Detecting methylated mammalian nucleic acid in stool

• US 7,785,772 B2
• Filed: 01/23/2008
• Issued: 08/31/2010
• Est. Priority Date: 01/23/2007
• Status: Expired due to Fees

• Current Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic)
• Original Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic)
• Inventors: Zou, Hongzhi, Ahlquist, David A.
• Primary Examiner(s): Aeder; Sean E
• Application Number:
  US12/018,273
• Publication Number:
  US 20080220433A1
• Time in Patent Office: 951 Days
• Field of Search: None
• US Class Current: 435/4
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/154 Methylation markers

Abstract

This document includes methods and materials for enriching and detecting cancer markers. For example, this document includes methods and materials for enriching methylated mammalian nucleic acid from stool samples.

Claims

1. A method for enriching methylated mammalian nucleic acid if present in a stool sample, wherein said method comprises:

(a) contacting nucleic acid from a stool sample with an MBD polypeptide to form a nucleic acid-MBD polypeptide complex;

(b) eluting bacterial nucleic acid from said MBD polypeptide of the formed nucleic acid-MBD polypeptide complex using a buffer with less than or equal to 0.6 M NaCl, and

(c) eluting methylated mammalian nucleic acid from said complex if said stool sample contained methylated mammalian nucleic acid using a buffer with greater than 0.6 M NaCl, thereby forming an enriched, methylated mammalian nucleic acid sample.

2. The method of claim 1, wherein said MBD polypeptide is a human or rat MBD polypeptide.

3. The method of claim 1, wherein said enriched, methylated mammalian nucleic acid sample comprises a higher ratio of methylated mammalian nucleic acid to methylated bacterial nucleic acid than the ratio of methylated mammalian nucleic acid to methylated bacterial nucleic acid present in said stool sample.

4. A method for detecting a colorectal or supracolonic aerodigestive neoplasm in a mammal, said method comprising:

(a) contacting nucleic acid from a stool sample from said mammal with an MBD polypeptide to form a nucleic acid-MBD polypeptide complex;

(b) eluting bacterial nucleic acid from said MBD polypeptide of the formed nucleic acid-MBD polypeptide complex using a buffer with less than or equal to 0.6 M NaCl;

(c) eluting methylated mammalian nucleic acid from said complex if said stool sample contained methylated mammalian nucleic acid using a buffer with greater than 0.6 M NaCl, thereby forming an enriched, methylated mammalian nucleic acid sample if said stool sample contained methylated mammalian nucleic acid; and

(d) determining whether or not said enriched, methylated mammalian nucleic acid sample comprises a methylated mammalian nucleic acid marker indicative of said colonic or supracolonic aerodigestive neoplasm, wherein the presence of said marker indicates that said mammal has said colonic or supracolonic aerodigestive neoplasm.

5. The method of claim 4, wherein said neoplasm comprises a premalignant neoplasm.

6. The method of claim 4, wherein said neoplasm comprises a malignant neoplasm.

7. The method of claim 4, wherein said neoplasm is a colorectal neoplasm.

8. The method of claim 4, wherein said neoplasm is a supracolonic aerodigestive neoplasm.

9. The method of claim 8, wherein said supracolonic aerodigestive neoplasm is selected from the group consisting of small intestine, gall bladder, bile duct, pancreas, liver, stomach, esophagus, lung, tracheal-bronchial, and naso-oro-pharyngeal neoplasms.

10. A method for enriching methylated mammalian nucleic acid if present in a stool sample, wherein said method comprises:

(a) contacting nucleic acid from a stool sample with an MBD polypeptide to form a nucleic acid-MBD polypeptide complex;

(b) eluting bacterial nucleic acid from said MBD polypeptide of the formed nucleic acid-MBD polypeptide complex using a buffer with less than or equal to 0.6 M NaCl; and

(c) eluting methylated mammalian nucleic acid from said complex if said stool sample contained methylated mammalian nucleic acid to obtain an enriched, methylated mammalian nucleic acid sample comprising a higher ratio of methylated mammalian nucleic acid to methylated bacterial nucleic acid than the ratio of methylated mammalian nucleic acid to methylated bacterial nucleic acid present in said stool sample, wherein said eluting step comprises eluting said methylated mammalian nucleic acid from said complex using a buffer with greater than 0.6 M NaCl.

11. The method of claim 10, wherein said MBD polypeptide is a human or rat MBD polypeptide.