Methods for detecting nucleic acid sequence variation
AssigneeThe Government of the U.S.A. as represented by the Secretary of the Dept. of Health & Human Services
InventorsRobert H. Lipsky, Chiara M. Mazzanti, David Goldman
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Abstract
Sequence variations, such as polymorphisms, are detected by detecting differences between or among melting profiles of plural nucleic acids. Melting profiles are produced by observing the nucleic acids during a temperature change over a period of time. If the nucleic acids are diluted into solutions for analysis, the nucleic acid concentrations between or among the solutions can be substantially the same. The melting profiles and sequence variation can be used to identify changes in a wild-type nucleic acid sequence, such as a single-nucleotide polymorphism (SNP), a small insertion or deletion, or a small inversion.
Claims45
A method of detecting a nucleic acid sequence variation, comprising:
- denaturing a first double-stranded nucleic acid of at least 105 bp in length comprising a first solution;
- denaturing a second double-stranded nucleic acid of at least 105 bp in length comprising a second solution, wherein the nucleic acid concentration of the first solution is substantially the same as the nucleic acid concentration of the second solution;
- allowing the first and second nucleic acids to reanneal;
- determining the melting profiles of the first and second nucleic acids;
- and
- detecting a difference between the melting profile of the first nucleic acid and the melting profile of the second nucleic acid, the difference between melting profiles indicating a sequence variation between the first and second nucleic acids.
A method of detecting a nucleic acid sequence variation, comprising:
- amplifying a first nucleic acid to produce a first amplicon having a length greater than 104 bp and amplifying a second nucleic acid to produce a second amplicon having a length greater than 104 bp;
- diluting the first amplicon in a first solution and diluting the second amplicon in a second solution, wherein the nucleic acid concentrations of the first and second solutions are substantially the same;
- determining melting profiles for the first and second amplicons, wherein a difference between the melting profiles indicates a difference in the nucleic acid sequences of the amplicons.
A method of detecting a nucleic acid sequence variation within an array of nucleic acids, comprising:
- denaturing plural double-stranded nucleic acids in an array, wherein each nucleic acid is at least 105 bp in length and each nucleic acid comprises a nucleic acid solution, and wherein the nucleic acid concentrations of the solutions are substantially the same;
- allowing the nucleic acids to reanneal;
- determining the melting profiles of the nucleic acids;
- and
- detecting a difference among the melting profiles of the nucleic acids, the difference among melting profiles indicating a sequence variation.
Dependencies are inferred from the claim text.