Multiplexing methods for indentifying nucleic acids using denaturing liquid chromatography
OwnerJames A. Apffel JR., Karen M. Hahnenberger +2inventors
InventorsJames A. Apffel JR., Karen M. Hahnenberger, Mel N. Kronick, Martin Verhoef
Life of this patent
Estimating the term…
Abstract
A method is provided for the separation of nucleic acid samples. The method includes: providing a test mixture of a plurality of nucleic acid samples, wherein each sample is labeled with a spectrally detectable tag; applying the test mixture to a reversed phase solid support; eluting the mixture under partially denaturing conditions; and detecting spectrally resolved signals produced by the nucleic acid samples labeled with the detectable tags.
Claims30
A method for separating nucleic acid samples in a test mixture, the method comprising:
- providing a test mixture comprising a plurality of nucleic acid samples, wherein each sample is labeled with a spectrally detectable tag;
- applying the test mixture to a reversed phase support;
- eluting the test mixture under partially denaturing conditions to separate at least one of the nucleic acid samples from the test mixture;
- and
- detecting spectrally resolved signals produced by the nucleic acid samples labeled with the detectable tags.
A method for detecting genotypic variations, the method comprising:
- providing a pre-mixture comprising one unlabeled nucleic acid sample and two or more reference genotypes comprising labeled nucleic acid, wherein each labeled nucleic acid is labeled with a different detectable tag and the unlabeled nucleic acid is present in an excess amount relative to the total amount of labeled nucleic acid;
- denaturing and reannealing the pre-mixture to form a test mixture comprising labeled/unlabeled nucleic acid duplexes;
- applying the test mixture to a reversed phase support;
- eluting the test mixture under partially denaturing conditions to separate at least one of the labeled/unlabeled nucleic acid duplexes from the test mixture;
- detecting spectrally resolved signals produced by the labeled nucleic acid to define an elution profile for each tag;
- and
- deducing from the elution profiles the composition of the unlabeled nucleic acid sample.
A method for determining haplotypes, the method comprising:
- providing a pre-mixture comprising one unlabeled nucleic acid sample and four or more reference haplotypes comprising labeled nucleic acid, wherein each labeled nucleic acid sample includes two or more alleles, each reference haplotype is labeled with a different detectable tag, and the unlabeled nucleic acid is present in an excess amount relative to the total amount of labeled nucleic acid;
- denaturing and reannealing the pre-mixture to form a test mixture comprising labeled/unlabeled nucleic acid duplexes;
- applying the test mixture to a reversed phase support;
- eluting the test mixture under partially denaturing conditions to separate at least one of the labeled/unlabeled nucleic acid duplexes from the test mixture;
- detecting spectrally resolved signals produced by the labeled nucleic acid to define an elution profile for each tag;
- and
- deducing from the elution profiles the composition of the unlabeled nucleic acid sample.
Dependencies are inferred from the claim text.