US 5,856,093 AGranted

Method of determining zygosity by ligation and cleavage

AssigneeLynx Therapeutics, Inc.

InventorSydney Brenner

Life of this patent

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Abstract

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.

Claims21

Dependent claims

A method of determining the zygosity of an individual at a predetermined genetic locus having a plurality of allelic forms of DNA, the method comprising the steps of:

(a) providing a sample of the DNA from the predetermined genetic locus such that the sample of DNA comprises polynucleotides, each polynucleotide of the sample having a protruding strand and a recessed strand;
(b) ligating a probe having a protruding strand and a nuclease recognition site to an end of each polynucleotide to form one or more ligated complexes, the one or more ligated complexes being formed only from those probes whose protruding strands form perfectly matched duplexes with the protruding strands of the polynucleotides of the sample, and the nuclease recognition site being of a nuclease whose cleavage site is separate from its recognition site;
(c) identifying the kind and relative abundance of nucleotides in the protruding strand of the polynucleotide, by the identity of the probe ligated thereto or by extending a strand of the polynucleotide or probe;
(d) cleaving the ligated complexes with said nuclease that recognizes said nuclease recognition site and cuts the ligated complexes to give an augmented probe and a new protruding strand on the polynucleotide;
and
(e) repeating steps (b) through (d) until the nucleotide sequences of the polynucleotides of the genetic locus are determined, thereby determining the zygosity of the individual.

3 more elements

Dependencies are inferred from the claim text.

Facts

Application: 08/478,239Filed 7 Jun 1995 · continuation
Priority: 4 Apr 1994
Granted: 5 Jan 1999
Term ends: Not estimatedThe source records don’t support an estimate.

Classification6 codes

C12Q 1/6869
Involving nucleic acids: methods for sequencing
C12Q 1/6862
Nucleic acid amplification reactions: ligase chain reaction [LCR]
C12Q 1/6874
Methods for sequencing: involving nucleic acid arrays, e.g. sequencing by hybridisation
Y02P 20/582
Improvements relating to the production of bulk chemicals: recycling of unreacted starting or intermediate materials

Family

Serial222,300Parent of this continuation-in-part · filed 4 Apr 1994
Serial280,441Parent of this continuation-in-part · filed 25 Jul 1994
Serial410,116Parent of this continuation · filed 24 Mar 1995
US 5,552,278 AParent of this continuation-in-part
US 5,856,093 AThis patent

From the US filing history. Foreign family members aren’t listed.