ANALYSIS OF HETEROGENEOUS NUCLEIC ACID SAMPLES

• US 20090325153A1
• Filed: 04/21/2006
• Published: 12/31/2009
• Est. Priority Date: 04/21/2005
• Status: Active Grant

• Current Assignee: Esoterix Genetic Laboratories LLC (Laboratory Corporation Of America Holdings)
• Original Assignee: Exact Sciences Corporation
• Inventors: Shuber, Anthony P.
• Granted Patent: US 9,777,314 B2
• Time in Patent Office: Days
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation

Abstract

Methods for capturing and characterizing low frequency nucleic acid molecules indicative of diseases such as cancer (e.g. adenomas or early stage cancers) are provided. In some aspects, a low complexity capture technique is combined with a high complexity analytical technique. In some aspects, samples may be analyzed using a digital analysis and/or a single molecule sequencing technique.

Claims

1. A method of detecting an altered or mutant nucleic acid molecule that is present at low frequency in a biological sample comprising

isolating a threshold number of genome equivalents of a target nucleic acid of low genomic complexity from a biological sample;

and

analyzing a statistically significant number of molecules of the isolated target nucleic acid using a technique useful for analyzing nucleic acid samples of high genomic complexity,

wherein the analyzing provides information about the presence or absence of an altered or mutant nucleic acid molecule in the biological sample.

2. The method of claim 1, wherein the biological sample is stool, whole blood, serum, plasma, tears, saliva, nasal fluid, sputum, ear fluid, genital fluid, breast fluid, milk, colostrum, placental fluid, amniotic fluid, perspirate, synovial fluid, ascites fluid, cerebrospinal fluid, bile, gastric fluid, aqueous humor, vitreous humor, gastrointestinal fluid, exudate, transudate, pleural fluid, pericardial fluid, semen, upper airway fluid, peritoneal fluid, fluid harvested from a site of an immune response, fluid harvested from a pooled collection site, bronchial lavage, urine, biopsy material, a nucleated cell sample, a fluid associated with a mucosal surface, hair, or skin.

3. The method of claim 1, wherein the mutant or altered nucleic acid molecule is suspected to be present in the biological sample at a frequency of about 0.0001%, about 0.001%, about 0.01%, about 0.1% or about 1.0%.

4. The method of claim 1, wherein the mutant of altered nucleic acid molecule is present in the biological sample at a frequency of about 1.0%.

5. The method of claim 1, wherein the presence of a mutant or altered nucleic acid molecule is indicative of an adenoma, early stage cancer, or other disease or condition in a subject from which the biological sample was obtained.

6. The method of claim 1, wherein the target nucleic acid molecules are at least 70 base pairs long.

7. The method of claim 1, wherein the target nucleic acid molecules are at least 200 base pairs long.

8. The method of claim 1, wherein the analyzing comprises single nucleic acid molecule sequencing, high speed parallel molecular nucleic acid sequencing, or high throughput nucleic acid sequencing.

9. The method of claim 1, wherein the mutant or altered nucleic acid molecule is suspected to be present in the biological sample at a frequency of x %, and 100/x or more genome equivalents of the target nucleic acid are analyzed.

10. The method of claim 9, wherein each genome equivalent of the target nucleic acid is sequenced.

11. The method of claim 10, wherein a portion of each genome equivalent of the target nucleic acid is sequenced.

12. The method of claim 10 or 11, wherein a single molecule sequencing technique is used to obtain sequence information for each genome equivalent of the target nucleic acid.