U.S. patent application number 10/105877 was filed with the patent office on 2002-11-07 for methods for stool sample preparation.
Invention is credited to Lapidus, Stanley N., Radcliffe, Gail E., Shuber, Anthony P..
Application Number | 20020164631 10/105877 |
Document ID | / |
Family ID | 26893464 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020164631 |
Kind Code |
A1 |
Shuber, Anthony P. ; et
al. |
November 7, 2002 |
Methods for stool sample preparation
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.
Inventors: |
Shuber, Anthony P.;
(Milford, MA) ; Lapidus, Stanley N.; (Bedford,
NH) ; Radcliffe, Gail E.; (Worcester, MA) |
Correspondence
Address: |
TESTA, HURWITZ & THIBEAULT, LLP
HIGH STREET TOWER
125 HIGH STREET
BOSTON
MA
02110
US
|
Family ID: |
26893464 |
Appl. No.: |
10/105877 |
Filed: |
March 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10105877 |
Mar 25, 2002 |
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09862167 |
May 21, 2001 |
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6406857 |
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09862167 |
May 21, 2001 |
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09198083 |
Nov 23, 1998 |
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6268136 |
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09198083 |
Nov 23, 1998 |
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08876638 |
Jun 16, 1997 |
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Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
C12Q 1/6827 20130101;
C12Q 2565/519 20130101; C12Q 2535/131 20130101; C12Q 2527/125
20130101; C12Q 1/6886 20130101; C12Q 1/6806 20130101; C12Q 2600/156
20130101; C12Q 1/6806 20130101; C12Q 1/6806 20130101; C12Q 1/6806
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
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; enriching said homogenized sample for human
DNA; and analyzing said human DNA for characteristics of
disease.
2. The method of claim 1 wherein the solvent volume to stool mass
ratio is from about 10:1 to about 30:1.
3. The method of claim 2 wherein the solvent volume to stool mass
ratio is about 10:1 to about 20:1.
4. The method of claim 2 wherein the solvent volume to stool mass
ratio is about 10:1.
5. The method of claim 1 wherein the solvent comprises a
physiologically compatible buffer.
6. The method of claim 5 wherein the buffer comprises
Tris-EDTA-NaCl.
7. The method of claim 6 wherein the 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.
8. The method of claim 1 wherein the solvent comprises guanidine
isothiocyanate buffer.
9. The method of claim 8 wherein the guanidine isothiocyanate
buffer comprises a final concentration of from about 1 to about 5
M.
10. The method of claim 9 wherein the guanidine isothiocyanate
buffer comprises a final concentration of about 3 M.
11. The method of claim 1 wherein said enriching step comprises
contacting said DNA with a sequence-specific capture probe.
12. The method of claim 1 wherein said solvent comprises a
detergent and a proteinase.
13. The method of claim 1 wherein said DNA is human DNA.
14. 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 the 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; enriching said sample for a target human DNA; and
analyzing the target human DNA for DNA characteristics indicative
of the presence of said colorectal cancerous or pre-cancerous
lesion.
15. The method of claim 14 wherein said analyzing step comprises
amplifying the DNA with a polymerase chain reaction.
16. The method of claim 14 wherein said DNA characteristics
comprise a loss of heterozygosity encompassing a polymorphic
locus.
17. The method of claim 14 wherein said DNA characteristic is a
mutation.
18. The method of claim 17 wherein said mutation is selected from
the group consisting of loss of heterozygosity and microsatellite
instability.
19. The method of claim 14 wherein said DNA characteristics
comprise a deletion in a tumor suppressor allele.
20. The method of claim 14 wherein said analyzing step comprises
determining whether a difference exists in said sample between a
number X of a first allele known or suspected to be mutated in a
subpopulation of cells in the sample and a number Y of a second
allele that is known or suspected not to be mutated in a
subpopulation of cells in the sample, the presence of a
statistically-significant difference being indicative of a mutation
in a subpopulation of cells in the sample and the potential
presence of a cancerous or precancerous lesion.
21. The method of claim 14 wherein said analyzing step comprises
determining whether a difference exists between a number of a
target tumor suppressor allele in the sample and a number of a
non-cancer-associated reference allele in the sample, the presence
of a statistically-significant difference being indicative of a
deletion of the target tumor suppressor allele in a subpopulation
of cells in the sample and the potential presence of a cancerous or
precancerous lesion.
22. The method of claim 14 wherein said analyzing step further
comprises the steps of: a) detecting an amount of a maternal allele
at a polymorphic locus in the sample; b) detecting an amount of a
paternal allele at the polymorphic locus in the sample; and c)
determining whether a difference exists between the amounts of
maternal and paternal allele, the presence of a
statistically-significant difference being indicative of a deletion
at the polymorphic locus in a subpopulation of cells in the sample
and the potential presence of a lesion.
23. The method of claim 22 wherein said polymorphic locus is a
single base polymorphism and is heterozygous between said maternal
and paternal alleles.
24. The method of claim 22 wherein said detecting steps comprise,
a) hybridizing probe to a portion of said polymorphic locus on both
maternal and paternal alleles that is immediately adjacent to said
single-base polymorphism; b) exposing said sample to a mixture of
detectably-labeled dideoxy nucleoside triphosphates under
conditions which allow appropriate binding of said dideoxy
nucleoside triphosphates to said single-base polymorphism; c)
washing the sample; and d) counting an amount of each
detectably-labeled dideoxy nucleoside triphosphate remaining for
the sample.
25. The method of claim 24 wherein said detectable label is
selected from the group consisting of radioisotopes, fluorescent
compounds, and particles.
26. The method of claim 14 wherein said analyzing step comprises a
method for detecting heterozygosity at a single-nucleotide
polymorphic locus, comprising the steps of. a) hybridizing probes
to a sequence immediately adjacent to a single-base polymorphism;
b) exposing the sample to a plurality of different labeled dideoxy
nucleotides c) washing the sample; d) determining which of said
dideoxy nucleotides are incorporated into said probes; and e)
detecting heterozygosity at the single-nucleotide polymorphic site
as the detection of two dideoxy nucleotides having been
incorporated into the probe.
27. The method of claim 14 wherein said analyzing step comprises:
(a) exposing the sample to a plurality of a first oligonucleotide
probe and to a plurality of a second oligonucleotide probe under
hybridization conditions, thereby to hybridize (1) said first
oligonucleotide probes to copies of a first polynucleotide segment
characteristic of wild-type cells of the organism, and (2) said
second oligonucleotide probes to copies of a second polynucleotide
segment characteristic of a wild-type genomic region suspected to
be deleted or mutated in colorectal cancer cells; (b) detecting a
first number of duplexes formed between said first probe and said
first segment and a second number of duplexes formed between said
second probe and said second segment; and (c) determining whether
there is a difference between the number of duplexes formed between
said first probe and said first segment and the number of duplexes
formed between said second probe and said second segment, the
presence of a statistically-significant difference being indicative
of the presence in said sample of a colorectal cancer or
precancerous lesion.
28. The method of claim 27 wherein said first and second
oligonucleotide probes each are coupled to a distinct detectable
label.
29. The method of claim 27 wherein said first oligonucleotide
probes are attached to a first particle in a ratio of one first
oligonucleotide probe to one particle and said second
oligonucleotide probes are attached to a second particle detectably
distinct from said first particle in a ratio of one second
oligonucleotide probe to one second particle, wherein said
detecting step comprises separating hybridized from unhybridized
first and second oligonucleotide probes and subsequently passing
hybridized first and second oligonucleotide probes through a
detector to determine said first and second numbers.
30. The method of claim 29 wherein said first and second particles
are of detectably different sizes.
31. The method of claim 29 wherein said first and second particles
are of detectably different colors.
32. The method of claim 27 further comprising, prior to step a) the
steps of converting double-stranded DNA in said sample to
single-stranded DNA and removing complement to said first and
second polynucleotide segments.
33. The method of claim 32 wherein said removing step comprises
hybridizing said complement to a nucleic acid probe attached to a
magnetic particle and subsequently removing said magnetic particle
from the sample.
34. The method of claim 14 wherein said analyzing step comprises a
method for detecting a nucleic acid sequence change in a target
allele in the sample, comprising the steps of: (a) determining (i)
an amount of wild-type target allele in the sample, and (ii) an
amount of a reference allele in the sample; and (b) detecting a
nucleic acid sequence change in the target allele in the sample, a
statistically significant difference in the amount wild-type target
allele and the amount of reference allele obtained in said
determining step being indicative of a nucleic acid sequence
change.
35. The method according to claim 34 wherein said determining step
comprises exposing said sample to a first oligonucleotide probe
capable of hybridizing with a portion of said wild-type allele and
to a second oligonucleotide probe capable of hybridizing to a
portion of said reference allele, and removing from said sample any
unhybridized first or second oligonucleotide probe.
36. 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 the 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; ensuring that said sample have 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.
37. 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 containing of least a
cross-sectional portion of a stool voided by the patient;
homogenizing the 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; enriching said homogenized sample for target human DNA;
ensuring that said sample have 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.
38. The method of claim 36 wherein said analyzing step comprises
amplifying the DNA with a polymerase chain reaction.
39. The method of claim 37 wherein said analyzing step comprises
amplifying the DNA with a polymerase chain reaction.
40. The method of claim 37 wherein said enriching step comprises
contacting said DNA with a sequence-specific capture probe.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 08/876,638, filed Jun. 15, 1997.
FIELD OF THE INVENTION
[0002] This invention relates to methods for the early detection of
colon cancer in patients, and more particularly to methods for
preparing stool samples in order to increase the yield of nucleic
acids.
BACKGROUND OF THE INVENTION
[0003] Stool samples frequently must be prepared for medical
diagnostic analysis. Stool samples may be analyzed for diagnosis of
medical conditions ranging from parasitic, bacterial or viral
infections to inflammatory bowel disease and colorectal cancer.
[0004] Colorectal cancer is a leading cause of death in Western
society. However, if diagnosed early, it may be treated effectively
by removal of the cancerous tissue. Colorectal cancers originate in
the colorectal epithelium and typically are not extensively
vascularized (and therefore not invasive) during the early stages
of development. Colorectal cancer is thought to result from the
clonal expansion of a single mutant cell in the epithelial lining
of the colon or rectum. The transition to a highly vascularized,
invasive and ultimately metastatic cancer which spreads throughout
the body commonly takes ten years or longer. If the cancer is
detected prior to invasion, surgical removal of the cancerous
tissue is an-effective cure. However,colorectal cancer is often
detected only upon manifestation of clinical symptoms, such as pain
and black tarry stool. Generally, such symptoms are present only
when the disease is well established, and often after metastasis
has occurred. Early detection of colorectal cancer therefore is
important in order to significantly reduce its morbidity.
[0005] Invasive diagnostic methods such as endoscopic examination
allow for direct visual identification, removal, and biopsy of
potentially cancerous growths. Endoscopy is expensive,
uncomfortable, inherently risky, and therefore not a practical tool
for screening populations to identify those with colorectal cancer.
Non-invasive analysis of stool samples for characteristics
indicative of the presence of colorectal cancer or precancer is a
preferred alternative for early diagnosis, but no known diagnostic
method is available which reliably achieves this goal.
[0006] Current non-invasive screening methods involve assaying
stool samples for the presence of fecal occult blood or for
elevated levels of carcinoembryonic antigen, both of which are
suggestive of the presence of colorectal cancer. Additionally,
recent developments in molecular biology provide methods of great
potential for detecting the presence of a range of DNA mutations or
alterations indicative of colorectal cancer. The presence of such
mutations can be detected in DNA found in stool samples during
various stages of colorectal cancer. However, stool comprises cells
and cellular debris from the patient, from microorganisms, and from
food, resulting in a heterogeneous population of cells. This makes
detection of small, specific subpopulations difficult to detect
reliably.
[0007] Use of the polymarase chain reaction (PCR) has made
detection of nucleic acids more routine, but any PCR is limited by
the amount of DNA present in a sample. A minimum amount of material
must be present for specific analysis and this limitation becomes
more relevant when one seeks to detect a nucleic acid that is
present in a sample in small proportion relative to other nucleic
acids in the sample, which is often the case when analyzing stool
sample for detecting DNA characteristics of colorectal cancer. If a
low-frequency mutant strand is not amplified in the first few
rounds of PCR, any signal obtained from the mutant strand in later
rounds will be obscured by background or by competing signal from
amplification of ubiquitous wild-type strand.
[0008] An additional problem encounter in preparation of stool
sample for detection of colorectal cancer is the difficulty of
extracting sufficient quantities of relevant DNA from the stool.
Stool samples routinely contain cell debris, enzymes, bacteria (and
associated nucleic acids), and various other compounds that can
interfere with traditional DNA extraction procedures and reduce DNA
yield. Furthermore, DNA in stool often appears digested or
partially digested, which can reduce the efficiency of extraction
methods.
SUMMARY OF THE INVENTION
[0009] It has now been appreciated that the yield of nucleic acid
from a stool sample is increased by providing an optimal ratio of
solvent volume to stool mass in the sample. Accordingly, the
invention provides stool sample preparation protocols for
increasing sample nucleic acid yield.
[0010] In a preferred embodiment, methods of the invention comprise
homogenizing a representative stool 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, then enriching the homogenized sample
for the target (human) DNA. The human DNA may then be analyzed for
the characteristics of disease. Providing an optimal solvent volume
to stool mass ratio increases the yield of nucleic acid obtained
from the sample. An especially-preferred ratio of solvent volume to
stool mass is between about 10:1 and about 30:1, more preferably
from about 10:1 to about 20:1, and most preferably 10:1.
[0011] A preferred solvent for preparing stool samples according to
the invention is a physiologically-compatible buffer such as a
buffer comprising Tris-EDTA-NaCl. A preferred buffer is a
Tris-EDTA-NaCl buffer comprising about 50 to about 100 mM Tris,
about 10 to about 20 mM EDTA, and about 5 to about 15 mM NaCl at
about pH 9.0. A particularly preferred buffer is 50 mM Tris, 16 mM
EDTA and 10 mM NaCl at pH 9.0. Another preferred solvent is
guanidine isothiocyanate (GITC). A preferred GITC buffer has a
concentration of about 1 M to about 5 M. A particularly preferred
GITC buffer has a concentration of about 3 M.
[0012] Also in a preferred embodiment, methods further comprise the
step of enriching the homogenized sample mixture for human DNA by,
for example, using sequence-specific nucleic acid probes
hybridizing to target human DNA.
[0013] In an alternative preferred embodiment, the methods of the
invention comprise homogenizing a, stool sample in a
physiologically-acceptable solvent for DNA in order to form a
homogenized sample mixture having a solvent volume to stool mass
ratio of at least 5:1; ensuring that the homogenized sample has at
least a minimum number N of total DNA molecules to facilitate
detection of a low-frequency target DNA molecule; and analyzing the
target DNA for the characteristics of disease, preferably by
amplifying the target DNA with a polymerase chain reaction.
[0014] In another embodiment, the present invention provides
methods for analyzing DNA extracted from stool which comprise
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; enriching the homogenized sample for human
DNA; ensuring that the enriched homogenized sample has at least a
minimum number N of total DNA molecules to provide for detection of
a low-frequency target DNA molecule; and analyzing the target DNA
for DNA characteristics indicative of disease.
[0015] Methods of the invention are useful to screen for the
presence in a stool sample of nucleic acids indicative of
colorectal cancer. Such methods comprise obtaining a representative
stool sample (i.e., at least a cross-section); homogenizing the
sample in a solvent having a solvent volume to stool mass ratio of
at least 5:1; enriching the sample for target human DNA; and
analyzing the DNA for characteristics of colorectal cancer. Various
methods of analysis of DNA characteristics exist, such as those
disclosed in co-owned, copending U.S. patent application Ser. No.
08/700,583, incorporated by reference herein.
[0016] Methods of the invention also comprise obtaining a
representative (i.e., cross-sectional) sample of stool and
homogenizing the stool in a buffer, such as a buffer comprising a
detergent and a proteinase and optionally a DNase inhibitor.
[0017] The methods of the invention are especially and most
preferably useful for detecting DNA characteristics indicative of a
subpopulation of transformed cells in a representative stool
sample. The DNA characteristics may be, for example, mutations,
including point mutations, deletions, additions, translocations,
substitutions, and loss of heterozygosity. Methods of the invention
may further comprise a visual examination of the colon. Finally,
surgical resection of abnormal tissue may be done in order to
prevent the spread of cancerous or precancerous tissue.
[0018] Accordingly, methods of the invention provide means for
screening for the presence of a cancerous or precancerous
subpopulation of cells in a heterogeneous sample, such as a stool
sample. Methods of the invention reduce morbidity and mortality
associated with lesions of the colonic epithelium. Moreover,
methods of the invention comprise more accurate and convenient
screening methods than are currently available in the art, because
such methods take advantage of the increased yield of relevant
DNA.
[0019] Methods of the invention thus provide unexpected and
enhanced detection and analysis of low-frequency DNA in a
heterogeneous sample is facilitated through application of the
methods described herein. That is, homogenization of stool sample
in solvent at a ratio of at least 5:1 (volume to mass) alone, or in
combination with methods for sample enrichment disclosed herein,
provides a reliable method for obtaining a sufficient number of DNA
molecules for effective and efficient analysis, even if the target
molecule is a low-frequency DNA molecule. Further aspects and
advantages of the invention are contained in the following detailed
description thereof.
DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a representation of a partial nucleotide sequence
of the kras gene (base pairs 62826571) and the positions of capture
probe CP1, PCR primer A1, and PCR primer B1, in relation to the
kras nucleotide sequence.
[0021] FIG. 2 is an image produced using a Stratagene Eagle Eye II
Still Video System (Stratagene, La Jolla, Calif.), of the results
of a gel electrophoresis run with the uncut DNA extracted as
described in Example 2.
[0022] FIG. 3 is an image produced using a Stratagene Eagle Eye II
Still Video System (Stratagene, La Jolla, Calif.), of the results
of a gel electrophoresis run with the DNA extracted as described in
Example 3.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The invention provides improved methods for extraction and
analysis of nucleic acids from stool. According to methods of the
invention, the yield of nucleic acids extracted from stool is
increased by homogenizing the stool in a buffer at optimal ratio of
buffer volume to stool mass. Yield is further improved by enriching
for human DNA. Improved nucleic acid yields allow nucleic acid
analysis of stool samples to be conducted more efficiently with
less stool volume.
[0024] In preferred methods of the invention a stool sample
obtained for analysis comprises at least a cross-section of a whole
stool. As provided in U.S. Pat. No. 5,741,650, incorporated by
reference herein, cells and cellular debris from the colonic
epithelium is deposited onto and into stool in a longitudinal
streak. Obtaining at least a cross-section of a stool ensures that
a representative sampling of colonic epithelial cells and cellular
debris is analyzed.
[0025] Once the stool sample is collected, it is homogenized in a
physiologically acceptable solvent. A preferred means of
homogenization employs agitation with glass beads. Physiologically
acceptable solvents include those solvents generally known to those
skilled in the art as suitable for dispersion of biological sample
material. Such solvents include phosphate-buffered saline
comprising a salt, such as 20-100 mM NaCl or KCl, and optionally a
detergent, such as 1-10% SDS or Triton.TM., and/or a proteinase,
such as proteinase K (at, e.g., about 20 mg/ml). A preferred
solvent is a physiologically-compatible buffer comprising, for
example, 1 M Tris, 0.5M EDTA, 5M NaCl and water to a final
concentration of 500 mM Tris, 16 mM EDTA and 10 mM NaCl at pH 9.
The buffer acts as a solvent to disperse the solid stool sample
during homogenization. Applicants have discovered that increasing
the-volume of solvent in relation to solid mass of the sample
results in increased yields of DNA.
[0026] According to methods of the invention, solvent (buffer) is
added to the solid sample in a solvent volume to solid mass ratio
of at least about 5:1. The solvent volume to solid mass ratio is
preferably in the range of about 10:1 to about 30:1, and more
preferably in the range of about 10:1 to about 20:1. Most
preferably, the solvent volume to solid mass ratio is about 10:1.
Typically, solvent volume may be measured in milliliters, and solid
mass measured in milligrams, but the practitioner will appreciate
that the ratio of volume to mass remains constant, regardless of
scale up or down of the particular mass and volume units. That is,
solvent volume to solid mass ratios may be measured as liters:grams
or .mu.l: .mu.g.
[0027] In a preferred embodiment of the present invention, the
homogenized sample is enriched for the target (human) DNA. In the
context of the present invention, "enrichment" of the sample means
manipulating the sample to decrease the amount of undesired,
non-human DNA in the sample relative to the amount of target human
DNA. Enrichment techniques include sequence-specific capture of
target DNA or removal of bacterial nucleic acids.
[0028] In a preferred embodiment of the invention, the enrichment
step is carried out in a physiologically compatible buffer, such as
guanidine isothiocyanate (GITC). Capture probes are then added to
the mixture to hybridize to target DNA in order to facilitate
selective removal of target DNA from the sample.
[0029] Sequence specific capture of target DNA can be accomplished
by initially denaturing sample DNA to form single-stranded DNA.
Then, a sufficient quantity of sequence specific oligonucleotide
probe that is complementary to at least a portion of a target
polynucleotide (e.g., a sequence in or near the p53 allele) is
added. The probe sequence (labeled with biotin) is allowed to
hybridize to the complementary target DNA sequence. Beads coated
with avidin or streptavidin are then added and attach to the
biotinylated hybrids by affinity-binding. The beads may be
magnetized to facilitate isolation.
[0030] After separation of probe-target hybrids, the resultant DNA
is washed repeatedly to remove inhibitors, including those commonly
introduced via the capture probe technique. In the methods of the
present invention, washes are preferably carried out approximately
four times with 1M GITC and 0.1% detergent, such as Igepal (Sigma).
The initial washes are then preferably followed by two washes with
a standard wash buffer (such as Tris-EDTA-NaCl) to remove the GITC
from the mix, since GITC is a known inhibitor of DNA polymerases,
including those associated with PCR.
[0031] Finally, the target DNA is eluted into a small volume of
distilled water by heating. Assays using polymerase chain reaction
(PCR), restriction fragment length polymorphism (RFLP) analysis or
other nucleic acid analysis methods may be used to detect DNA
characteristics indicative of a disorder, such as colorectal cancer
or pre-cancer. Several particularly useful analytical techniques
are described in co-pending U.S. applications Ser. Nos. 08/700,583,
08/815,576 and 08/877,333, the disclosures of which are
incorporated herein by reference.
[0032] In an alternative embodiment, the homogenized sample is
examined to determine that the sample has at least a minimum number
(N) of total DNA molecules to provide for detection of a
low-frequency target DNA molecule. The number of molecules analyzed
in a sample determines the ability of the analysis to detect
low-frequency events. In the case of PCR, the number of input
molecules must be about 500 if the PCR efficiency is close to 100%.
As PCR efficiency goes down, the required number of input molecules
goes up. Analyzing the minimum number of input molecules reduces
the probability that a low-frequency event is not detected in PCR
because it is not amplified in the first few rounds. Methods of the
invention therefore include determining a threshold number of
sample molecules that must be analyzed in order to detect a
low-frequency molecular event at a prescribed level of
confidence.
[0033] As is more fully described in copending U.S. application
Ser. No. ______ [Atty Docket No. EXT-021], which is incorporated
herein by reference, the determination of a minimum number N of DNA
molecules that must be present in a sample to permit amplification
and analysis of a low-frequency target DNA molecule is based upon a
model of stochastic processes in PCR. Utilizing pre-set or
predetermined values for PCR efficiency and mutant DNA to wild-type
DNA ratio in the sample, the model predicts the number of molecules
that must be presented to the PCR in order to ensure, within a
defined level of statistical confidence, that a low-frequency
molecule will be amplified.
[0034] The skilled practitioner will appreciate that determination
of the minimum number N of molecules present in the sample may be
used in lieu of, or in addition to, the enrichment techniques
detailed above, to ensure reliable results in the methods of the
present invention.
[0035] Alternatively, methods of the invention may also be used to
isolate total DNA from stool homogenate. The homogenized mixture is
centrifuged to form a pellet made up of cell debris and stool
matter, and a supernatant containing nucleic acid and associated
proteins, lipids, etc. The supernatant is treated with a detergent,
such as 20% SDS, and enzymes capable of degrading protein (e.g.,
Proteinase K). The supernatant is then Phenol-Chloroform extracted.
The resulting purified nucleic acids are then precipitated by means
known in the art. A variety of techniques in the art can then be
employed to manipulate the resulting nucleic acids, including
further purification or isolation of specific nucleic acids.
[0036] Methods of the invention are also useful for analysis of
pooled DNA samples. As described in more detail in U.S. application
Ser. No. 09/098,180, and U.S. Pat. No. 5,670,325, both of which are
incorporated by reference herein, enumerative analysis of pooled
genomic DNA samples is used to determine the presence or likelihood
of disease. Pooled genomic DNA from healthy members of a population
and pooled genomic DNA from diseased members of a population are
obtained. The number or amount of each variant at a
single-nucleotide polymorphic site is determined in each sample.
The numbers or amounts are analyzed to determine if there is a
statistically-significant difference between the variant(s) present
in the sample obtained from the healthy population and those
present in the sample obtained from the diseased population. A
statistically-significant difference indicates that the polymorphic
locus is a marker for disease.
[0037] These methods may be used to identify a nucleic acid (e.g.,
a polymorphic variant) associated with a disease. Such methods
comprise counting the number or determining the amount of a nucleic
acid, preferably a single base, in members of a diseased
population, and counting numbers or determining amounts of the same
nucleic acid in members of a healthy population. A
statistically-significant difference in the numbers of the nucleic
acid between the two populations is indicative that the
interrogated locus is associated with a disease.
[0038] Once the polymorphic locus is identified, either by methods
of the invention or by consulting an appropriate database, such
methods are useful to determine which variant at the polymorphic
locus is associated with a disease. In this case, enumerative
methods are used to determine whether there is a
statistically-significant difference between the number of a fist
variant in members of a diseased population, and the number of a
second variant at the same locus in members of a healthy
population. A statistically-significant difference is indicative
that the variant in members of the diseased population is useful as
a marker for disease. Using this information, patients are screened
for the presence of the variant that is thought to be associated
with disease, the presence such a variant being indicative of the
presence of disease, or a predisposition for a disease.
[0039] Methods of the present invention are particularly useful for
isolation and analysis of nucleic acids that encompass genes that
have mutations implicated in colorectal cancer, such as kras. The
kras gene has a length of more than 30 kbp and codes for a 189
amino acid protein characterized as a low-molecular weight
GTP-binding protein. The gene acquires malignant properties by
single point mutations, the most common of which occurs at the 12th
amino acid. Several studies have confirmed that approximately 40%
of primary colorectal adenocarcinoma cells in humans contain a
mutated form of the kras gene. Accordingly, the kras gene is a
particularly suitable target for the methods of colorectal cancer
detection of the present invention.
[0040] Toward this end, applicants have constructed a suitable
exemplary capture probe directed to the kras nucleotide sequence.
The capture probe, designated CP1, has the following sequence: 5'
GCC TGC TGA AM TGA CTG AAT ATA AAC TTG TGG TAG T 3' (SEQ, ID NO:
1), and is preferably biotinylated at the 5' end in order to
facilitate isolation. As illustrated more fully below, CP1 is
effective in the sequence specific capture of kras DNA.
[0041] Suitable PCR primers for the analysis of extracted kras DNA
sequence have also been determined. Primer A1 has the sequence:
5.degree. C. CTG CTG AAA ATG ACT GAA 3' (SEQ ID NO: 2), and Primer
B1 has the sequence: 5.degree. CAT GM MT GGT CAG AGA M 3' (SEQ ID
NO: 3). The PCR primers A1 and B1, as well as capture probe CP1,
are depicted in FIG. 1, showing their relation to the kras
nucleotide sequence, base pairs 6282-6571 (SEQ ID NO: 4). One
skilled in the art can construct other suitable capture probes and
PCR primers for kras or other target genes or nucleotide sequences,
using techniques well known in the art.
[0042] Accordingly, the methods of the present invention, which
involve homogenizing stool sample in a volume of solvent such that
the ratio of solvent volume to stool mass is at least 5:1, and/or
enriching the sample for human DNA, provide a means for obtaining a
sample having a minimum number N of total DNA molecules to
facilitate detection of a low-frequency target DNA molecule. These
methods thus provide the unexpected result that one is now able to
reliably detect a small portion of low-frequency DNA in a
heterogeneous sample.
[0043] The following examples provide further details of methods
according to the invention. However, numerous additional aspects of
the invention will become apparent upon consideration of the
following examples.
EXAMPLE 1
[0044] Stool Sample Preparation
[0045] Voided stool was collected from a patient and a
cross-sectional portion of the stool was removed for use as a
sample. After determining the mass of the sample, an approximately
10.times.volume of Tris-EDTA-NaCl lysis buffer was added to the
solid sample in a test tube. The final concentration of the buffer
was 500 mM Tris, 16 mM EDTA and 10 mM NaCl, at a pH of about 9.0.
Four 10 mm glass balls were placed in the tube and the tube and
contents were homogenized in an Exactor II shaker for 15 minutes.
The homogenized mixture was then allowed to stand 5 minutes at room
temperature. The tube was then centrifuged for 5 minutes at 10,000
rpm in a Sorvall Centrifuge, and the supernatant was transferred to
a clean test tube. A 20% SDS solution was added to the tube to a
final concentration of 0.5%. Proteinase K was also added to the
tube to a final concentration of 500 mg/ml. The tube was then
incubated overnight at 37.degree. C.
[0046] After incubation, the contents of the tube were extracted
with an equal volume of phenol/chloroform and centrifuged at 3500
rpm for 3 minutes. The aqueous layer was then transferred to a new
tube and extracted three (3) times with equal volumes of chloroform
and centrifuged at 3500 rpm for 3 minutes. The aqueous layer was
then transferred to a new tube and 0.1.times.volume of 3M NaOAc was
added to the aqueous portion, which was then extracted with an
equal volume of isopropanol, and centrifuged for 5 minutes at
12,000 rpm. The supernatant was discarded, and the pellet was
washed with 10 ml of 70% ethanol, and centrifuged at 12,000 rpm for
5 minutes. The supernatant was discarded and the pellet containing
isolated DNA was dried by inverting the tube.
EXAMPLE 2
[0047] A comparative analysis of solvent volume to mass ratios was
conducted. Three separate stool samples were prepared as described
above. A first sample, designated SS88-3.times., was homogenized in
buffer at a volume to mass ratio of 3:1. A second sample,
designated SS88-5.times., was homogenized at a ratio of 5:1; and a
third sample, designated SS88-10.times., was homogenized at a ratio
of 10:1.
[0048] Total DNA from each sample was resuspended in 100 ul of 100
mM Tris, 10 mM EDTA buffer and 10 ul aliquots were loaded onto a 4%
agarose gel for electrophoresis at 125 V constant voltage for about
one hour. The results are shown in FIG. 2. As shown in FIG. 2, the
yield of total DNA increased as the ratio of solvent to mass
increased from 3.times.to 10.times..
EXAMPLE 3
[0049] A second set of four equivalent samples was prepared from a
single stool sample. Each of the four samples was of equal mass,
and was homogenized as described in Example 1 at a solvent volume
to stool mass ratio of 5:1, 10:1, 20:1, and 30:1, respectively.
After homogenization each sample was subdivided into 8 aliquots, 4
treated with RNase, and 4 untreated. Total DNA was then isolated as
described above and analyzed on agarose gels.
[0050] The results are shown in FIG. 3. As shown, a ratio of 10:1
produced the greatest yield of nucleic acids. FIG. 3 also shows the
effect of RNase treatment on the yield of DNA from each stool
sample. As shown in the Figure, RNase treatment virtually
eliminates RNA from the sample, but leaves DNA intact. The results
indicate that optimal solvent volume to stool mass ratios greatly
increase DNA yield from stool samples.
EXAMPLE 4
[0051] Sequence-Specific Capture of Target DNA.
[0052] Once extracted from stool, specific nucleic acids are
isolated using sequence-specific capture probes. Total DNA was
extracted from a stool sample according to the methods described in
Example 1. The pelletized DNA was resuspended in 1 ml of TE buffer.
A 100 .mu.l aliquot of this solution was removed to a new tube and
100 .mu.l of 6M guanidine isothiocyanate (GITC) was added to a
final concentration of 3M GITC. A vast excess of biotinylated kras
capture probe CP1 was the added to the sample. The mixture was
heated to 95.degree. C. for 5 minutes to denature the DNA, then
cooled to 37.degree. C. for 5 minutes. Finally, probe and target
DNA were allowed to hybridize for 30 minutes at room temperature.
Streptavidin-coated magnetized beads (320 mg) (Dynal Corp.) were
suspended in 400 .mu.l distilled water and added to the mixture.
After briefly mixing, the tube was maintained at room temperature
for 30 minutes.
[0053] Once the affinity binding was completed, a magnetic field
was applied to the sample to draw the magnetized isolation beads
(both with and without hybridized complex out of the sample. The
beads were then washed four (4) times in 1M GITC/0.1% Igepal
(Sigma, St. Louis, Mo.) solution for 15 minutes, followed by two
(2) washes with wash buffer (TE with 1M NaCl) for 15 minutes in
order to isolate complexed streptavidin. Finally, 10 .mu.l
distilled water was added to the beads and heated at 95.degree. C.
for 3 minutes to elute the DNA. Sequencing and/or gel
electrophoresis enable confirmation of the capture of kras-specific
DNA.
[0054] Accordingly, methods of the invention produce increased
yields of DNA from stool, thereby allowing more efficient
sequence-specific capture of target nucleic acid. Methods of the
invention provide improvements in the ability to detect
disease-related nucleic acid mutations present in stool. The
skilled artisan will find additional applications and embodiments
of the invention useful upon inspection of the foregoing
description of the invention. Therefore, the invention is limited
only by the scope of the appended claims.
Sequence CWU 1
1
4 1 37 DNA Artificial Sequence misc_feature Description of
Artificial Sequence CP1 kras capture probe 1 gcctgctgaa aatgactgaa
tataaacttg tggtagt 37 2 19 DNA Artificial Sequence misc_feature
Description of Artificial Sequence kras PCR Primer A1 2 cctgctgaaa
atgactgaa 19 3 20 DNA Artificial Sequence misc_feature Description
of Artificial Sequence kras PCR primer B1 3 catgaaaatg gtcagagaaa
20 4 307 DNA homo sapiens misc_feature Partial nucleotide sequence
of the kras gene 4 gtactggtgg agtatttgat agtgtattaa ccttatgtgt
gacatgttct aatatagtca 60 cattttcatt atttttatta taaggcctgc
tgaaaatgac tgaatataaa cttgtggtag 120 ttggagctgg tggcgtaggc
aagagtgcct tgacgataca gctaattcag aatcattttg 180 tggacgaata
tgatccaaca atagaggtaa atcttgtttt aatatgcata ttactggtgc 240
aggaccattc tttgatacag ataaaggttt ctctgaccat tttcatgtac agaagtcctt
300 gctaaga 307
* * * * *