Method for separating homoduplexed and heteroduplexed nucleic acids
View Patent ↗The invention includes methods and apparatus for separating heteroduplexed nucleic acids from homoduplexed nucleic acids having similar sequences and being at a much higher concentration. The heteroduplexed nucleic acids may be separated through the application of a time varying driving field and a time-varying mobility field to a sample of heteroduplexed and homoduplexed nucleic acids in a separation medium. Once the heteroduplexed nucleic acids are isolated and recovered, it is straightforward to analyze the sequences of the heteroduplexed nucleic acids, e.g., using sequencing or hybrid assays.
1. A method for separating a mixture of homoduplexed and heteroduplexed nucleic acids, comprising;
loading a sample comprising a mixture of homoduplexed and heteroduplexed nucleic acids on a separation medium;
applying a time-varying driving field to the separation medium; and
applying a time-varying mobility varying field to the separation medium,
thereby causing the homoduplexed nucleic acids to be separated from the heteroduplexed nucleic acids.
2. The method of claim 1 , wherein the time-varying mobility varying field is temperature.
3. The method of claim 1 , wherein the time-varying driving field comprises two non-collinear electric fields.
4. The method of claim 1 , wherein the time-varying driving field comprises three non-collinear electric fields.
5. The method of claim 1 , wherein the heteroduplexed nucleic acids comprise a first sequence and a second sequence, and either the first or the second sequence is known.
6. The method of claim 5 , wherein the known sequence comprises a sequence that is associated with an oncogene.
7. The method of claim 6 , wherein the oncogene is selected from a RAS-related oncogene, a p53-related tumor suppressor gene, and a WNT-related oncogene.
8. The method of claim 1 , wherein the heteroduplexed nucleic acids comprise a first sequence and a second sequence, and neither the first nor the second sequence is known.
9. The method of claim 1 , wherein the heteroduplexed nucleic acids comprise a first sequence and a second sequence and the first and second sequences are complementary strand paired except for a mutation selected from a nucleotide polymorphism, a base deletion, or a base insertion.
10. The method of claim 9 , wherein the mutation is a single nucleotide polymorphism (SNP).
11. The method of claim 1 , wherein the heteroduplexed nucleic acids comprise a first sequence and a second sequence and the first and second sequences are complementary strand paired except for a chemical modification selected from differential methylation or differential acetylation.
12. The method of claim 1 , further comprising:
obtaining a sample comprising nucleic acids;
denaturing the nucleic acids to produce single-stranded oligomers; and
reannealing the single-stranded oligomers to create a sample comprising a mixture of homoduplexed and heteroduplexed nucleic acids.
13. The method of claim 12 , wherein the sample comprising nucleic acids comprises a dominant nucleic acid sequence comprising a majority of the nucleic acid sequences in the sample.
14. The method of claim 13 , wherein at least a portion of the heteroduplexed nucleic acids comprises a pairing strand having the dominant nucleic acid sequence and a non-dominant nucleic acid sequence.
15. The method of claim 1 , further comprising recovering the heteroduplexed nucleic acids.
16. The method of claim 15 , further comprising amplifying the recovered heteroduplexed nucleic acids.
17. The method of claim 15 , further comprising sequencing the recovered heteroduplexed nucleic acids.
18. The method of claim 12 , wherein the sample comprising nucleic acids comprises PCR amplicons.
19. The method of claim 18 , wherein the PCR amplicons comprise primers with a GC clamp region.
20. A method for determining a mutation in a nucleic acid sample, comprising:
amplifying a plurality of non-identical nucleic acids in a sample to create a plurality of non-identical amplicons;
denaturing and reannealing the non-identical amplicons to produce a sample comprising a mixture of homoduplexed and heteroduplexed nucleic acids;
loading the sample comprising a mixture of homoduplexed and heteroduplexed nucleic acids on a separation medium;
applying both a time-varying driving field and a time-varying mobility varying field to the separation medium, thereby causing the homoduplexed nucleic acids to be separated from the heteroduplexed nucleic acids; and
recovering the heteroduplexed nucleic acids.