNA (1) immobilized on a substrate and (2) comprising a modified nucleotide with (ii) a ssDNA cleaving enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate (after cleavage) a first ssDNA fragment having a 3′OH and a second ssDNA fragment comprising the modified nucleotide) ; and (b) cleaving the immobilized ssDNA to release the second single stranded DNA fragment from the substrate. At least 95% (m/m) of an ssDNA comprising a modified nucleotide may be cleaved in less than 60 minutes.