Methods for stool sample preparation

• US 6,406,857 B1
• Filed: 05/21/2001
• Issued: 06/18/2002
• Est. Priority Date: 06/16/1997
• Status: Expired due to Term

• Current Assignee: Exact Sciences Corporation
• Original Assignee: Exact Sciences Corporation
• Inventors: Shuber, Anthony P., Lapidus, Stanley N., Radcliffe, Gail E.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/862,167
• Time in Patent Office: 393 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 436/63, 436/64
• US Class Current: 435/6.14
• 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 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention provides methods for the preparation of stool samples to increase the yield of relevant DNA, and further provides methods for isolating and analyzing target DNA for characteristics indicative of colorectal cancer. Methods for screening patients for the presence of cancerous or pre-cancerous colorectal lesions are also provided.

Claims

1. A method for analyzing DNA extracted from stool, comprising:

homogenizing a stool sample in a solvent for DNA in order to form a homogenized sample mixture having a solvent volume to stool mass ratio of at least 5;

1;

allowing said homogenized sample mixture to stand at room temperature;

enriching said homogenized sample mixture for human DNA; and

analyzing said human DNA for characteristics of disease.

2. The method of claim 1 wherein said solvent volume to stool mass ratio is from about 10:

1 to about 30;

1.

3. The method of claim 1 wherein said solvent comprises a buffer that disperses biologic material.

4. The method of claim 1 wherein said allowing step is performed for a period of time.

5. The method of claim 4 wherein said period of time is 5 minutes.

6. The method of claim 4 further comprising obtaining said stool sample.

7. The method of claim 6 wherein said stool sample comprises at least a cross-sectional portion of a stool voided by a patient.

8. The method of claim 1 wherein said enriching step comprises contacting said DNA with a sequence-specific capture probe.

9. The method of claim 1 wherein said solvent comprises a detergent and a proteinase.

10. The method of claim 1 wherein said DNA is human DNA.

11. The method of claim 1 wherein said solvent comprises between about 10 mM EDTA and about 20 mM EDTA.

12. The method of claim 1 wherein said solvent comprises about 16 mM EDTA.

13. The method of claim 1 wherein said solvent comprises guanidine isothiocyanate buffer.

14. The method of claim 13 wherein said guanidine isothiocyanate buffer comprises a final concentration of from about 1 to about 5 M.

15. The method of claim 14 wherein said guanidine isothiocyanate buffer comprises a final concentration of about 3 M.

16. The method of claim 2 wherein said solvent volume to stool mass ratio is about 10:

1 to about 20;

1.

17. The method of claim 2 wherein said solvent volume to stool mass ratio is about 10:

1.

18. The method of claim 3 wherein said buffer comprises Tris-EDTA-NaCl.

19. The method of claim 18 wherein said Tris-EDTA-NaCl buffer comprises a final concentration of about 50 mM Tris, about 16 mM EDTA and about 10 mM NaCl at about pH 9.0.

20. A method of screening for the presence of a colorectal cancerous or pre-cancerous lesion in a patient, the method comprising the steps of:

obtaining a sample comprising at least a cross-sectional portion of a stool voided by the patient;

homogenizing said sample in a solvent in order to form a homogenized sample mixture having a solvent volume to stool mass ratio of at least 5;

1;

allowing said homogenized sample mixture to stand at room temperature;

enriching said sample for a target human DNA; and

analyzing said target human DNA for DNA characteristics indicative of the presence of said colorectal cancerous or pre-cancerous lesion.

21. The method of claim 20 wherein said analyzing step comprises amplifying the DNA with a polymerase chain reaction.

22. The method of claim 20 wherein said DNA characteristics comprise a loss of heterozygosity encompassing a polymorphic locus.

23. The method of claim 20 wherein said DNA characteristic is a mutation.

24. The method of claim 20 wherein said DNA characteristics comprise a deletion in a tumor suppressor allele.

25. The method of claim 20 wherein said solvent comprises about 16 mM EDTA.

26. The method of claim 20 wherein said solvent comprises between about 10 mM EDTA and about 20 mM EDTA.

27. The method of claim 23 wherein said mutation is selected from the group consisting of loss of heterozygosity and microsatellite instability.

28. A method for screening for the presence of a colorectal cancerous or precancerous lesion in a patient, the method comprising the steps of:

obtaining a sample comprising at least a cross-sectional portion of a stool voided by the patient;

homogenizing said sample in a solvent in order to form a homogenized sample mixture having a solvent volume to stool mass ratio of at least 5;

1;

allowing said homogenized sample mixture to stand at room temperature;

determining if said sample has at least a minimum number N of total DNA molecules to provide for detection of a low-frequency target DNA molecule;

analyzing the target DNA for DNA characteristics indicative of the presence of said colorectal cancerous or pre-cancerous lesion.

29. The method of claim 28 wherein said solvent comprises between about 10 mM EDTA and about 20 mM EDTA.

30. The method of claim 28 wherein said solvent comprises about 16 mM EDTA.

31. A method for screening for the presence of a colorectal cancerous or precancerous lesion in a patient, the method comprising the steps of:

obtaining a sample comprising at least a cross-sectional portion of a stool voided by the patient;

homogenizing said sample in a solvent in order to form a homogenized sample mixture having a solvent volume to stool mass ratio of at least 5;

1;

allowing said homogenized sample mixture to stand at room temperature;

enriching said homogenized sample mixture for target human DNA;

determining if said sample has at least a minimum number N of total DNA molecules to provide for detection of a low-frequency target DNA molecule;

analyzing the target human DNA for DNA characteristics indicative of the presence of said colorectal cancerous or pre-cancerous lesion.

32. The method of claim 31 wherein said solvent comprises between about 10 mM EDTA and about 20 mM EDTA.

33. The method of claim 31 wherein said solvent comprises about 16 mM EDTA.