Dna sequencing by stepwise ligation and cleavage

Details Dna sequencing by stepwise ligation and cleavage Dna sequencing by stepwise ligation and cleavage

C - Chemistry, Metallurgy

12

Q

C12Q 1/68 (2006.01)

Patent

CA 2163662

The invention provides a method of nucleic acid sequence analysis based on repeated cycles of ligation to and cleavage of probes at the terminus of a target polynucleotide. At each such cycle one or more terminal nucleotides are identified and one or more nucleotides are removed from the end of the target polynucleotide, such that further cycles of ligation and cleavage can take place. At each cycle the target sequence is shortened by one or more nucleotides until the nucleotide sequence of the target polynucleotide is determined. The method obviates electrophoretic separation of similarly sized DNA fragments and eliminates the difficulties associated with the detection and analysis of spatially overlapping bands of DNA fragments in a gel, or like medium. The invention further obviates the need to generate DNA fragments from long single stranded templates with a DNA polymerase.

Procédé d'analyse de séquences d'acides nucléiques basé sur des cycles répétés de ligature et de clivage de sondes situées à la terminaison d'un polynucléotide cible. A chacun de ces cycles, un ou plusieurs nucléotides de terminaison sont identifiés et un ou plusieurs nucléotides sont éliminés de la terminaison du polynucléotide cible, si bien que de nouveaux cycles de ligature et de clivage peuvent avoir lieu. A chaque cycle, la séquence cible est raccourcie d'un ou plusieurs nucléotides jusqu'à ce que la séquence nucléotidique du polynucléotide cible soit déterminée. Le présent procédé rend superflue la séparation électrophorétique de fragments d'ADN de taille similaire et élimine les difficultés associées à la détection et à l'analyse de bandes se chevauchant spatialement de fragments d'ADN dans un gel, ou milieu analogue. La présente invention rend en outre superflu le besoin de produire des fragments d'ADN à partir de matrices longues à simple brin à l'aide d'une ADN polymérase.

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