Provided are a method for constructing a library based on an RNA sample and uses thereof. The method includes: step 1 of subjecting the RNA sample to a reverse transcription reaction to obtain DNA-RNA hybrid strands; step 2 of performing reaction of the DNA-RNA hybrid strands with an endoribonuclease, a first DNA polymerase, a second DNA polymerase, and dATPs to obtain a double-stranded DNA added with dA-tail, where the first DNA polymerase has a 5′-3′ exonuclease activity and a 3′-5′ exonuclease activity, and the second DNA polymerase has no 3′-5′ exonuclease activity; step 3 of ligating the double-stranded DNA added with dA-tail and a sequencing adaptor to obtain a ligated product; and step 4 of subjecting the ligated product to PCR amplification to obtain a sequencing library.

A method and system have been provided to perform high speed nucleic acid melting analysis while still obtaining accurate melting curve sufficient for genotyping. This rapid ability to interrogate DNA should be useful whenever time to result is important, such as in molecular point of care testing. Specifically, microfluidics enables genotyping by melting analysis at rates up to 50° C./s, requiring less than is to acquire an entire melting curve. High speed melting reduces the time for melting analysis, decreases errors, and improves genotype discrimination of small amplicons.

A method and system have been provided to perform high speed nucleic acid melting analysis while still obtaining accurate melting curve sufficient for genotyping. This rapid ability to interrogate DNA should be useful whenever time to result is important, such as in molecular point of care testing. Specifically, microfluidics enables genotyping by melting analysis at rates up to 50° C./s, requiring less than is to acquire an entire melting curve. High speed melting reduces the time for melting analysis, decreases errors, and improves genotype discrimination of small amplicons.

Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.

Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.

Disclosed herein are methods, reagent kits, and compositions for performing a bisulfite conversion of DNA directly from a biological sample including a patient's urine sample or a slide-mounted FFPE tissue sample. For example, a method of performing a bisulfite conversion of DNA can comprise certain preliminary steps for processing the biological sample and transferring a portion of the processed sample into a reaction vessel containing a bisulfite mixture. The method can further comprise heating the reaction vessel containing the biological sample and the bisulfite mixture at several heating temperatures and subsequently holding the reaction vessel at a holding temperature for a holding period. The method can also comprise certain bisulfite removal steps, desulfonation steps, and removal of the desulfonation solution. A final elution step can yield the converted DNA for further downstream sequencing and analysis.

Disclosed herein are methods, reagent kits, and compositions for performing a bisulfite conversion of DNA directly from a biological sample including a patient's urine sample or a slide-mounted FFPE tissue sample. For example, a method of performing a bisulfite conversion of DNA can comprise certain preliminary steps for processing the biological sample and transferring a portion of the processed sample into a reaction vessel containing a bisulfite mixture. The method can further comprise heating the reaction vessel containing the biological sample and the bisulfite mixture at several heating temperatures and subsequently holding the reaction vessel at a holding temperature for a holding period. The method can also comprise certain bisulfite removal steps, desulfonation steps, and removal of the desulfonation solution. A final elution step can yield the converted DNA for further downstream sequencing and analysis.

The invention relates to a method for the duplication of nucleic acids by means of a polymerase chain reaction, in the case of which a cycle consisting of the steps of denaturing, annealing and elongation is repeatedly performed. In one embodiment, in at least one passage of the cycle, the quotient of the duration of effect t.sub.A and the reaction volume V.sub.r irradiated by the energy source is less than 1 seconds per microliter. In another embodiment, in at least one passage of the cycle, the ratio of the duration of effect (t.sub.A) and the duration of the PCR cycle (t.sub.c) is less than 20%. In certain embodiments, the yield (g) of nucleic acids at the end of at least one of the passages of the cycle is less than 80% of the nucleic acids present at the start of the passage.

The invention relates to a method for the duplication of nucleic acids by means of a polymerase chain reaction, in the case of which a cycle consisting of the steps of denaturing, annealing and elongation is repeatedly performed. In one embodiment, in at least one passage of the cycle, the quotient of the duration of effect t.sub.A and the reaction volume V.sub.r irradiated by the energy source is less than 1 seconds per microliter. In another embodiment, in at least one passage of the cycle, the ratio of the duration of effect (t.sub.A) and the duration of the PCR cycle (t.sub.c) is less than 20%. In certain embodiments, the yield (g) of nucleic acids at the end of at least one of the passages of the cycle is less than 80% of the nucleic acids present at the start of the passage.

Disclosed herein is a method for providing a preparation for detecting a target nucleic acid sequence in a specimen. According to an embodiment, conventional nucleic acid extraction processes performed in many steps can be omitted, whereby the shortage of nucleic acid extraction reagents can be solved and a preparation for detecting target nucleic acid sequence in a specimen can be supplied in an inexpensive and simple manner.