Method for isolating target nucleic acid using heteroduplex binding proteins

Patent metadata

Field	Value
Publication number	US-11130986-B2
Application number	US-201615575699-A
Country	US
Kind code	B2
Filing date	May 19, 2016
Priority date	May 20, 2015
Publication date	Sep 28, 2021
Grant date	Sep 28, 2021

Abstract

The invention includes methods and apparatus for separating mutations, especially rare and unknown mutations, using heteroduplex binding proteins. Nucleic acids may optionally be nicked at or near the mutation in order to promote heteroduplex binding protein recognition and binding. In particular, using the disclosed methods, it is possible to separate heteroduplexed nucleic acid strand pair from homoduplexed nucleic acid strand pairs having similar sequences and being at a much higher concentration. 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.

First claim

What is claimed is:

1. A method for separating target heteroduplex nucleic acid from background homoduplex nucleic acid, comprising:
obtaining a sample comprising nucleic acid;
denaturing the nucleic acid to produce single-stranded nucleic acid;
reannealing the single-stranded nucleic acid with reference nucleic acids to create a mixture of target heteroduplex nucleic acid and background homoduplex nucleic acid;
nicking the target heteroduplex nucleic acid and background homoduplex nucleic acid;
and then loading the mixture of target heteroduplex nucleic acid and background homoduplex nucleic acid on a separation medium comprising a heteroduplex-binding protein, wherein the target heteroduplex nucleic acid and the background homoduplex nucleic acid differ by at least one base, wherein the ratio of target nucleic acid to background nucleic acid is less than 1:10,000;
applying a time-varying driving field and a time-varying mobility varying field to the separation medium,
thereby causing the target heteroduplex nucleic acid to be separated from the background homoduplex nucleic acid.

2. The method of claim 1 , wherein the heteroduplex-binding protein comprises a mismatch recognition domain.

3. The method of claim 1 , wherein the heteroduplex-binding protein is a MutS protein or a modified MutS protein.

4. The method of claim 1 , wherein the amino acid sequence of the heteroduplex-binding protein is at least 85% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

5. The method of claim 1 , wherein the time-varying driving field comprises two non-collinear electric fields.

6. The method of claim 5 , wherein the time-varying driving field comprises three non-collinear electric fields.

7. The method of claim 1 , wherein the background homoduplex nucleic acid comprises wild-type nucleic acid and the target heteroduplex nucleic acid comprises a mutation.

8. The method of claim 1 , wherein the target heteroduplex nucleic acid and homoduplex background nucleic acid are recovered from a biological sample selected from whole blood, serum, plasma, sputum, tissue, sweat, tears, urine, or aspirate.

9. The method of claim 8 , further comprising identifying a ratio of target heteroduplex nucleic acid to background homoduplex nucleic acid in the biological sample.

10. The method of claim 1 , wherein the ratio of target heteroduplex nucleic acid to background nucleic acid ratio is from 1:10,000 to 1:100,000.

11. The method of claim 4 , wherein the amino acid sequence of the heteroduplex-binding protein is at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 3.

12. The method of claim 1 , wherein nicking the target heteroduplex nucleic acid and background homoduplex nucleic acid is performed using an enzyme.

13. The method of claim 12 , further comprising ligating the homoduplex nucleic acid after nicking the target heteroduplex nucleic acid and background homoduplex nucleic acid.

14. A method for determining a mutation in a target nucleic acid, 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 in the presence of reference nucleic acids to create a mixture of homoduplex and heteroduplex nucleic acids;
nicking the target heteroduplex nucleic acid and background homoduplex nucleic acid;
and then loading the mixture of homoduplex and heteroduplex nucleic acids on a separation medium comprising a heteroduplex-binding protein;
applying both a time-varying driving field and a time-varying mobility varying field to the separation medium, thereby causing the homoduplex nucleic acids to be separated from the heteroduplex nucleic acids;
recovering the heteroduplex nucleic acids; and
sequencing the heteroduplex nucleic acids to determine a mutation in the target nucleic acid.

15. The method of claim 14 , wherein the amino acid sequence of the heteroduplex-binding protein is at least 95% identical to SEQ ID NO: 2 or 3.

16. The method of claim 14 , wherein the amino acid sequence of the heteroduplex-binding protein is SEQ ID NO: 2.

17. The method of claim 14 , wherein the amino acid sequence of the heteroduplex-binding protein is SEQ ID NO: 3.

18. The method of claim 14 , further comprising ligating the homoduplex nucleic acid after nicking the target heteroduplex nucleic acid and background homoduplex nucleic acid.

Assignees

• Quantum Si Inc

Inventors

• Marziali Andrea
• Despotovic Milenko
• Wiggin Matthew
• Pel Joel

Classifications

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q1/6811 Primary

  Selection methods for production or design of target specific oligonucleotides or binding molecules · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title