Academia Sinica

Kuo Ping Chiu

Sheng-Chung Chen

Yi Hung Chou

Yu-Shin Nai

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&#x2032; end; and (2) the second oligonucleotide (T&#x2dc;oligo) with an extra 3&#x2032;-T and without a 5&#x2032;-phosphate. The P-oligo and T-oligo are complementary to each other except at the 3&#x2032;-T (thymine) in the T-oligo. The adaptor is ligated to nucleic acids of unknown sequences which have an extra A in the 3&#x2032; end (3&#x2032;&#x2dc;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.

Methods for full-length amplification of double-stranded linear nucleic acids of unknown sequences

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.

Multiplex barcoded paired-end diTag (mbPED) sequencing approach and ITS application in fusion gene identification

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&#x2032;-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.

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Taipei, Taiwan

Kuo Ping Chiu