Abstract: A method for amplifying free nucleic acids (NAs) directly from one drop unpurified crude sample. The sample comprises nucleic acid fragments. The method includes (a) mixing an unpurified sample with a buffer to form an uniformly mixed solution; (b) adding the mixed solution to a tube then heating the tube to denature the proteins in the mixed solution, and cooling to room temperature; (c) subjecting he nucleic acid fragments in the mixture to a processing reaction required for adaptor-dependent PCR; (d) performing a ligation reaction between the processed nucleic acid fragments and adapters, forming nucleic acid fragments ligated with adapter in both ends, wherein the adapter is a complementary double-stranded nucleic acid (dsNA) fragment, one of which is an oligonucleotide with 5?-phosphate and the other is an oligonucleotide with thymine (T) or uracil (U); and (e) performing the adapter-dependent PCR to the nucleic acid fragments in the mixed solution.

Abstract: The present invention provides a method, entitled gene net-digital polymerase chain reaction (gn-dPCR), for the analysis of nucleic acids in a sample.

Abstract: The present invention relates to a method for amplifying and detecting ribonucleic acid (RNA) fragments. In particular, the method of the present invention comprises conversion of RNA fragments to cDNA and DNA amplification. The present invention also provides a kit for performing the method as described herein.

Abstract: The present invention relates to a method for detecting nucleic acid (NA) molecules in samples. More particularly, the present invention relates to an improved digital PCR-based method for detecting specific nucleic acid sequence(s). The present invention is useful for research and diagnostic applications with increased sensitivity and accuracy. The present invention also provides a kit for performing the method for assessingnucleic acids in samples as described herein.

Abstract: An adaptor for use in amplifying all linear, double-stranded nucleic acid molecules of unknown sequences in a sample is disclosed. The adaptor consists of: (1) the first oligonucleotide (P-oligo) with a phosphate at the 5? end and without an additional thymine nucleotide at the 3? end; and (2) the second oligonucleotide (T˜oligo) with an extra 3?-T and without a 5?-phosphate. The P-oligo and T-oligo are complementary to each other except at the 3?-T (thymine) in the T-oligo. The adaptor is ligated to nucleic acids of unknown sequences which have an extra A in the 3? end (3?˜A overhang) to form adaptor-ligated target nucleic acids. The T-oligo is then employed as a single primer for T-oligo-primed polymerase chain reaction (TOP-PCR) and amplifies the nucleic acids of unknown sequences in full-length.

Abstract: An adaptor for use in amplifying all linear, double-stranded nucleic acid molecules of unknown sequences in a sample is disclosed. The adaptor consists of: (1) the first oligonucleotide (P-oligo) with a phosphate at the 5? end and without an additional thymine nucleotide at the 3? end; and (2) the second oligonucleotide (T˜oligo) with an extra 3?-T and without a 5?-phosphate. The P-oligo and T-oligo are complementary to each other except at the 3?-T (thymine) in the T-oligo. The adaptor is ligated to nucleic acids of unknown sequences which have an extra A in the 3? end (3?˜A overhang) to form adaptor-ligated target nucleic acids. The T-oligo is then employed as a single primer for T-oligo-primed polymerase chain reaction (TOP-PCR) and amplifies the nucleic acids of unknown sequences in full-length.

Abstract: A method of generating a barcoded Paired-End Ditag (bPED) nucleic acid fragment is disclosed. The method comprises: a) performing a first ligation by ligating a half-adaptor with one or two 3?-overhanging ends to a target nucleic acid to obtain a nucleic acid fragment with two ends each attached to one of the half-adaptor, the half adaptor comprising a half-barcode and a restriction enzyme (RE) recognition site; b) performing a second ligation by ligating two of the half-adaptor at the two ends of the nucleic acid fragment to form a circularized nucleic acid construct, wherein the circularized nucleic acid construct comprises a full-size barcoded adaptor; and c) digesting the circularized nucleic acid construct with a RE that cleaves at a defined distance from the RE recognition site, and thereby generating the bPED nucleic acid fragment.

Abstract: Multiplex barcoded Paired-End Ditag (mbPED) library construction for ultra high throughput sequencing is disclosed. The mbPED library comprises multiple types of barcoded Paired-End Ditag (bPED) nucleic acid fragment constructs, each of which comprises a unique barcoded adaptor, a first tag, and a second tag linked to the first tag via the barcoded adaptor. The two tags are the 5?- and 3?-ends of a nucleic acid molecule from which they originate. The barcoded adaptor comprises a barcode, a first polynucleotide sequence comprising a first restriction enzyme (RE) recognition site, and a second polynucleotide sequence comprising a second RE recognition site and covalently linked to the first polynucleotide sequence via the barcode. The two REs lead to cleavage of a nucleic acid at a defined distance from their recognition sites. The length of the adaptor is set so that the bPED nucleic acid fragment fits one-step sequencing.

Abstract: There is provided a method and system for processing and/or mapping ditag nucleotide sequence(s) to a genome, the ditag sequence comprising the 5? terminal tag and the 3? terminal tag of a nucleic acid molecule or fragment thereof or genomic fragment. The method of processing comprises preparing a database or file comprising at least one ditag sequence. The method of mapping comprises preparing a database or file of ditag(s), and mapping the ditag sequence(s) to the genome, comprising matching the 5? and the 3? terminal tags of the ditag sequence to at least a portion of the genome.

Abstract: A method of generating a barcoded Paired-End Ditag (bPED) nucleic acid fragment is disclosed. The method comprises: a) performing a first ligation by ligating a half-adaptor with one or two 3?-overhanging ends to a target nucleic acid to obtain a nucleic acid fragment with two ends each attached to one of the half-adaptor, the half adaptor comprising a half-barcode and a restriction enzyme (RE) recognition site; b) performing a second ligation by ligating two of the half-adaptor at the two ends of the nucleic acid fragment to form a circularized nucleic acid construct, wherein the circularized nucleic acid construct comprises a full-size barcoded adaptor; and c) digesting the circularized nucleic acid construct with a RE that cleaves at a defined distance from the RE recognition site, and thereby generating the bPED nucleic acid fragment.

Abstract: Multiplex barcoded Paired-End Ditag (mbPED) library construction for ultra high throughput sequencing is disclosed. The mbPED library comprises multiple types of barcoded Paired-End Ditag (bPED) nucleic acid fragment constructs, each of which comprises a unique barcoded adaptor, a first tag, and a second tag linked to the first tag via the barcoded adaptor. The two tags are the 5?- and 3?-ends of a nucleic acid molecule from which they originate. The barcoded adaptor comprises a barcode, a first polynucleotide sequence comprising a first restriction enzyme (RE) recognition site, and a second polynucleotide sequence comprising a second RE recognition site and covalently linked to the first polynucleotide sequence via the barcode. The two REs lead to cleavage of a nucleic acid at a defined distance from their recognition sites. The length of the adaptor is set so that the bPED nucleic acid fragment fits one-step sequencing.