U.S. patent application number 17/188018 was filed with the patent office on 2021-10-28 for methods and materials for detecting genetic or epigenetic elements.
The applicant listed for this patent is Cascade Biosystems, Inc.. Invention is credited to Mariya Smit, Kenneth D. Smith, Nina Yazvenko.
Application Number | 20210332435 17/188018 |
Document ID | / |
Family ID | 1000005705234 |
Filed Date | 2021-10-28 |
United States Patent
Application |
20210332435 |
Kind Code |
A1 |
Smith; Kenneth D. ; et
al. |
October 28, 2021 |
METHODS AND MATERIALS FOR DETECTING GENETIC OR EPIGENETIC
ELEMENTS
Abstract
This document provides methods and materials for detecting
genetic and/or epigenetic elements. For example, methods and
materials for detecting the presence or absence of target nucleic
acid containing a genetic or epigenetic element, methods and
materials for detecting the amount of target nucleic acid
containing a genetic or epigenetic element within a sample, kits
for detecting the presence or absence of target nucleic acid
containing a genetic or epigenetic element, kits for detecting the
amount of target nucleic acid containing a genetic or epigenetic
element present within a sample, and methods for making such kits
are provided.
Inventors: |
Smith; Kenneth D.; (Colfax,
WI) ; Yazvenko; Nina; (Vancouver, WA) ; Smit;
Mariya; (Vancouver, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cascade Biosystems, Inc. |
Colfax |
WI |
US |
|
|
Family ID: |
1000005705234 |
Appl. No.: |
17/188018 |
Filed: |
March 1, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16448784 |
Jun 21, 2019 |
10968483 |
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17188018 |
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|
15604112 |
May 24, 2017 |
10329619 |
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16448784 |
|
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|
14841282 |
Aug 31, 2015 |
9689037 |
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15604112 |
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|
14046389 |
Oct 4, 2013 |
9150921 |
|
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14841282 |
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13027887 |
Feb 15, 2011 |
8551701 |
|
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14046389 |
|
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61304793 |
Feb 15, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/683 20130101;
C12Q 2600/154 20130101; C12Q 1/6876 20130101; C12Q 2600/156
20130101; C12Q 1/6883 20130101 |
International
Class: |
C12Q 1/6883 20060101
C12Q001/6883; C12Q 1/683 20060101 C12Q001/683; C12Q 1/6876 20060101
C12Q001/6876 |
Claims
1. (canceled)
2. A device for point of care or home use assessment of an organism
for a genetic or epigenetic element, wherein said device comprises:
(a) a sample chamber configured to receive a sample from a user,
wherein said sample was obtained from said organism and (i)
comprises fragments of single stranded nucleic acid or (ii) is
prepared to contain fragments of single stranded nucleic acid
within said sample chamber; (b) a recognition chamber configured to
receive said fragments from said sample chamber, wherein said
recognition chamber comprises (i) probe nucleic acid comprising an
amplifying restriction endonuclease and a nucleotide sequence
complementary to a sequence of a target nucleic acid containing
said genetic or epigenetic element and (ii) a recognition
restriction endonuclease, wherein, if said target nucleic acid is
present in said fragments, at least a portion of said target
nucleic acid hybridizes to at least a portion of said probe nucleic
acid to form a double-stranded portion of nucleic acid comprising a
restriction endonuclease cut site of said recognition restriction
endonuclease and said recognition restriction endonuclease cleaves
said double-stranded portion of nucleic acid at said restriction
endonuclease cut site, thereby separating a portion of said probe
nucleic acid comprising said amplifying restriction endonuclease
from at least another portion of said probe nucleic acid, (c) an
amplification chamber configured to receive said portion of said
probe nucleic acid comprising said amplifying restriction
endonuclease from said recognition chamber, wherein said
amplification chamber comprises a reporter nucleic acid comprising
a label, an amplifying restriction endonuclease, and a
double-stranded portion of nucleic acid comprising a restriction
endonuclease cut site of said amplifying restriction endonuclease
of said probe nucleic acid, wherein, if said portion of said probe
nucleic acid comprising said amplifying restriction endonuclease is
received from said recognition chamber, said amplifying restriction
endonuclease of said probe nucleic acid cleaves said
double-stranded portion of nucleic acid of said reporter nucleic
acid at said restriction endonuclease cut site, thereby separating
a portion of said reporter nucleic acid comprising said label from
at least another portion of said reporter nucleic acid, and (d) a
detection chamber configured to receive said portion of said
reporter nucleic acid comprising said label from said amplification
chamber, wherein said detection chamber provides said user with an
indication of the presence or absence of said label, thereby
indicating the presence or absence of said genetic or epigenetic
element within said organism.
3. The device of claim 2, wherein said organism is a mammal.
4. The device of claim 2, wherein said organism is a human.
5. The device of claim 2, wherein said device is for assessment of
said epigenetic element.
6. The device of claim 5, wherein said epigenetic element is a
methylated DNA sequence.
7. The device of claim 2, wherein said sample is selected from the
group consisting of blood samples, hair samples, skin samples,
throat swab samples, cheek swab samples, tissue samples, cellular
samples, and tumor samples.
8. The device of claim 2, wherein said recognition restriction
endonuclease comprises the ability to recognize a methylated
nucleotide.
9. The device of claim 2, wherein said recognition restriction
endonuclease is a DpnI, GlaI, HpaII, MspI, AciI, HhaI, or SssI
restriction endonuclease.
10. The device of claim 2, wherein said label is a fluorescent
label.
11. The device of claim 2, wherein said label is a radioactive
label.
12. The device of claim 2, wherein said label is an enzyme
label.
13. The device of claim 12, wherein said enzyme label is horse
radish peroxidase, alkaline phosphatase, laccase, galactosidase, or
luciferase.
14. The device of claim 2, wherein said label is a redox label.
15. The device of claim 2, wherein said device is for point of
care, and wherein said user is a medical, laboratory, or
veterinarian personnel.
16. The device of claim 4, wherein said device is for home use, and
wherein said user is said human.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 16/448,784, filed Jun. 21, 2019, which is a continuation of
U.S. application Ser. No. 15/604,112, filed May 24, 2017 (now U.S.
Pat. No. 10,329,619), which is a continuation of U.S. application
Ser. No. 14/841,282, filed Aug. 31, 2015 (now U.S. Pat. No.
9,689,037), which is a continuation of U.S. application Ser. No.
14/046,389, filed Oct. 4, 2013 (now U.S. Pat. No. 9,150,921), which
is a continuation of U.S. application Ser. No. 13/027,887, filed
Feb. 15, 2011 (now U.S. Pat. No. 8,551,701), which claims the
benefit of priority of U.S. Provisional Application Ser. No.
61/304,793, filed Feb. 15, 2010. The disclosure of the prior
application is considered part of (and is incorporated by reference
in) the disclosure of this application.
BACKGROUND
1. Technical Field
[0002] This document relates to methods and materials involved in
detecting genetic and/or epigenetic elements. For example, this
document relates to methods and materials involved in using an
enzymatic amplification cascade of restriction endonucleases to
detect genetic and/or epigenetic elements present within an
organism (e.g., a human).
2. Background
[0003] Many aspects of an organism's phenotype are controlled by
the genotype of that organism. In other words, the genetic makeup
of an organism can control the traits of that organism. Thus, the
presence or absence of certain genetic elements such as single
nucleotide polymorphisms (SNPs), sequence deletions, or sequence
additions present within an organism's genome can provide important
information about the organism's health and/or susceptibilities to
certain diseases or disorders. Likewise, epigenetic elements such
as methylated DNA can control or influence an organism's phenotype.
Thus, the presence or absence of certain epigenetic elements such
as methylated DNA present within an organism can provide important
information about the organism's health and/or susceptibilities to
certain diseases or disorders.
SUMMARY
[0004] This document provides methods and materials for detecting
genetic and/or epigenetic elements. For example, this document
relates to methods and materials involved in using an enzymatic
amplification cascade of restriction endonucleases to detect
genetic and/or epigenetic elements present within an organism
(e.g., a human). Information about an organism's genotype can be
important for understanding that organism's health and/or
susceptibilities to certain diseases or disorders. For example, the
presence of certain genetic elements in a human's genome (e.g.,
genetic markers such as single nucleotide polymorphisms (SNPs),
sequence deletions, or sequence additions) can indicate that that
particular human has an enzyme deficiency or is susceptible to
developing a certain disease. Likewise, information about
epigenetic elements that may influence the phenotype of an organism
can be important for understanding that organism's health and/or
susceptibilities to certain diseases or disorders. For example, the
presence of certain epigenetic elements such as methylated DNA can
indicate that a human has a particular type of cancer.
[0005] In some cases, this document provides methods and materials
for detecting target nucleic acid that contains a genetic element
or epigenetic element. For example, this document provides methods
and materials for detecting the presence or absence of target
nucleic acid (e.g., target nucleic acid containing a particular
genetic element) in an organism's genome, methods and materials for
detecting the presence or absence of target nucleic acid that
contains an epigenetic element (e.g., methylated DNA) in a cell of
an organism, kits for detecting the presence or absence of target
nucleic acid (e.g., target nucleic acid containing a particular
genetic element) in an organism's genome, kits for detecting the
presence or absence of target nucleic acid that contains an
epigenetic element (e.g., methylated DNA) in a cell of an organism,
and methods for making such kits.
[0006] In general, the methods and materials provided herein can
include performing an enzymatic amplification cascade of
restriction endonucleases as described herein to detect target
nucleic acid indicative of a genetic and/or epigenetic element in a
manner that is rapid, inexpensive, sensitive, and specific. For
example, a sample (e.g., a sample of genomic nucleic acid or a
sample of nucleic acid from a cell or tissue) can be obtained from
an organism (e.g., a human) and/or processed such that target
nucleic acid, if present within the sample, is capable of
hybridizing to probe nucleic acid of an enzymatic amplification
cascade of restriction endonucleases described herein. In some
cases, such an obtained and/or processed sample can be assessed for
the presence, absence, or amount of target nucleic acid using an
enzymatic amplification cascade of restriction endonucleases
described herein without using a nucleic acid amplification
technique (e.g., a PCR-based nucleic acid technique). Assessing
samples (e.g., biological samples) for the presence, absence, or
amount of target nucleic acid using an enzymatic amplification
cascade of restriction endonucleases described herein without using
a nucleic acid amplification technique can allow patients as well
as medical, laboratory, or veterinarian personnel (e.g.,
clinicians, physicians, physician's assistants, laboratory
technicians, research scientists, and veterinarians) to test
organisms for possible genetic and/or epigenetic elements using a
nucleic acid-based assay without the need for potentially expensive
thermal cycling devices and potentially time consuming thermal
cycling techniques. In addition, the methods and materials provided
herein can allow patients as well as medical, laboratory, or
veterinarian personnel to detect any type of genetic and/or
epigenetic element suspected of being present within an organism
(e.g., a mammal such as a human). For example, the methods and
materials provided herein can be used to detect the presence or
absence of a single nucleotide polymorphism within the genome of a
human.
[0007] In general, one aspect of this document features a method
for assessing an organism for a genetic or epigenetic element. The
method comprises, or consists essentially of, (a) contacting a
sample from the organism with a probe nucleic acid comprising an
amplifying restriction endonuclease and a nucleotide sequence
complementary to a sequence of a target nucleic acid containing the
genetic or epigenetic element under conditions wherein, if the
target nucleic acid is present in the sample, at least a portion of
the target nucleic acid hybridizes to at least a portion of the
probe nucleic acid to form a double-stranded portion of nucleic
acid comprising a restriction endonuclease cut site, (b) contacting
the double-stranded portion of nucleic acid with a recognition
restriction endonuclease having the ability to cut the
double-stranded portion of nucleic acid at the restriction
endonuclease cut site under conditions wherein the recognition
restriction endonuclease cleaves the double-stranded portion of
nucleic acid at the restriction endonuclease cut site, thereby
separating a portion of the probe nucleic acid comprising the
amplifying restriction endonuclease from at least another portion
of the probe nucleic acid, (c) contacting the portion of the probe
nucleic acid comprising the amplifying restriction endonuclease
with a reporter nucleic acid comprising a double-stranded portion
of nucleic acid comprising a restriction endonuclease cut site of
the amplifying restriction endonuclease under conditions wherein
the amplifying restriction endonuclease cleaves the reporter
nucleic acid at the restriction endonuclease cut site of the
amplifying restriction endonuclease, thereby separating a portion
of the reporter nucleic acid from at least another portion of the
reporter nucleic acid, and (d) determining the presence or absence
of the portion of the reporter nucleic acid, wherein the presence
of the portion of the reporter nucleic acid indicates that the
sample contains the target nucleic acid, thereby indicating that
the organism contains the genetic or epigenetic element, and
wherein the absence of the portion of the reporter nucleic acid
indicates that the sample does not contain the target nucleic acid,
thereby indicating that the organism does not contain the genetic
or epigenetic element. The organism can be a human. The organism
can be a mammal. The mammal can be selected from the group
consisting of bovine, porcine, and equine species. The organism can
be a plant. The plant can be selected from the group consisting of
trees, flowers, shrubs, grains, grasses, and legumes. The method
comprises assessing the organism for the genetic element. The
genetic element can be an allelic variant known to exist in the
species of the organism. The genetic element can be a single
nucleotide polymorphism. The method can comprise assessing the
organism for the epigenetic element. The epigenetic element can be
a methylated DNA sequence. The sample can be selected from the
group consisting of blood samples, hair samples, skin samples,
throat swab samples, cheek swab samples, tissue samples, cellular
samples, and tumor samples. Prior to step (a), the sample can be a
sample that was processed to remove non-nucleic acid material from
the sample, thereby increasing the concentration of nucleic acid,
if present, within the sample. The sample can be a sample that was
subjected to a nucleic acid extraction technique. Prior to step
(a), the sample can be a sample that was subjected to a nucleic
acid amplification technique to increase the concentration of one
or more nucleic acids, if present, within the sample. The sample
can be a sample that was subjected to a PCR-based technique
designed to amplify the target nucleic acid. Prior to step (a), the
method can comprise removing non-nucleic acid material from the
sample, thereby increasing the concentration of nucleic acid, if
present, within the sample. The removing can comprise performing a
nucleic acid extraction technique. Prior to step (a), the method
can comprise performing a nucleic acid amplification technique to
increase the concentration of one or more nucleic acids, if
present, within the sample. The nucleic acid amplification
technique can comprise a PCR-based technique designed to amplify
the target nucleic acid. Prior to step (a), the method can comprise
removing non-nucleic acid material from the sample, thereby
increasing the concentration of nucleic acid, if present, within
the sample, and performing a nucleic acid amplification technique
to increase the concentration of one or more nucleic acids, if
present, within the sample. The probe nucleic acid can be
single-stranded probe nucleic acid. The probe nucleic acid can be
attached to a solid support. The probe nucleic acid can be directly
attached to a solid support. The portion of the probe nucleic acid
comprising the amplifying restriction endonuclease can be released
from the solid support via the step (b). Step (a) and step (b) can
be performed in the same compartment, or step (a), step (b), and
step (c) can be performed in the same compartment, or step (a),
step (b), step (c), and step (d) can be performed in the same
compartment. Step (a) and step (b) can be performed in a first
compartment, and step (c) can be performed in a second compartment.
Step (a) and step (b) can be performed by adding the sample to a
compartment comprising the probe nucleic acid and the recognition
restriction endonuclease. The probe nucleic acid can comprise (i) a
single-stranded portion comprising the nucleotide sequence
complementary to the sequence of the target nucleic acid and (ii) a
double-stranded portion. The probe nucleic acid can comprise a
first nucleic acid strand comprising the nucleotide sequence
complementary to the sequence of the target nucleic acid hybridized
to a second nucleic acid strand comprising the amplifying
restriction endonuclease. The first nucleic acid strand can be
attached to a solid support. The first nucleic acid strand can be
directly attached to a solid support. A portion of the second
nucleic acid strand can hybridize with the first nucleic acid
strand to form the double-stranded portion. The portion of the
probe nucleic acid comprising the amplifying restriction
endonuclease that is separated from the at least another portion of
the probe nucleic acid in step (b) can comprise a portion of the
first nucleic acid strand and all of the second strand. The portion
of the probe nucleic acid comprising the amplifying restriction
endonuclease that is separated from the at least another portion of
the probe nucleic acid in step (b) can comprise at least a portion
of the target nucleic acid.
[0008] In some cases, the method can comprise using a plurality of
the probe nucleic acid in the step (a). The method can comprise
using a plurality of the reporter nucleic acid in the step (c). The
reporter nucleic acid in the step (c) can be in molar excess of the
portion of the probe nucleic acid comprising the amplifying
restriction endonuclease from the step (b). The number of molecules
of the portion of the probe nucleic acid comprising the amplifying
restriction endonuclease that is separated from the at least
another portion of the probe nucleic acid in step (b) can be in an
essentially linear relationship to the number of molecules of the
target nucleic acid present in the sample. The reporter nucleic
acid can be attached to a solid support. The reporter nucleic acid
can be directly attached to a solid support. The reporter nucleic
acid can comprise a single-stranded portion of nucleic acid. The
reporter nucleic acid can comprise a label. The label can be a
fluorescent label, a radioactive label, an enzyme label, or a redox
label. The portion of the reporter nucleic acid that is separated
from the at least another portion of the reporter nucleic acid can
comprise the label. The reporter nucleic acid can comprise a first
nucleic acid strand comprising the label hybridized to a second
nucleic acid strand. The second nucleic acid strand can be attached
to a solid support. The second nucleic acid strand can be directly
attached to a solid support. A portion of the first nucleic acid
strand can hybridize with the second nucleic acid strand to form
the double-stranded portion of nucleic acid comprising the
restriction endonuclease cut site of the amplifying restriction
endonuclease. The reporter nucleic acid can comprise a third
nucleic acid strand. The third nucleic acid strand can hybridize
with the second nucleic acid strand to form the double-stranded
portion of nucleic acid comprising the restriction endonuclease cut
site of the amplifying restriction endonuclease. The reporter
nucleic acid can be attached to a solid support, and the portion of
the reporter nucleic acid that is separated from the at least
another portion of the reporter nucleic acid and that comprises the
label can be released from the solid support via the step (c). The
determining step (d) can comprise detecting the label. The label
can be a fluorescent label, and the determining step (d) comprises
detecting the fluorescent label. The determining step (d) can
comprise detecting the portion of the reporter nucleic acid
separated from the at least another portion of the reporter nucleic
acid using a capillary electrophoresis technique. The steps (a),
(b), and (c) can be performed without nucleic acid amplification,
or the steps (a), (b), (c), and (d) can be performed without
nucleic acid amplification. The determining step can comprise
determining the amount of the target nucleic acid present within
the sample.
[0009] In another aspect, this document features a method for an
organism for a genetic or epigenetic element. The method comprises,
or consists essentially of, (a) contacting a sample from the mammal
with a probe nucleic acid comprising an initial amplifying
restriction endonuclease and a nucleotide sequence complementary to
a sequence of a target nucleic acid containing the genetic or
epigenetic element under conditions wherein, if the target nucleic
acid is present in the sample, at least a portion of the target
nucleic acid hybridizes to at least a portion of the probe nucleic
acid to form a double-stranded portion of nucleic acid comprising a
restriction endonuclease cut site, (b) contacting the
double-stranded portion of nucleic acid with a recognition
restriction endonuclease having the ability to cut the
double-stranded portion of nucleic acid at the restriction
endonuclease cut site under conditions wherein the recognition
restriction endonuclease cleaves the double-stranded portion of
nucleic acid at the restriction endonuclease cut site, thereby
separating a portion of the probe nucleic acid comprising the
initial amplifying restriction endonuclease from at least another
portion of the probe nucleic acid, (c) contacting the portion of
the probe nucleic acid comprising the initial amplifying
restriction endonuclease with a first nucleic acid comprising a
secondary amplifying restriction endonuclease and a double-stranded
portion of nucleic acid comprising a restriction endonuclease cut
site of the initial amplifying restriction endonuclease under
conditions wherein the initial amplifying restriction endonuclease
cleaves the first nucleic acid at the restriction endonuclease cut
site of the initial amplifying restriction endonuclease, thereby
separating a portion of the first nucleic acid comprising the
secondary amplifying restriction endonuclease from at least another
portion of the first nucleic acid, (d) contacting the portion of
the first nucleic acid comprising the secondary amplifying
restriction endonuclease with a second nucleic acid comprising the
initial amplifying restriction endonuclease and a double-stranded
portion of nucleic acid comprising a restriction endonuclease cut
site of the secondary amplifying restriction endonuclease under
conditions wherein the secondary amplifying restriction
endonuclease cleaves the second nucleic acid at the restriction
endonuclease cut site of the secondary amplifying restriction
endonuclease, thereby separating a portion of the second nucleic
acid comprising the initial amplifying restriction endonuclease
from at least another portion of the second nucleic acid, (e)
contacting the portion of the second nucleic acid comprising the
initial amplifying restriction endonuclease with a reporter nucleic
acid comprising a double-stranded portion of nucleic acid
comprising a restriction endonuclease cut site of the initial
amplifying restriction endonuclease under conditions wherein the
initial amplifying restriction endonuclease cleaves the reporter
nucleic acid at the restriction endonuclease cut site of the
initial amplifying restriction endonuclease, thereby separating a
portion of the reporter nucleic acid from at least another portion
of the reporter nucleic acid, and (f) determining the presence or
absence of the portion of the reporter nucleic acid, wherein the
presence of the portion of the reporter nucleic acid indicates that
the sample contains the target nucleic acid, thereby indicating
that the organism contains the genetic or epigenetic element, and
wherein the absence of the portion of the reporter nucleic acid
indicates that the sample does not contain the target nucleic acid,
thereby indicating that the organism does not contain the genetic
or epigenetic element. The organism can be a human. The organism
can be a mammal. The mammal can be selected from the group
consisting of bovine, porcine, and equine species. The organism can
be a plant. The plant can be selected from the group consisting of
trees, flowers, shrubs, grains, grasses, and legumes. The method
can comprise assessing the organism for the genetic element. The
genetic element can be an allelic variant known to exist in the
species of the organism. The genetic element can be a single
nucleotide polymorphism. The method can comprise assessing the
organism for the epigenetic element. The epigenetic element can be
a methylated DNA sequence. The sample can be selected from the
group consisting of blood samples, hair samples, skin samples,
throat swab samples, cheek swab samples, tissue samples, cellular
samples, and tumor samples. Prior to step (a), the sample can be a
sample that was processed to remove non-nucleic acid material from
the sample, thereby increasing the concentration of nucleic acid,
if present, within the sample. The sample can be a sample that was
subjected to a nucleic acid extraction technique. Prior to step
(a), the sample can be a sample that was subjected to a nucleic
acid amplification technique to increase the concentration of one
or more nucleic acids, if present, within the sample. The sample
can be a sample that was subjected to a PCR-based technique
designed to amplify the target nucleic acid. Prior to step (a), the
method can comprise removing non-nucleic acid material from the
sample, thereby increasing the concentration of nucleic acid, if
present, within the sample. The removing can comprise performing a
nucleic acid extraction technique. Prior to step (a), the method
can comprise performing a nucleic acid amplification technique to
increase the concentration of one or more nucleic acids, if
present, within the sample. The nucleic acid amplification
technique can comprise a PCR-based technique designed to amplify
the target nucleic acid. Prior to step (a), the method can comprise
removing non-nucleic acid material from the sample, thereby
increasing the concentration of nucleic acid, if present, within
the sample, and performing a nucleic acid amplification technique
to increase the concentration of one or more nucleic acids, if
present, within the sample. The probe nucleic acid can be
single-stranded probe nucleic acid. The probe nucleic acid can be
attached to a solid support. The probe nucleic acid can be directly
attached to a solid support. The portion of the probe nucleic acid
comprising the initial amplifying restriction endonuclease can be
released from the solid support via the step (b). Step (a) and step
(b) can be performed in the same compartment, step (a), step (b),
and step (c) can be performed in the same compartment, step (a),
step (b), step (c), and step (d) can be performed in the same
compartment, step (a), step (b), step (c), step (d), and step (e)
can be performed in the same compartment, or step (a), step (b),
step (c), step (d), step (e), and step (f) can be performed in the
same compartment. Step (c) and step (d) can be performed in the
same compartment. Step (a) and step (b) can be performed in a first
compartment, and step (c) and step (d) can be performed in a second
compartment. Step (a) and step (b) can be performed by adding the
sample to a compartment comprising the probe nucleic acid and the
recognition restriction endonuclease. Step (c) and step (d) can be
performed by adding the portion of the probe nucleic acid
comprising the initial amplifying restriction endonuclease to a
compartment comprising the first nucleic acid and the second
nucleic acid. The probe nucleic acid can comprise (i) a
single-stranded portion comprising the nucleotide sequence
complementary to the sequence of the target nucleic acid and (ii) a
double-stranded portion. The probe nucleic acid can comprise a
first nucleic acid strand comprising the nucleotide sequence
complementary to the sequence of the target nucleic acid hybridized
to a second nucleic acid strand comprising the initial amplifying
restriction endonuclease. The first nucleic acid strand can be
attached to a solid support. The first nucleic acid strand can be
directly attached to a solid support. A portion of the second
nucleic acid strand can hybridize with the first nucleic acid
strand to form the double-stranded portion. The portion of the
probe nucleic acid comprising the initial amplifying restriction
endonuclease that is separated from the at least another portion of
the probe nucleic acid in step (b) can comprise a portion of the
first nucleic acid strand and all of the second strand. The portion
of the probe nucleic acid comprising the initial amplifying
restriction endonuclease that is separated from the at least
another portion of the probe nucleic acid in step (b) can comprise
at least a portion of the target nucleic acid.
[0010] In some cases, the method can comprise using a plurality of
the probe nucleic acid in the step (a). The method can comprise
using a plurality of the reporter nucleic acid in the step (e). The
reporter nucleic acid in the step (e) can be in molar excess of the
portion of the probe nucleic acid comprising the initial amplifying
restriction endonuclease from the step (b). The number of molecules
of the portion of the probe nucleic acid comprising the initial
amplifying restriction endonuclease that is separated from the at
least another portion of the probe nucleic acid in step (b) can be
in an essentially linear relationship to the number of molecules of
the target nucleic acid present in the sample. The first nucleic
acid and the second nucleic acid can be attached to a solid
support. The first nucleic acid and the second nucleic acid can be
directly attached to a solid support. The first nucleic acid and
the second nucleic acid can be attached to a solid support in the
same compartment. The portion of the first nucleic acid comprising
the secondary amplifying restriction endonuclease can be released
from the solid support via the step (c). The portion of the second
nucleic acid comprising the initial amplifying restriction
endonuclease can be released from the solid support via the step
(d). The first nucleic acid can comprise a first nucleic acid
strand comprising the secondary amplifying restriction endonuclease
hybridized to a second nucleic acid strand to form the
double-stranded portion of nucleic acid comprising the restriction
endonuclease cut site of the initial amplifying restriction
endonuclease. The first nucleic acid strand can be attached to a
solid support. The first nucleic acid strand can be directly
attached to a solid support. The second nucleic acid strand can be
attached to a solid support. The second nucleic acid strand can be
directly attached to a solid support. The second nucleic acid can
comprise a first nucleic acid strand comprising the initial
amplifying restriction endonuclease hybridized to a second nucleic
acid strand to form the double-stranded portion of nucleic acid
comprising the restriction endonuclease cut site of the secondary
amplifying restriction endonuclease. The first nucleic acid strand
can be attached to a solid support. The first nucleic acid strand
can be directly attached to a solid support. The second nucleic
acid strand can be attached to a solid support. The second nucleic
acid strand can be directly attached to a solid support. The
reporter nucleic acid can be attached to a solid support. The
reporter nucleic acid can be directly attached to a solid support.
The reporter nucleic acid can comprise a single-stranded portion of
nucleic acid. The reporter nucleic acid can comprise a label. The
label can be a fluorescent label, a radioactive label, an enzyme
label, or a redox label. The portion of the reporter nucleic acid
that is separated from the at least another portion of the reporter
nucleic acid can comprise the label. The reporter nucleic acid can
comprise a first nucleic acid strand comprising the label
hybridized to a second nucleic acid strand. The second nucleic acid
strand can be attached to a solid support. The second nucleic acid
strand can be directly attached to a solid support. A portion of
the first nucleic acid strand can hybridize with the second nucleic
acid strand to form the double-stranded portion of nucleic acid
comprising the restriction endonuclease cut site of the initial
amplifying restriction endonuclease. The reporter nucleic acid can
comprise a third nucleic acid strand. The third nucleic acid strand
can hybridize with the second nucleic acid strand to form the
double-stranded portion of nucleic acid comprising the restriction
endonuclease cut site of the initial amplifying restriction
endonuclease. The reporter nucleic acid can be attached to a solid
support, and the portion of the reporter nucleic acid that is
separated from the at least another portion of the reporter nucleic
acid and that comprises the label can be released from the solid
support via the step (e). The determining step (f) can comprise
detecting the label. The label can be a fluorescent label, and the
determining step (f) can comprise detecting the fluorescent label.
The determining step (f) can comprise detecting the portion of the
reporter nucleic acid separated from the at least another portion
of the reporter nucleic acid using a capillary electrophoresis
technique. Steps (a), (b), (c), (d), and (e) can be performed
without nucleic acid amplification, or steps (a), (b), (c), (d),
(e), and (f) can be performed without nucleic acid amplification.
The determining step can comprise determining the amount of the
target nucleic acid present within the sample.
[0011] In another aspect, this document features a method for
assessing an organism for a genetic or epigenetic element. The
method comprises, or consists essentially of, (a) contacting a
sample from the mammal with a probe nucleic acid comprising an
initial amplifying restriction endonuclease and a nucleotide
sequence complementary to a sequence of a target nucleic acid
containing the genetic or epigenetic element under conditions
wherein, if the target nucleic acid is present in the sample, at
least a portion of the target nucleic acid hybridizes to at least a
portion of the probe nucleic acid to form a double-stranded portion
of nucleic acid comprising a restriction endonuclease cut site, (b)
contacting the double-stranded portion of nucleic acid with a
recognition restriction endonuclease having the ability to cut the
double-stranded portion of nucleic acid at the restriction
endonuclease cut site under conditions wherein the recognition
restriction endonuclease cleaves the double-stranded portion of
nucleic acid at the restriction endonuclease cut site, thereby
separating a portion of the probe nucleic acid comprising the
initial amplifying restriction endonuclease from at least another
portion of the probe nucleic acid, (c) contacting the portion of
the probe nucleic acid comprising the initial amplifying
restriction endonuclease with a first reporter nucleic acid
comprising a secondary amplifying restriction endonuclease and a
double-stranded portion of nucleic acid comprising a restriction
endonuclease cut site of the initial amplifying restriction
endonuclease under conditions wherein the initial amplifying
restriction endonuclease cleaves the first reporter nucleic acid at
the restriction endonuclease cut site of the initial amplifying
restriction endonuclease, thereby separating a portion of the first
nucleic acid comprising the secondary amplifying restriction
endonuclease from at least another portion of the first nucleic
acid, (d) contacting the portion of the first reporter nucleic acid
comprising the secondary amplifying restriction endonuclease with a
second reporter nucleic acid comprising the initial amplifying
restriction endonuclease and a double-stranded portion of nucleic
acid comprising a restriction endonuclease cut site of the
secondary amplifying restriction endonuclease under conditions
wherein the initial amplifying restriction endonuclease cleaves the
second nucleic acid at the restriction endonuclease cut site of the
secondary amplifying restriction endonuclease, thereby separating a
portion of the second nucleic acid comprising the initial
amplifying restriction endonuclease from at least another portion
of the second nucleic acid, and (e) determining the presence or
absence of the portion of the first reporter nucleic acid, the
second reporter nucleic acid, or both the first reporter nucleic
acid and the second reporter nucleic acid, wherein the presence
indicates that the sample contains the target nucleic acid, thereby
indicating that the organism contains the genetic or epigenetic
element, and wherein the absence indicates that the sample does not
contain the target nucleic acid, thereby indicating that the
organism does not contain the genetic or epigenetic element. The
organism can be a human. The organism can be a mammal. The mammal
can be selected from the group consisting of bovine, porcine, and
equine species. The organism can be a plant. The plant can be
selected from the group consisting of trees, flowers, shrubs,
grains, grasses, and legumes. The method can comprise assessing the
organism for the genetic element. The genetic element can be an
allelic variant known to exist in the species of the organism. The
genetic element can be a single nucleotide polymorphism. The method
can comprise assessing the organism for the epigenetic element. The
epigenetic element can be a methylated DNA sequence. The sample can
be selected from the group consisting of blood samples, hair
samples, skin samples, throat swab samples, cheek swab samples,
tissue samples, cellular samples, and tumor samples. Prior to step
(a), the sample can be a sample that was processed to remove
non-nucleic acid material from the sample, thereby increasing the
concentration of nucleic acid, if present, within the sample. The
sample can be a sample that was subjected to a nucleic acid
extraction technique. Prior to step (a), the sample can be a sample
that was subjected to a nucleic acid amplification technique to
increase the concentration of one or more nucleic acids, if
present, within the sample. The sample can be a sample that was
subjected to a PCR-based technique designed to amplify the target
nucleic acid. Prior to step (a), the method can comprise removing
non-nucleic acid material from the sample, thereby increasing the
concentration of nucleic acid, if present, within the sample. The
removing can comprise performing a nucleic acid extraction
technique. Prior to step (a), the method can comprise performing a
nucleic acid amplification technique to increase the concentration
of one or more nucleic acids, if present, within the sample. The
nucleic acid amplification technique can comprise a PCR-based
technique designed to amplify the target nucleic acid. Prior to
step (a), the method can comprise removing non-nucleic acid
material from the sample, thereby increasing the concentration of
nucleic acid, if present, within the sample, and performing a
nucleic acid amplification technique to increase the concentration
of one or more nucleic acids, if present, within the sample. The
probe nucleic acid can be single-stranded probe nucleic acid. The
probe nucleic acid can be attached to a solid support. The probe
nucleic acid can be directly attached to a solid support. The
portion of the probe nucleic acid comprising the initial amplifying
restriction endonuclease can be released from the solid support via
the step (b). Step (a) and step (b) can be performed in the same
compartment, step (a), step (b), and step (c) can be performed in
the same compartment, step (a), step (b), step (c), and step (d)
can be performed in the same compartment, or step (a), step (b),
step (c), step (d), and step (e) can be performed in the same
compartment. Step (c) and step (d) can be performed in the same
compartment. Step (a) and step (b) can be performed in a first
compartment, and step (c) and step (d) can be performed in a second
compartment. Step (a) and step (b) can be performed by adding the
sample to a compartment comprising the probe nucleic acid and the
recognition restriction endonuclease. Step (c) and step (d) can be
performed by adding the portion of the probe nucleic acid
comprising the initial amplifying restriction endonuclease to a
compartment comprising the first reporter nucleic acid and the
second reporter nucleic acid. The probe nucleic acid can comprise
(i) a single-stranded portion comprising the nucleotide sequence
complementary to the sequence of the target nucleic acid and (ii) a
double-stranded portion. The probe nucleic acid can comprise a
first nucleic acid strand comprising the nucleotide sequence
complementary to the sequence of the target nucleic acid hybridized
to a second nucleic acid strand comprising the initial amplifying
restriction endonuclease. The first nucleic acid strand can be
attached to a solid support. The first nucleic acid strand can be
directly attached to a solid support. A portion of the second
nucleic acid strand can hybridize with the first nucleic acid
strand to form the double-stranded portion. The portion of the
probe nucleic acid comprising the initial amplifying restriction
endonuclease that is separated from the at least another portion of
the probe nucleic acid in step (b) can comprise a portion of the
first nucleic acid strand and all of the second strand. The portion
of the probe nucleic acid comprising the initial amplifying
restriction endonuclease that is separated from the at least
another portion of the probe nucleic acid in step (b) can comprise
at least a portion of the target nucleic acid.
[0012] In some cases, the method can comprise using a plurality of
the probe nucleic acid in the step (a). The method can comprise
using a plurality of the first reporter nucleic acid in the step
(c). The first reporter nucleic acid in the step (c) can be in
molar excess of the portion of the probe nucleic acid comprising
the initial amplifying restriction endonuclease from the step (b).
The method can comprise using a plurality of the second reporter
nucleic acid in the step (d). The second reporter nucleic acid in
the step (d) can be in molar excess of the portion of the probe
nucleic acid comprising the initial amplifying restriction
endonuclease from the step (b). The number of molecules of the
portion of the probe nucleic acid comprising the initial amplifying
restriction endonuclease that is separated from the at least
another portion of the probe nucleic acid in step (b) can be in an
essentially linear relationship to the number of molecules of the
target nucleic acid present in the sample. The first reporter
nucleic acid and the second reporter nucleic acid can be attached
to a solid support. The first reporter nucleic acid and the second
reporter nucleic acid can be directly attached to a solid support.
The first reporter nucleic acid and the second reporter nucleic
acid can be attached to a solid support in the same compartment.
The portion of the first reporter nucleic acid comprising the
secondary amplifying restriction endonuclease can be released from
the solid support via the step (c). The portion of the second
reporter nucleic acid comprising the initial amplifying restriction
endonuclease can be released from the solid support via the step
(d). The first reporter nucleic acid can comprise a label. The
label can be a fluorescent label, a radioactive label, an enzyme
label, or a redox label. The second reporter nucleic acid can
comprise a label. The label can be a fluorescent label, a
radioactive label, an enzyme label, or a redox label. The first
reporter nucleic acid and the second reporter nucleic acid can
comprise a label. The first reporter nucleic acid and the second
reporter nucleic acid can comprise the same label. The label can be
a fluorescent label, a radioactive label, an enzyme label, or a
redox label. The first reporter nucleic acid can be attached to a
solid support, the portion of the first reporter nucleic acid that
is separated from the at least another portion of the first
reporter nucleic acid can comprise a label, and the portion of the
first reporter nucleic acid that is separated from the at least
another portion of the first reporter nucleic acid and that
comprises the label can be released from the solid support via the
step (c). The first reporter nucleic acid can comprise a first
nucleic acid strand comprising the secondary amplifying restriction
endonuclease hybridized to a second nucleic acid strand to form the
double-stranded portion of nucleic acid comprising the restriction
endonuclease cut site of the initial amplifying restriction
endonuclease. The first nucleic acid strand can be attached to a
solid support. The first nucleic acid strand can be directly
attached to a solid support. The second nucleic acid strand can be
attached to a solid support. The second nucleic acid strand can be
directly attached to a solid support. The first nucleic acid strand
can comprise a label. The label can be a fluorescent label, a
radioactive label, an enzyme label, or a redox label. The second
nucleic acid strand can comprise a label. The label can be a
fluorescent label, a radioactive label, an enzyme label, or a redox
label. The second reporter nucleic acid can be attached to a solid
support, the portion of the second reporter nucleic acid that is
separated from the at least another portion of the second reporter
nucleic acid can comprise a label, and the portion of the second
reporter nucleic acid that is separated from the at least another
portion of the second reporter nucleic acid and that comprises the
label can be released from the solid support via the step (d). The
second reporter nucleic acid can comprise a first nucleic acid
strand comprising the initial amplifying restriction endonuclease
hybridized to a second nucleic acid strand to form the
double-stranded portion of nucleic acid comprising the restriction
endonuclease cut site of the secondary amplifying restriction
endonuclease. The first nucleic acid strand can be attached to a
solid support. The first nucleic acid strand can be directly
attached to a solid support. The second nucleic acid strand can be
attached to a solid support. The second nucleic acid strand can be
directly attached to a solid support. The first nucleic acid strand
can comprise a label. The label can be a fluorescent label, a
radioactive label, an enzyme label, or a redox label. The second
nucleic acid strand can comprise a label. The label can be a
fluorescent label, a radioactive label, an enzyme label, or a redox
label. The portion of the first reporter nucleic acid separated
from the at least another portion of the first reporter nucleic
acid can comprise a fluorescent label, the portion of the second
reporter nucleic acid separated from the at least another portion
of the second reporter nucleic acid can comprise a fluorescent
label, and the determining step (e) can comprise detecting the
fluorescent label. The determining step (e) can comprise detecting
the portion of the first reporter nucleic acid separated from the
at least another portion of the first reporter nucleic acid using a
capillary electrophoresis technique. The determining step (e) can
comprise detecting the portion of the second reporter nucleic acid
separated from the at least another portion of the second reporter
nucleic acid using a capillary electrophoresis technique. Steps
(a), (b), (c), and (d) can be performed without nucleic acid
amplification, or steps (a), (b), (c), (d), and (e) can be
performed without nucleic acid amplification. The determining step
can comprise determining the amount of the target nucleic acid
present within the sample.
[0013] In another aspect, this document features a kit for
assessing an organism for a genetic or epigenetic element. The kit
comprises, or consists essentially of, a probe nucleic acid
comprising an amplifying restriction endonuclease and a nucleotide
sequence complementary to a sequence of a target nucleic acid
containing the genetic or epigenetic element, wherein at least a
portion of the target nucleic acid is capable of hybridizing to at
least a portion of the probe nucleic acid to form a double-stranded
portion of nucleic acid comprising a restriction endonuclease cut
site. The probe nucleic acid can be single-stranded probe nucleic
acid. The kit can comprise a solid support, and the probe nucleic
acid can be attached to the solid support. A portion of the probe
nucleic acid comprising the amplifying restriction endonuclease can
be releasable from the solid support via cleavage with a
recognition restriction endonuclease having the ability to cleave
at the restriction endonuclease cut site. The kit can further
comprise the recognition restriction endonuclease. The probe
nucleic acid can comprise (i) a single-stranded portion comprising
the nucleotide sequence complementary to the sequence of the target
nucleic acid and (ii) a double-stranded portion. The probe nucleic
acid can comprise a first nucleic acid strand comprising the
nucleotide sequence complementary to the sequence of the target
nucleic acid hybridized to a second nucleic acid strand comprising
the amplifying restriction endonuclease. The kit can further
comprise a reporter nucleic acid comprising a double-stranded
portion of nucleic acid comprising a restriction endonuclease cut
site of the amplifying restriction endonuclease. The kit can
comprise a solid support, and the reporter nucleic acid can be
attached to the solid support. The reporter nucleic acid can be
directly attached to the solid support. The reporter nucleic acid
can comprise a single-stranded portion of nucleic acid. The
reporter nucleic acid can comprise a label. The label can be a
fluorescent label, a radioactive label, an enzyme label, or a redox
label. A portion of the reporter nucleic acid comprising the label
can be capable of being separated from at least another portion of
the reporter nucleic acid via cleavage by the amplifying
restriction endonuclease. The reporter nucleic acid can comprise a
first nucleic acid strand comprising the label hybridized to a
second nucleic acid strand. The kit can further comprise: (a) a
first signal expansion nucleic acid comprising a secondary
amplifying restriction endonuclease and a double-stranded section
having a restriction endonuclease cut site for the amplifying
restriction endonuclease, and (b) a second signal expansion nucleic
acid comprising the amplifying restriction endonuclease and a
double-stranded section having a restriction endonuclease cut site
for the secondary amplifying restriction endonuclease. The probe
nucleic acid can be lyophilized. All the ingredients of the kit can
be lyophilized or dry.
[0014] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used to practice the invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references (e.g., GenBank.RTM.
records) mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0015] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic depicting an exemplary method for
detecting target nucleic acid using probe nucleic acid, a
recognition restriction endonuclease, and reporter nucleic
acid.
[0017] FIG. 2 is a schematic of an exemplary configuration of probe
nucleic acid that can be used with the methods and materials
provided herein for detecting target nucleic acid.
[0018] FIG. 3 is a schematic depicting an exemplary method for
detecting target nucleic acid using probe nucleic acid, a
recognition restriction endonuclease, first signal expansion
nucleic acid, second signal expansion nucleic acid, and reporter
nucleic acid.
[0019] FIG. 4 is a schematic of an exemplary configuration of first
signal expansion nucleic acid and second signal expansion nucleic
acid that can be used with the methods and materials provided
herein for detecting target nucleic acid. Such first signal
expansion nucleic acid and second signal expansion nucleic acid can
be used with or without reporter nucleic acid. When used without a
separate reporter nucleic acid step, such signal expansion nucleic
acid can be referred to as reporter nucleic acid.
[0020] FIG. 5 is a schematic of an exemplary configuration of first
signal expansion nucleic acid and second signal expansion nucleic
acid that can be used with the methods and materials provided
herein for detecting target nucleic acid. Such first signal
expansion nucleic acid and second signal expansion nucleic acid can
be used with or without reporter nucleic acid. When used without a
separate reporter nucleic acid step, such signal expansion nucleic
acid can be referred to as reporter nucleic acid.
[0021] FIG. 6 contains line graphs demonstrating the effect of
target oligonucleotide concentration (A) and recognition
restriction endonuclease concentration (B) on the cleavage of
HRP-labeled nucleic acid as detected by the formation of colored
reaction product.
[0022] FIG. 7 is a schematic of an exemplary configuration for a
single-use, pen-style point of care device.
[0023] FIG. 8 is a diagram of an example of a method that can be
used to detect methylated target DNA. In this example, target DNA
is hybridized with the same probe in two different compartments
(e.g., wells). Then, a recognition restriction endonuclease (Rr1),
which is methylation-sensitive, is added to the compartment 1. Rr1
can only cleave its corresponding restriction site if it is
unmodified by methylation (unmethylated C). Another recognition
restriction endonuclease (Rr2), which is methylation-insensitive,
is added to the compartment 2. Rr2 can cleave both methylated and
unmethylated versions of the target DNA. Signal detection for both
compartments is then completed as described herein. The resultant
signal from compartment 1 corresponds exclusively to unmethylated
target, while the resultant signal from compartment 2 corresponds
to all target DNA notwithstanding its methylation state. Thus, the
amount of methylated target can be calculated by subtracting the
compartment 1 signal from the compartment 2 signal. This example
uses a pair of methylation sensitive and methylation insensitive
restriction endonucleases that recognize and cut the same
site/sequence.
[0024] FIG. 9 is a diagram of an example of a method that can be
used to detect methylated target DNA using two different
methylation sensitive recognition restriction endonucleases (Rr1
and Rr1) that have different cut sites (cut site A and cut site
B).
[0025] FIG. 10 is a diagram demonstrating how target nucleic acid
that perfectly matches (perfect match, PM) the probe nucleic acid
at the generated cut site is cleaved by the recognition restriction
endonuclease, while nucleic acid lacking the perfect match
(mismatch, MM) with the probe nucleic acid at the generated cut
site is not cleaved by the recognition restriction
endonuclease.
[0026] FIG. 11 contains a double stranded section of DNA (SEQ ID
NO:1) that contains the recognition site and cleavage site for a
FokI restriction endonuclease.
[0027] FIG. 12 is a diagram of an example of a method that can be
used to differentiate between any target nucleic acid that
perfectly matches a portion of probe nucleic acid and any nucleic
acid that does not perfectly match that portion of probe nucleic
acid. Panel A is a diagram demonstrating how target nucleic acid
that perfectly matches (perfect match, PM) the probe nucleic acid
generates a cut site, of a recognition restriction endonuclease
that has separate recognition and cleavage sites (e.g., an FokI
restriction endonuclease), and together with the probe nucleic acid
is cleaved by the recognition restriction endonuclease. Panel B is
a diagram demonstrating how nucleic acid lacking a perfect match
(e.g., nucleic acid containing a SNP) with the probe nucleic acid
does not generate a cut site, of a recognition restriction
endonuclease that has separate recognition and cleavage sites
(e.g., an FokI restriction endonuclease), and together with the
probe nucleic acid is not cleaved by the recognition restriction
endonuclease.
[0028] FIG. 13 depicts two probe nucleic acids, one designed to
detect target nucleic acid of an un-mutated version of an
thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) enzyme (A; SEQ
ID NO:13 linked to SEQ ID NO:11), and one designed to detect target
nucleic acid of a mutated version of TPMT carrying a SNP in the
codon 154 (B; SEQ ID NO:13 linked to SEQ ID NO:12).
DETAILED DESCRIPTION
[0029] This document provides methods and materials for detecting
genetic and/or epigenetic elements. For example, this document
relates to methods and materials involved in using an enzymatic
amplification cascade of restriction endonucleases to detect
genetic and/or epigenetic elements present within an organism
(e.g., a human). In some cases, this document provides methods and
materials for detecting target nucleic acid that contains a genetic
element or epigenetic element. For example, this document provides
methods and materials for detecting the presence or absence of
target nucleic acid (e.g., target nucleic acid containing a
particular genetic element) in an organism's genome, methods and
materials for detecting the presence or absence of target nucleic
acid that contains an epigenetic element (e.g., methylated DNA) in
a cell of an organism, kits for detecting the presence or absence
of target nucleic acid (e.g., target nucleic acid containing a
particular genetic element) in an organism's genome, kits for
detecting the presence or absence of target nucleic acid that
contains an epigenetic element (e.g., methylated DNA) in a cell of
an organism, and methods for making such kits.
[0030] Any type of organism (e.g., plant or animal) can be assessed
using the methods and materials provided herein to determine
whether or not the organism contains a genetic and/or epigenetic
element. Examples of organisms that can be assessed using the
methods and materials provided herein to determine whether or not
the organism contains a genetic and/or epigenetic element include,
without limitation, plants (e.g., trees, flowers, shrubs, grains,
grasses, and legumes), mammals (e.g., humans, dogs, cats, cows,
horses, pigs, sheep, goats, monkeys, buffalo, bears, whales, and
dolphins), avian species (e.g., chickens, turkeys, ostrich, emus,
cranes, and falcons), and non-mammalian animals (e.g., mollusks,
frogs, lizards, snakes, and insects). For example, plant crops such
as corn, soybeans, wheat, and rice can be assessed for the presence
or absence of genetic elements such as possible introduced
transgenes, transposable elements, or polymorphisms.
[0031] Any type of biological sample can be used with the methods
and materials provided herein to assess an organism for a
particular genetic or epigenetic element. For example, any type of
biological sample that is obtained from an organism to be tested
and that contains the organism's nucleic acid (e.g., potentially
methylated nucleic acid or genomic DNA) can be used as described
herein. Examples of samples that can be used as described herein
include, without limitation, blood samples (e.g., 50 mL collection
of a patient's blood), serum samples, hair samples, skin samples,
tissue samples (e.g., tissue biopsy samples), bone marrow samples,
tumor samples, amniotic fluid samples, throat or cheek swab samples
(e.g., a buccal smear sample), and mouthwash samples. In some
cases, a sample used herein can be a serum sample prepared from
whole blood such that circulating DNA is present in the sample as
described elsewhere (Sunami et al., Methods Mol. Biol., 507:349-56
(2009)).
[0032] The methods and materials provided herein can be used to
assess an organism for any type of genetic or epigenetic element.
Examples of possible genetic elements that can be assessed using
the methods and materials provided herein include, without
limitation, wild-type or common standard allele sequences of an
organism's species, mutant or uncommon allele sequences of an
organism's species, sequence insertions, sequence deletions,
sequence substitutions, polymorphisms, SNPs, or combinations
thereof. Examples of possible epigenetic elements that can be
assessed using the methods and materials provided herein include,
without limitation, methylated DNA. In some cases, an organism can
be assessed for one or more of the genetic or epigenetic elements
listed in Table 1 using the methods and materials provided herein.
When designing a method for detecting a genetic or epigenetic
element listed in Table 1, a probe nucleic acid can be designed
that is complementary to a portion of any of the indicated
sequences from Table 1. For example, when designing a method for
detecting a single nucleotide polymorphism (SNP) in the human
thiopurine S-methyltransferase gene (Ensemble gene ID
ENSG00000137364; Chromosome 6: 18,128,542-18,155,305 reverse
strand), a probe nucleic acid can be designed that is complementary
to a portion that includes positions 18143896-18143901 of the
sequence. In some cases, one or more of the DNA methylation events
described elsewhere (e.g., Szyf, Ageing Res. Rev., 2(3):299-328
(2003); Di Gioia et al., BMC Cancer, 6:89 (2006); Maruya et al.,
Clin. Cancer Res., 10:3825 (2004); Fischer et al., Lung Cancer,
56:115-123 (2007); Yeo et al., Pathology, 37:125-130 (2005); Allen
Chan et al., Clin. Chem., 10:1373 (2008); Wang et al., Lung Cancer,
56:289-294 (2007); and Widschwendter et al., PLoS ONE, 3(7):e2656
(2008)) can be detected using the methods and materials provided
herein, for example, to provide information about conditions such
as carcinogenesis or tumor metastasis.
TABLE-US-00001 TABLE 1 Types of genetic or epigenetic elements that
can be detected. Possible Associated Organism Genetic or Epigenetic
Element Condition Human C825T polymorphism of the GNB3 Glioblastoma
Human RASSF2A Cancers Human MGMT Cancers Human Cyclins D1 and D2
(CCDN1 and CCDN2) Cancers Human HOXA10 Ovarian cancer Human
thiopurine S-methyltransferase Drug toxicity Human APP, PS1, and
PS2 Alzheimer's Disease inheritance of the APOE4 allele confers
increased risk for AD Human MTCYB gene Parkinson disease Human
Congenital heart disease (CHD) Human CHDS1, Chromosome: 16;
Location: 16pter-p13, Coronary heart disease GeneID: 338334 Human
PARK7, Chromosome: 1; Location: 1p36.23, GeneID: Parkinson disease
11315 Human BRCA1, BRCA2 Breast cancer Human BCL2 Cancers Human IL2
Cancers Human CFTR Cystic Fibrosis Human TSC2 Tuberous sclerosis
Human APP Alzheimer's Disease Human NPPB Cardiovascular disease
Human LEP Obesity Human OSM Leukemia Human MCM6 lactose intolerance
Horse LAMA3 skin blistering disease Cow DGAT1 Milk Production Dog
EPM2A Epilepsy
[0033] In one embodiment, a method for assessing an organism for a
genetic or epigenetic element can include determining whether or
not a biological sample obtained from the organism contains a
target nucleic acid having the genetic or epigenetic element of
interest. For example, a biological sample (e.g., a blood sample to
be tested) can be placed in contact with probe nucleic acid. The
probe nucleic acid can be designed to have a single-stranded
portion with a nucleotide sequence that is complementary to at
least a portion of the target nucleic acid to be detected. In this
case, target nucleic acid present within the sample can hybridize
with the complementary sequence of this single-stranded portion of
the probe nucleic acid to form a double-stranded section with one
strand being target nucleic acid and the other strand being probe
nucleic acid. In addition, the single-stranded portion of the probe
nucleic acid having the nucleotide sequence that is complementary
to at least a portion of the target nucleic acid to be detected can
be designed such that hybridization with the target nucleic acid
creates a restriction endonuclease cut site. Thus, target nucleic
acid present within the sample can hybridize with the complementary
sequence of the single-stranded portion of the probe nucleic acid
to form a double-stranded section that creates a cut site for a
restriction endonuclease. This cut site created by the
hybridization of target nucleic acid to probe nucleic acid can be
referred to as a recognition restriction endonuclease cut site. In
addition, a restriction endonuclease that cleaves nucleic acid at
such a recognition restriction endonuclease cut site can be
referred to as a recognition restriction endonuclease.
[0034] The probe nucleic acid also can be designed to contain a
restriction endonuclease. This restriction endonuclease, which can
be a component of the probe nucleic acid, can be referred to as an
amplifying restriction endonuclease. An amplifying restriction
endonuclease is typically a different restriction endonuclease than
the restriction endonuclease that is used as a recognition
restriction endonuclease. For example, when an EcoRI restriction
endonuclease is used as a recognition restriction endonuclease, a
restriction endonuclease other than an EcoRI restriction
endonuclease (e.g., a Hind III restriction endonuclease) is used as
an amplifying restriction endonuclease. Thus, in general, probe
nucleic acid is designed to contain an amplifying restriction
endonuclease and to have a nucleotide sequence such that the target
nucleic acid can hybridize to the probe nucleic acid and create a
recognition restriction endonuclease cut site for a recognition
restriction endonuclease. In some cases, the probe nucleic acid can
be attached to a solid support (e.g., a well of a microtiter
plate). For example, the probe nucleic acid can be attached to a
solid support such that cleavage at the recognition restriction
endonuclease cut site via the recognition restriction endonuclease
releases a portion of the probe nucleic acid that contains the
amplifying restriction endonuclease.
[0035] After contacting the sample (e.g., a biological sample) that
may or may not contain target nucleic acid with the probe nucleic
acid that is attached to a solid support, the target nucleic acid,
if present in the sample, can hybridize to the probe nucleic acid
and create the recognition restriction endonuclease cut site. At
this point, the recognition restriction endonuclease, whether added
to the reaction or already present in the reaction, can cleave the
probe nucleic acid at the recognition restriction endonuclease cut
sites that are formed by the hybridization of target nucleic acid
to the probe nucleic acid, thereby releasing the portion of the
probe nucleic acid that contains the amplifying restriction
endonuclease from the solid support. The number of amplifying
restriction endonuclease-containing portions of the probe nucleic
acid that are released from the solid support can be in an
essentially linear relationship (e.g., essentially a one-for-one
relationship) with the number of target nucleic acid molecules that
hybridize with the probe nucleic acid to form the recognition
restriction endonuclease cut site.
[0036] The portions of the probe nucleic acid containing the
amplifying restriction endonuclease that were released from the
solid support can be collected and placed in contact with reporter
nucleic acid. For example, the released portions of the probe
nucleic acid, if present, can be transferred from one well of a
microtiter plate (e.g., a 96-well plate) that contained the probe
nucleic acid to another well of a microtiter plate that contains
the reporter nucleic acid. The reporter nucleic acid can be
designed to have a double-stranded portion with a restriction
endonuclease cut site for the amplifying restriction endonuclease
of the probe nucleic acid. This restriction endonuclease cut site
for the amplifying restriction endonuclease can be referred to as
an amplifying restriction endonuclease cut site. If portions of the
probe nucleic acid containing the amplifying restriction
endonuclease are present and placed in contact with the reporter
nucleic acid, then the reporter nucleic acid can be cleaved at the
amplifying restriction endonuclease cut site by the amplifying
restriction endonuclease. Since the amplifying restriction
endonucleases of the released portions of the probe nucleic acid
are free to carry out repeated cleavage events, the number of
reporter nucleic acid molecules that are cleaved can greatly exceed
the number of amplifying restriction endonucleases present in the
reaction. For example, the number of cleaved reporter nucleic acid
molecules can greatly exceed (e.g., exponentially exceed) the
number of amplifying restriction endonucleases present in the
reaction and therefore can greatly exceed (e.g., exponentially
exceed) the number of target nucleic acid molecules that were
present in the sample contacted with the probe nucleic acid. Such a
greatly expanded relationship (e.g., an exponential relationship)
can allow very small amounts of target nucleic acid present in the
sample to be readily detected.
[0037] After the released portions of the probe nucleic acid, if
present, are contacted with the reporter nucleic acid, the presence
or absence of cleaved reporter nucleic acid can be determined. The
presence of cleaved reporter nucleic acid can indicate that the
sample contained the target nucleic acid, thereby indicating that
the sample contained the target genetic or epigenetic element for
which the sample is being tested, while the absence of cleaved
reporter nucleic acid can indicate that the sample lacked the
target nucleic acid, thereby indicating that the sample lacked the
target genetic or epigenetic element for which the sample is being
tested. In some cases, the amount of cleaved reporter nucleic acid
can be determined. In such cases, the amount of cleaved reporter
nucleic acid can indicate the amount of target nucleic acid present
in the sample, which can indicated the relative amount of the
genetic or epigenetic element present in the organism being tested.
A standard curve using known amounts of target nucleic acid can be
used to aid in the determination of the amount of target nucleic
acid present within a sample. For example, genomic DNA from known
heterozygous and/or homozygous (e.g., homozygous for a particular
genetic element being tested for) organisms can be included in an
assay to determine whether a genomic DNA sample from an organism
being tested contains zero, one, or two copies of a particular
target nucleic acid for which the organism is being tested based on
the amount of cleaved reporter nucleic acid.
[0038] In some cases, the reporter nucleic acid can contain a label
to aid in the detection of cleaved reporter nucleic acid. For
example, reporter nucleic acid can contain a fluorescent label and
a quencher such that cleaved reporter nucleic acid provides a
fluorescent signal and uncleaved reporter nucleic acid does not
provide a fluorescent signal. In some cases, the reporter nucleic
acid can contain a label (e.g., a colorimetric label, a fluorescent
label or an enzyme (e.g., a redox enzyme) such as horse radish
peroxidase) and can be attached to a solid support (e.g., a well of
a microtiter plate). For example, the reporter nucleic acid can be
attached to a solid support such that cleavage at the amplifying
restriction endonuclease cut site by the amplifying restriction
endonuclease releases a portion of the reporter nucleic acid that
contains the label. The resulting reaction mixture can be collected
and assessed for the presence, absence, or amount of released
portions of the reporter nucleic acid using the label. For example,
the released portions of the reporter nucleic acid, if present, can
be transferred from one well of a microtiter plate (e.g., a 96-well
plate) that contained the reporter nucleic acid to another well of
a microtiter plate, where the transferred material can be assessed
for a signal from the label.
[0039] One example of a method of detecting target nucleic acid
that includes using probe nucleic acid and reporter nucleic acid is
set forth in FIG. 1. With reference to FIG. 1, first reaction
chamber 100 (e.g., a microtiter plate well) can contain probe
nucleic acid 101. Probe nucleic acid 101 can be attached (e.g.,
immobilized) to solid support 102 and can include amplifying
restriction endonuclease 103 (Ra). Probe nucleic acid 101 can be
attached to solid support 102 such that amplifying restriction
endonuclease 103 is released from solid support 102 upon cleavage
of a nucleic acid component of probe nucleic acid 101. Probe
nucleic acid 101 can have a single-stranded section having a
nucleotide sequence that is complementary to at least a portion of
target nucleic acid 104. Probe nucleic acid 101 can be contacted
with a sample that may or may not contain target nucleic acid 104.
If target nucleic acid 104 is present, at least a portion of target
nucleic acid 104 and probe nucleic acid 101 can hybridize to form a
double-stranded section of nucleic acid. Such a double-stranded
section can contain at least one recognition restriction
endonuclease cut site 105. Addition of recognition restriction
endonuclease 106 (Rr) to first reaction chamber 100 can result in
the cleave of probe nucleic acid 101 at recognition restriction
endonuclease cut site 105 formed by one strand of probe nucleic
acid and one strand of target nucleic acid, thereby releasing
portion 107 of probe nucleic acid 101 from solid support 102.
Portion 107 can include amplifying restriction endonuclease
103.
[0040] The reaction product from first reaction chamber 100
containing released portion 107, if target nucleic acid 104 was
present, can be transferred (e.g., manually or automatically) to
second reaction chamber 120. Second reaction chamber 120 can
contain reporter nucleic acid 121. Reporter nucleic acid 121 can be
attached (e.g., immobilized) to solid support 122 and can include
marker (e.g., a label) 123 (M). Reporter nucleic acid 121 can be
attached to solid support 122 such that marker 123 is released from
solid support 122 upon cleavage of a nucleic acid component of
reporter nucleic acid 121. Reporter nucleic acid 121 can have at
least one double-stranded portion that contains at least one
amplifying restriction endonuclease cut site 124. Addition of the
reaction product from first reaction chamber 100 to second reaction
chamber 120 can result in the cleavage of reporter nucleic acid 121
at amplifying restriction endonuclease cut site 124 if the reaction
product contains portion 107. Such cleavage of reporter nucleic
acid 121 can result in the release of portion 127 from solid
support 122. Portion 127 can include marker 123.
[0041] The reaction product from second reaction chamber 120 can be
assessed to determine the presence, absence, or amount of portion
127. The presence of portion 127 can indicate that the sample
contained target nucleic acid 104, while the absence of portion 127
can indicate that the sample lacked target nucleic acid 104. In
some cases, the amount of portion 127 can be determined. In such
cases, the amount of portion 127 can indicate the amount of target
nucleic acid 104 present in the sample. The presence, absence, or
amount of portion 127 can be determined using marker 123, and
portion 127 having marker 123 can be distinguished from uncleaved
reporter nucleic acid 121 having marker 123 since, in this example,
portion 127 is released from solid support 122, while uncleaved
reporter nucleic acid 121 remains attached to solid support 122.
For example, in some cases, the reaction product from second
reaction chamber 120 can be transferred to third reaction chamber
where the presence or absence of portion 127 via marker 123 is
assessed. If portion 127 is present, the amount of portion 127
present can be quantified.
[0042] Probe nucleic acid 101 and reporter nucleic acid 121 can
have various configurations. For example, with reference to FIG. 1,
probe nucleic acid 101 can be designed to have a single nucleic
acid strand such that the entire nucleic acid component of probe
nucleic acid 101 is single-stranded prior to contact with target
nucleic acid 104. In another example, with reference to FIG. 2,
probe nucleic acid 101 can be designed to have first strand 128 and
second strand 108. First strand 128 can be attached to solid
support 102 and can be designed to have a single-stranded section
having a nucleotide sequence that is complementary to at least a
portion of target nucleic acid 104. Second strand 108 can include
amplifying restriction endonuclease 103 and can have a
single-stranded section having a nucleotide sequence that can
hybridize to first strand 128. In some cases, first strand 128 and
second strand 108 can be synthesized or obtained separately and
then mixed together to form probe nucleic acid 101. For example,
first strand 128 can be synthesized, biotinylated, and attached to
a streptavidin-coated solid support. After synthesizing the nucleic
acid component of second strand 108 and attaching amplifying
restriction endonuclease 103 to the synthesized nucleic acid
component, second strand 108 can be incubated with first strand 128
to form nucleic acid probe 101. In some cases, probe nucleic acid
101 can contain more than two strands. For example, probe nucleic
acid can include first strand 150, second strand 152, and third
strand 154. In this case, first strand 150 can be attached to solid
support 102, second strand 152 can be hybridized to first strand
150 and can include a single-stranded section having a nucleotide
sequence that is complementary to at least a portion of target
nucleic acid 104, and third strand 154 can be hybridized to second
strand 152 and can be attached to amplifying restriction
endonuclease 103. Similar one, two, three, or more strand
configurations can be used to make reporter nucleic acid.
[0043] In another embodiment, a method for detecting target nucleic
acid can include contacting a sample (e.g., a biological sample to
be tested) with probe nucleic acid. The probe nucleic acid can be
designed to have a single-stranded portion with a nucleotide
sequence that is complementary to at least a portion of the target
nucleic acid to be detected. In this case, target nucleic acid
present within the sample can hybridize with the complementary
sequence of this single-stranded portion of the probe nucleic acid
to form a double-stranded section with one strand being target
nucleic acid and the other strand being probe nucleic acid. In
addition, the single-stranded portion of the probe nucleic acid
having the nucleotide sequence that is complementary to at least a
portion of the target nucleic acid to be detected can be designed
such that hybridization with the target nucleic acid creates a
recognition restriction endonuclease cut site. Thus, target nucleic
acid present within the sample can hybridize with the complementary
sequence of the single-stranded portion of the probe nucleic acid
to form a double-stranded section that creates a recognition
restriction endonuclease cut site for a recognition restriction
endonuclease. The probe nucleic acid also can be designed to
contain an amplifying restriction endonuclease. Since this method
includes the use of two or more different amplifying restriction
endonucleases, the amplifying restriction endonuclease that is a
component of the probe nucleic acid can be referred to as a first
or an initial amplifying restriction endonuclease, with additional
amplifying restriction endonucleases being referred to as second,
third, and so on or secondary, tertiary, and so on amplifying
restriction endonucleases. This initial amplifying restriction
endonuclease is typically a different restriction endonuclease than
the restriction endonuclease that is used as a recognition
restriction endonuclease. For example, when an EcoRI restriction
endonuclease is used as a recognition restriction endonuclease, a
restriction endonuclease other than an EcoRI restriction
endonuclease (e.g., a Hind III restriction endonuclease) is used as
an initial amplifying restriction endonuclease. Thus, in general,
probe nucleic acid is designed to contain an initial amplifying
restriction endonuclease and to have a nucleotide sequence such
that the target nucleic acid can hybridize to the probe nucleic
acid and create a recognition restriction endonuclease cut site for
a recognition restriction endonuclease. In some cases, the probe
nucleic acid can be attached to a solid support (e.g., a well of a
microtiter plate). For example, the probe nucleic acid can be
attached to a solid support such that cleavage at the recognition
restriction endonuclease cut site via the recognition restriction
endonuclease releases a portion of the probe nucleic acid that
contains the initial amplifying restriction endonuclease.
[0044] After contacting the sample that may or may not contain
target nucleic acid with the probe nucleic acid that is attached to
a solid support, the target nucleic acid, if present in the sample,
can hybridize to the probe nucleic acid and create the recognition
restriction endonuclease cut site. At this point, the recognition
restriction endonuclease, whether added to the reaction or already
present in the reaction, can cleave the probe nucleic acid at the
recognition restriction endonuclease cut sites that are formed by
the hybridization of target nucleic acid to the probe nucleic acid,
thereby releasing the portion of the probe nucleic acid that
contains the initial amplifying restriction endonuclease from the
solid support. The number of initial amplifying restriction
endonuclease-containing portions of the probe nucleic acid that are
released from the solid support can be in an essentially linear
relationship (e.g., essentially a one-for-one relationship) with
the number of target nucleic acid molecules that hybridize with the
probe nucleic acid to form the recognition restriction endonuclease
cut site.
[0045] The portions of the probe nucleic acid containing the
initial amplifying restriction endonuclease that were released from
the solid support can be collected and placed in contact with first
signal expansion nucleic acid and second signal expansion nucleic
acid. The first signal expansion nucleic acid can be designed to
have a double-stranded portion with a restriction endonuclease cut
site for the initial amplifying restriction endonuclease of the
probe nucleic acid. This restriction endonuclease cut site for the
initial amplifying restriction endonuclease can be referred to as
an initial amplifying restriction endonuclease cut site. The first
signal expansion nucleic acid also can be designed to contain a
secondary amplifying restriction endonuclease. The second signal
expansion nucleic acid can be designed to have a double-stranded
portion with a restriction endonuclease cut site for the secondary
amplifying restriction endonuclease of the first signal expansion
nucleic acid. This restriction endonuclease cut site for the
secondary amplifying restriction endonuclease can be referred to as
a secondary amplifying restriction endonuclease cut site. The
second signal expansion nucleic acid also can be designed to
contain an initial amplifying restriction endonuclease. For
example, when an EcoRI restriction endonuclease is used as a
recognition restriction endonuclease and a HindIII restriction
endonuclease is used as an initial amplifying restriction
endonuclease of the probe nucleic acid, a SmaI restriction
endonuclease can be used as a secondary amplifying restriction
endonuclease of the first signal expansion nucleic acid and a
HindIII restriction endonuclease can be used as the initial
amplifying restriction endonuclease of the second signal expansion
nucleic acid.
[0046] In some cases, the first signal expansion nucleic acid and
second signal expansion nucleic acid can be attached to a solid
support (e.g., a well of a microtiter plate). For example, the
first signal expansion nucleic acid can be attached to a solid
support such that cleavage at the initial amplifying restriction
endonuclease cut site via the initial amplifying restriction
endonuclease releases a portion of the first signal expansion
nucleic acid that contains the secondary amplifying restriction
endonuclease, and the second signal expansion nucleic acid can be
attached to a solid support such that cleavage at the secondary
amplifying restriction endonuclease cut site via the secondary
amplifying restriction endonuclease releases a portion of the
second signal expansion nucleic acid that contains the initial
amplifying restriction endonuclease. The first signal expansion
nucleic acid can be attached to the same solid support (e.g., two
different sub-compartments of a larger compartment) that contains
the second signal expansion nucleic acid provided that the
secondary amplifying restriction endonuclease of uncleaved first
signal expansion nucleic acid is unable to cleave the second signal
expansion nucleic acid and provided that the initial amplifying
restriction endonuclease of uncleaved second signal expansion
nucleic acid is unable to cleave the first signal expansion nucleic
acid. In some cases, the first signal expansion nucleic acid can be
attached to the same solid support within a joint compartment such
that the first signal expansion nucleic acid is within a first
compartment of the joint compartment and the second signal
expansion nucleic acid is within a second compartment of the joint
compartment. In such cases, the secondary amplifying restriction
endonuclease of uncleaved first signal expansion nucleic acid in
the first compartment is unable to cleave the second signal
expansion nucleic acid located in the second compartment, while the
secondary amplifying restriction endonuclease of cleaved first
signal expansion nucleic acid is capable of moving (e.g.,
diffusing) from the first compartment to the second compartment to
cleave the second signal expansion nucleic acid located in the
second compartment. In addition, the initial amplifying restriction
endonuclease of uncleaved second signal expansion nucleic acid in
the second compartment is unable to cleave the first signal
expansion nucleic acid located in the first compartment, while the
initial amplifying restriction endonuclease of cleaved second
signal expansion nucleic acid is capable of moving (e.g.,
diffusing) from the second compartment to the first compartment to
cleave the first signal expansion nucleic acid located in the first
compartment.
[0047] If portions of the probe nucleic acid containing the initial
amplifying restriction endonuclease are present and placed in
contact with the first signal expansion nucleic acid, then the
first signal expansion nucleic acid can be cleaved at the initial
amplifying restriction endonuclease cut site by the initial
amplifying restriction endonuclease, thereby releasing a portion of
the first signal expansion nucleic acid that contains the secondary
amplifying restriction endonuclease from the solid support. The
released portions of the first signal expansion nucleic acid
containing the secondary amplifying restriction endonuclease can be
free to cleave the second signal expansion nucleic acid at the
secondary amplifying restriction endonuclease cut site, thereby
releasing a portion of the second signal expansion nucleic acid
that contains the initial amplifying restriction endonuclease from
the solid support. Since the initial amplifying restriction
endonucleases of the released portions of the probe nucleic acid,
the initial amplifying restriction endonucleases of the released
portions of the second signal expansion nucleic acid, and the
secondary amplifying restriction endonucleases of the released
portions of the first signal expansion nucleic acid are free to
carry out repeated cleavage events, the number of released portions
containing the initial amplifying restriction endonucleases is
greatly increased from the number that were released by the
recognition restriction endonuclease. For example, the number of
cleaved first signal expansion nucleic acid molecules can greatly
exceed (e.g., exponentially exceed) the number of released portions
of the probe nucleic acid, and the number of cleaved second signal
expansion nucleic acid molecules can greatly exceed (e.g.,
exponentially exceed) the number of released portions of the probe
nucleic acid. Such a greatly expanded relationship (e.g., an
exponential relationship) can allow very small amounts of target
nucleic acid present in the sample to be readily detected.
[0048] In some cases, this method can be performed with the first
signal expansion nucleic acid being attached to a solid support
that is different from the solid support that contains the second
signal expansion nucleic acid. For example, the first signal
expansion nucleic acid can be attached to one well of a microtiter
plate, while the second signal expansion nucleic acid can be
attached to a different well of a microtiter plate. In this case,
the resulting reaction material from the well with the first signal
expansion nucleic acid can be collected and transferred to the well
containing the second signal expansion nucleic acid.
[0049] The portions of the second signal expansion nucleic acid
containing the initial amplifying restriction endonuclease that
were released from the solid support containing the second signal
expansion nucleic acid along with any other released portions in
this reaction (e.g., the released portions of the probe nucleic
acid containing the initial amplifying restriction endonuclease and
the released portions of the first signal expansion nucleic acid
containing the secondary amplifying restriction endonuclease) can
be collected and placed in contact with reporter nucleic acid. For
example, the released portions, if present, can be transferred from
one well of a microtiter plate (e.g., a 96-well plate) that
contained the second signal expansion nucleic acid to another well
of a microtiter plate that contains the reporter nucleic acid. The
reporter nucleic acid can be designed to have a double-stranded
portion with a restriction endonuclease cut site for the initial
amplifying restriction endonuclease. If released portions
containing the initial amplifying restriction endonuclease are
present and placed in contact with the reporter nucleic acid, then
the reporter nucleic acid can be cleaved at the initial amplifying
restriction endonuclease cut site by the initial amplifying
restriction endonuclease. Since the initial amplifying restriction
endonucleases of the released portions are free to carry out
repeated cleavage events, the number of reporter nucleic acid
molecules that are cleaved can greatly exceed the number of initial
amplifying restriction endonucleases present in the reaction. For
example, the number of cleaved reporter nucleic acid molecules can
greatly exceed (e.g., exponentially exceed) the number of initial
amplifying restriction endonucleases present in the reaction and
therefore can greatly exceed (e.g., exponentially exceed) the
number of target nucleic acid molecules that were present in the
sample contacted with the probe nucleic acid. Such a greatly
expanded relationship (e.g., an exponential relationship) can allow
very small amounts of target nucleic acid present in the sample to
be readily detected.
[0050] After the released portions containing the initial
amplifying restriction endonuclease, if present, are contacted with
the reporter nucleic acid, the presence or absence of cleaved
reporter nucleic acid can be determined. The presence of cleaved
reporter nucleic acid can indicate that the sample contained the
target nucleic acid, thereby indicating that the sample contained
the target genetic or epigenetic element for which the sample is
being tested, while the absence of cleaved reporter nucleic acid
can indicate that the sample lacked the target nucleic acid,
thereby indicating that the sample lacked the target genetic or
epigenetic element for which the sample is being tested.
[0051] In some cases, the amount of cleaved reporter nucleic acid
can be determined. In such cases, the amount of cleaved reporter
nucleic acid can indicate the amount of target nucleic acid present
in the sample, which can indicated the relative amount of the
genetic or epigenetic element present in the organism being tested.
A standard curve using known amounts of target nucleic acid can be
used to aid in the determination of the amount of target nucleic
acid present within a sample. For example, genomic DNA from known
heterozygous and/or homozygous (e.g., homozygous for a particular
genetic element being tested for) organisms can be included in an
assay to determine whether a genomic DNA sample from an organism
being tested contains zero, one, or two copies of a particular
target nucleic acid for which the organism is being tested based on
the amount of cleaved reporter nucleic acid.
[0052] In some cases, the reporter nucleic acid can contain a label
to aid in the detection of cleaved reporter nucleic acid. For
example, reporter nucleic acid can contain a fluorescent label and
a quencher such that cleaved reporter nucleic acid provides a
fluorescent signal and uncleaved reporter nucleic acid does not
provide a fluorescent signal. In some cases, the reporter nucleic
acid can contain a label (e.g., a colorimetric label, fluorescent
label or an enzyme such as horse radish peroxidase) and can be
attached to a solid support (e.g., a well of a microtiter plate).
For example, the reporter nucleic acid can be attached to a solid
support such that cleavage at the initial amplifying restriction
endonuclease cut site by the initial amplifying restriction
endonuclease releases a portion of the reporter nucleic acid that
contains the label. The resulting reaction mixture can be collected
and assessed for the presence, absence, or amount of released
portions of the reporter nucleic acid using the label. For example,
the released portions of the reporter nucleic acid, if present, can
be transferred from one well of a microtiter plate (e.g., a 96-well
plate) that contained the reporter nucleic acid to another well of
a microtiter plate, where the transferred material can be assessed
for a signal from the label.
[0053] In some cases, the presence or absence of cleaved first
signal expansion nucleic acid, cleaved second signal expansion
nucleic acid, or both can be determined. The presence of such
cleaved nucleic acid can indicate that the sample contained the
target nucleic acid, thereby indicating that the sample contained
the target genetic or epigenetic element for which the sample is
being tested, while the absence of such cleaved nucleic acid can
indicate that the sample lacked the target nucleic acid, thereby
indicating that the sample lacked the target genetic or epigenetic
element for which the sample is being tested. In some cases, the
amount of cleaved first signal expansion nucleic acid, cleaved
second signal expansion nucleic acid, or both can be determined. In
such cases, the amount of cleaved nucleic acid can indicate the
amount of target nucleic acid present in the sample, which can
indicated the relative amount of the genetic or epigenetic element
present in the organism being tested. In these cases, the use of
cleaved first signal expansion nucleic acid, cleaved second signal
expansion nucleic acid, or both to assess the sample for target
nucleic acid can be in addition to the use of a separate reporter
nucleic acid step or can replace the use of a separate reporter
nucleic acid step. In some cases, the first signal expansion
nucleic acid, the second signal expansion nucleic acid, or both can
be labeled in a manner similar to that described herein for the
reporter nucleic acid to aid in detection. When the presence,
absence, or amount of cleaved first signal expansion nucleic acid,
cleaved second signal expansion nucleic acid, or both are
determined to assess the sample for target nucleic acid, the first
signal expansion nucleic acid can be referred to as a first
reporter nucleic acid and the second signal expansion nucleic acid
can be referred to as a second reporter nucleic acid even though
they include amplifying restriction endonucleases. A standard curve
using known amounts of target nucleic acid can be used to aid in
the determination of the amount of target nucleic acid present
within a sample. For example, genomic DNA from known heterozygous
and/or homozygous (e.g., homozygous for a particular genetic
element being tested for) organisms can be included in an assay to
determine whether a genomic DNA sample from an organism being
tested contains zero, one, or two copies of a particular target
nucleic acid for which the organism is being tested based on the
amount of cleaved first signal expansion nucleic acid, cleaved
second signal expansion nucleic acid, or both.
[0054] Examples of a method of detecting target nucleic acid that
includes using probe nucleic acid, first signal expansion nucleic
acid, second signal expansion nucleic acid, and reporter nucleic
acid are set forth in FIGS. 3-5. With reference to FIG. 3, first
reaction chamber 200 (e.g., a microtiter plate well) can contain
probe nucleic acid 201. Probe nucleic acid 201 can be attached
(e.g., immobilized) to solid support 202 and can include initial
amplifying restriction endonuclease 203 (Ra). Probe nucleic acid
201 can be attached to solid support 202 such that initial
amplifying restriction endonuclease 203 is released from solid
support 202 upon cleavage of a nucleic acid component of probe
nucleic acid 201. Probe nucleic acid 201 can have a single-stranded
section having a nucleotide sequence that is complementary to at
least a portion of target nucleic acid 204. Probe nucleic acid 201
can be contacted with a sample that may or may not contain target
nucleic acid 204. If target nucleic acid 204 is present, at least a
portion of target nucleic acid 204 and probe nucleic acid 201 can
hybridize to form a double-stranded section of nucleic acid. Such a
double-stranded section can contain at least one recognition
restriction endonuclease cut site 205. Addition of recognition
restriction endonuclease 206 (Rr) to first reaction chamber 200 can
result in the cleavage of probe nucleic acid 201 at recognition
restriction endonuclease cut site 205 formed by one strand of probe
nucleic acid and one strand of target nucleic acid, thereby
releasing portion 207 of probe nucleic acid 201 from solid support
202. Portion 207 can include initial amplifying restriction
endonuclease 203.
[0055] The reaction product from first reaction chamber 200
containing released portion 207, if target nucleic acid 204 was
present, can be transferred (e.g., manually or automatically) to
second reaction chamber 220. Second reaction chamber 220 can
contain first signal expansion nucleic acid 226 and second signal
expansion nucleic acid 225. First signal expansion nucleic acid 226
can have at least one double-stranded portion that contains at
least one initial amplifying restriction endonuclease cut site 230.
First signal expansion nucleic acid 226 can be attached (e.g.,
immobilized) to solid support 222 and can include secondary
amplifying restriction endonuclease 223 (Rb). First signal
expansion nucleic acid 226 can be attached to solid support 222
such that portion 234 containing secondary amplifying restriction
endonuclease 223 is released from solid support 222 upon cleavage
of first signal expansion nucleic acid 226 at initial amplifying
restriction endonuclease cut site 230. For clarity, frame E of FIG.
3 omits depicting one strand from the cleaved versions of first
signal expansion nucleic acid 226 and second signal expansion
nucleic acid 225.
[0056] Second signal expansion nucleic acid 225 can have at least
one double-stranded portion that contains at least one secondary
amplifying restriction endonuclease cut site 232. Second signal
expansion nucleic acid 225 can be attached (e.g., immobilized) to
solid support 222 and can include initial amplifying restriction
endonuclease 224. Second signal expansion nucleic acid 225 can be
attached to solid support 222 such that portion 236 containing
initial amplifying restriction endonuclease 224 is released from
solid support 222 upon cleavage of second signal expansion nucleic
acid 225 at secondary amplifying restriction endonuclease cut site
232. Initial amplifying restriction endonuclease 203 of probe
nucleic acid 201 and initial amplifying restriction endonuclease
224 of second signal expansion nucleic acid 225 can be the same
restriction endonuclease. For example, both can be an EcoRI
restriction endonuclease.
[0057] Addition of the reaction product from first reaction chamber
200 to second reaction chamber 220 can result in the cleavage of
first signal expansion nucleic acid 226 at initial amplifying
restriction endonuclease cut site 230 if the reaction product
contains portion 207. Such cleavage of first signal expansion
nucleic acid 226 can result in the release of portion 234 from
solid support 222. Portion 234, which can include secondary
amplifying restriction endonuclease 223, can result in the cleavage
of second signal expansion nucleic acid 225 at secondary amplifying
restriction endonuclease cut site 232. Such cleavage of second
signal expansion nucleic acid 225 can result in the release of
portion 236 from solid support 222. Thus, this reaction can result
in the accumulation of released portions 234 and 236.
[0058] The reaction product from second reaction chamber 220
containing released portion 207, released portion 234, and released
portion 236, if target nucleic acid 204 was present, can be
transferred (e.g., manually or automatically) to third reaction
chamber 240. Third reaction chamber 240 can contain reporter
nucleic acid 241. Reporter nucleic acid 241 can be attached (e.g.,
immobilized) to solid support 242 and can include marker (e.g., a
label) 243 (M). Reporter nucleic acid 241 can be attached to solid
support 242 such that marker 243 is released from solid support 242
upon cleavage of a nucleic acid component of reporter nucleic acid
241. Reporter nucleic acid 241 can have at least one
double-stranded portion that contains at least one initial
amplifying restriction endonuclease cut site 246. Addition of the
reaction product from second reaction chamber 220 to third reaction
chamber 240 can result in the cleavage of reporter nucleic acid 241
at initial amplifying restriction endonuclease cut site 246 if the
reaction product contains portion 207 and portion 236. In some
cases, reporter nucleic acid 241 can include at least one
double-stranded portion that contains at least one cut site for
secondary amplifying restriction endonuclease 223. In such cases,
addition of the reaction product from second reaction chamber 220
to third reaction chamber 240 can result in the cleavage of
reporter nucleic acid 241 at the cut site for secondary amplifying
restriction endonuclease 223 if the reaction product contains
portion 234. Cleavage of reporter nucleic acid 241 can result in
the release of portion 247 from solid support 242. Portion 247 can
include marker 243.
[0059] The reaction product from third reaction chamber 240 can be
assessed to determine the presence, absence, or amount of portion
247. The presence of portion 247 can indicate that the sample
contained target nucleic acid 204, while the absence of portion 247
can indicate that the sample lacked target nucleic acid 204. In
some cases, the amount of portion 247 can be determined. In such
cases, the amount of portion 247 can indicate the amount of target
nucleic acid 204 present in the sample. The presence, absence, or
amount of portion 247 can be determined using marker 243, and
portion 247 having marker 243 can be distinguished from uncleaved
reporter nucleic acid 241 having marker 243 since, in this example,
portion 247 is released from solid support 242, while uncleaved
reporter nucleic acid 241 remains attached to solid support 242.
For example, in some cases, the reaction product from third
reaction chamber 24 can be transferred to fourth reaction chamber
where the presence or absence of portion 247 via marker 243 is
assessed. If portion 347 is present, the amount of portion 247
present can be quantified.
[0060] In some cases and with reference to FIGS. 4 and 5, first
signal expansion nucleic acid 226 can include marker (e.g., a
label) 243 (M) and second signal expansion nucleic acid 225 can
include marker (e.g., a label) 243 (M). In such cases, cleavage of
first signal expansion nucleic acid 226 and cleavage of second
signal expansion nucleic acid 225 can be assessed using marker 243
to determine the presence, absence, or amount of target nucleic
acid within a sample. For example, detector 250 can be used to
detect marker 243 released from solid support 222.
[0061] Probe nucleic acid 201, first signal expansion nucleic acid
226, second signal expansion nucleic acid 225, and reporter nucleic
acid 241 can have various configurations. For example, with
reference to FIG. 3, probe nucleic acid 201 can be designed to have
a single nucleic acid strand such that the entire nucleic acid
component of probe nucleic acid 201 is single-stranded prior to
contact with target nucleic acid 204. In another example, probe
nucleic acid 201 can be designed in a manner like probe nucleic
acid 101 to have two or more strands. See, e.g., FIG. 2. For
example, probe nucleic acid 201 can have a first strand and a
second strand. The first strand can be attached to a solid support
and can be designed to have a single-stranded section having a
nucleotide sequence that is complementary to at least a portion of
target nucleic acid. The second strand can include an initial
amplifying restriction endonuclease and can have a single-stranded
section having a nucleotide sequence that can hybridize to the
first strand. In some cases, the first strand and second strand can
be synthesized or obtained separately and then mixed together to
form probe nucleic acid 201. For example, the first strand can be
synthesized, biotinylated, and attached to a streptavidin-coated
solid support. After synthesizing the nucleic acid component of the
second strand and attaching an initial amplifying restriction
endonuclease to the synthesized nucleic acid component, the second
strand can be incubated with the first strand to form nucleic acid
probe 201. In some cases, probe nucleic acid 201 can contain more
than two strands. For example, probe nucleic acid can include a
first strand, a second strand, and a third strand. In this case,
the first strand can be attached to a solid support, the second
strand can be hybridized to the first strand and can include a
single-stranded section having a nucleotide sequence that is
complementary to at least a portion of target nucleic acid, and the
third strand can be hybridized to the second strand and can be
attached to an initial amplifying restriction endonuclease. Similar
one, two, three, or more strand configurations can be used to make
first signal expansion nucleic acid, second signal expansion
nucleic acid, or reporter nucleic acid. For example, first signal
expansion nucleic acid and second signal expansion nucleic acid can
be designed to have a configuration as shown in FIG. 4 or 5.
[0062] Probe nucleic acid described herein typically includes at
least one single-stranded DNA section that is designed to hybridize
with a desired target nucleic acid and thereby create a recognition
restriction endonuclease cut site. The other portions of the probe
nucleic acid can include DNA, RNA, or other molecules. For example,
probe nucleic acid can include biotin such that the probe nucleic
acid can be attached to a streptavidin-coated solid support. In
some cases, the single-stranded section of the probe nucleic acid
that is designed to hybridize with a desired target nucleic acid
and create a recognition restriction endonuclease cut site can be
RNA or a nucleic acid analog (e.g., a peptide nucleic acid (PNA))
provided that such a single-stranded section can (i) hybridize with
the desired target nucleic acid and (ii) create a recognition
restriction endonuclease cut site with the complementary target
nucleic acid sequence that is capable of being cleaved by the
recognition restriction endonuclease. Examples of restriction
endonucleases that can be used as recognition restriction
endonucleases to cleave a recognition restriction endonuclease cut
site that is created between an RNA section of the probe nucleic
acid and a DNA section of the target nucleic acid include, without
limitation, HhaI, AluI, TaqI, HaeIII, EcoRI, HindII, SalI, and MspI
restriction endonucleases.
[0063] Probe nucleic acid described herein can be any length
provided that the single-stranded section of the probe nucleic acid
that is designed to hybridize with a desired target nucleic acid is
capable of hybridizing to the target nucleic acid and provided that
the amplifying restriction endonuclease of the probe nucleic acid
is capable of cleaving its amplifying restriction endonuclease cut
site after the probe nucleic acid is cleaved by a recognition
restriction endonuclease. In general, the single-stranded section
of the probe nucleic acid that is designed to hybridize with a
desired target nucleic acid can be between about 10 and about 500
or more nucleotides (e.g., between about 10 and about 400
nucleotides, between about 10 and about 300 nucleotides, between
about 10 and about 200 nucleotides, between about 10 and about 100
nucleotides, between about 10 and about 50 nucleotides, between
about 10 and about 25 nucleotides, between about 20 and about 500
nucleotides, between about 30 and about 500 nucleotides, between
about 40 and about 500 nucleotides, between about 50 and about 500
nucleotides, between about 15 and about 50 nucleotides, between
about 15 and about 25 nucleotides, between about 20 and about 50
nucleotides, between about 18 and about 25 nucleotides, between
about 20 and about 60 nucleotides, between about 25 and about 55
nucleotides, between about 30 and about 50 nucleotides, between
about 35 and about 45 nucleotides, or between about 38 and about 42
nucleotides) in length. The recognition restriction endonuclease
cut site that will be created by the hybridization of target
nucleic acid to this single-stranded section of the probe nucleic
acid can be located at any position alone the single-stranded
section. For example, the recognition restriction endonuclease cut
site to be created can be towards the 5' end, towards the '3 end,
or near the center of the single-stranded section of the probe
nucleic acid. In general, the overall length of the probe nucleic
acid described herein can be between about 10 and about 2500 or
more nucleotides (e.g., between about 10 and about 2000
nucleotides, between about 10 and about 1000 nucleotides, between
about 10 and about 500 nucleotides, between about 10 and about 400
nucleotides, between about 10 and about 300 nucleotides, between
about 10 and about 200 nucleotides, between about 10 and about 100
nucleotides, between about 10 and about 50 nucleotides, between
about 10 and about 25 nucleotides, between about 20 and about 500
nucleotides, between about 30 and about 500 nucleotides, between
about 40 and about 500 nucleotides, between about 50 and about 500
nucleotides, between about 75 and about 500 nucleotides, between
about 100 and about 500 nucleotides, between about 150 and about
500 nucleotides, between about 15 and about 50 nucleotides, between
about 15 and about 25 nucleotides, between about 20 and about 50
nucleotides, between about 18 and about 25 nucleotides, between
about 20 and about 60 nucleotides, between about 25 and about 55
nucleotides, between about 30 and about 50 nucleotides, between
about 35 and about 45 nucleotides, or between about 38 and about 42
nucleotides) in length.
[0064] The recognition restriction endonuclease cut site to be
created by hybridization of target nucleic acid to the probe
nucleic acid can be a cut site of any type of restriction
endonuclease. In addition, any type of restriction endonuclease can
be used as a recognition restriction endonuclease to cleave probe
nucleic acid upon target nucleic acid hybridization. Examples of
restriction endonucleases that can be used as recognition
restriction endonucleases include, without limitation, EcoRI,
EcoRII, BamHI, HindIII, TaqI, NotI, HinfI, Sau3A, PovII, SmaI,
HaeIII, HgaI, AluI, EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI,
SphI, StuI, XbaI, AarI, BanII, BseGI, BspPI, CfrI, EcoNI, Hsp92II,
NlaIV, RsaI, TaiI, AasI, BbsI, BseJI, BspTI, ClaI, EcoO109I,
I-PpoI, NmuCI, RsrII, TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI, KasI,
Acc65I, BbvCI, BseMI, BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI,
AccB7I, BbvI, BseMII, BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI,
AccI, BceAI, BseNI, BsrGI, CspI, Esp3I, KspAI, NsiI, SapI, and TauI
restriction endonucleases. In some cases, nucleic acid encoding a
naturally-occurring restriction endonuclease can be genetically
engineered to create a modified restriction endonuclease that has
the ability to recognize a particular cut site. Common computer
algorithms can be used to locate restriction endonuclease cut sites
along the nucleotide sequence of any desired target nucleic acid.
Once located, the sequence of the restriction endonuclease cut site
along with additional flanking sequence (e.g., 5' flanking
sequence, 3' flanking sequence, or both 5' and 3' flanking
sequence) can be used to design the complementary sequence of the
probe nucleic acid that is used to hybridize to the target nucleic
acid and create the recognition restriction endonuclease cut site
upon target nucleic acid hybridization. In some cases, a probe
nucleic acid can be designed to have the restriction endonuclease
cut site located in the middle or near the middle such that the
restriction endonuclease cut site has both 5' and 3' flanking
sequences that are complementary to the target nucleic acid.
[0065] In some cases, the probe nucleic acid is designed to have a
single-stranded section that is designed to hybridize with desired
target nucleic acid and to form a recognition restriction
endonuclease cut site upon target nucleic acid hybridization such
that target nucleic acid containing the particular genetic or
epigenetic element being tested for hybridizes to the probe nucleic
acid and, together with the probe nucleic acid, is cleaved by the
recognition restriction endonuclease, thereby releasing a portion
of the probe nucleic acid, while nucleic acid lacking the
particular genetic or epigenetic element being tested for does not
result in the formation of cleaved probe nucleic acid even though
such nucleic acid lacking the genetic or epigenetic element may
hybridize with the probe nucleic acid. For example, a probe nucleic
acid can be designed to contain a single-stranded portion that is
designed to hybridize with a target nucleic acid containing a
particular SNP sequence and to form a recognition restriction
endonuclease cut site at the location of the particular SNP
sequence upon target nucleic acid hybridization. In such cases, the
target nucleic acid containing the particular SNP sequence being
tested for can hybridize to the probe nucleic acid and, together
with the probe nucleic acid, can be cleaved by the recognition
restriction endonuclease, thereby releasing a portion of the probe
nucleic acid, while nucleic acid lacking the particular SNP
sequence being tested for can fail to result in the formation of
cleaved probe nucleic acid even though such nucleic acid lacking
the particular SNP sequence may hybridize with the probe nucleic
acid (see, e.g., FIG. 10). In these cases, the recognition
restriction endonuclease can be used to differentiate between
target nucleic acid containing the particular genetic or epigenetic
element being tested for and other nucleic acids that lack the
particular genetic or epigenetic element being tested for even
though such other nucleic acids may hybridize with the probe
nucleic acid. In some cases, the difference between these other
nucleic acids and the target nucleic acid being tested for can be a
single nucleotide. For example, ApoI can be used as a recognition
restriction endonuclease to differentiate between a target nucleic
acid containing a 5'-AAATTC-3' sequence and other nucleic acids
that simply have a 5'-AAATTA-3' sequence in place of the
5'-AAATTC-3' sequence.
[0066] In some cases, probe nucleic acid can be designed for use
with a recognition restriction endonuclease that has separate
recognition and cleavage sites such as an FokI restriction
endonuclease (FIG. 11). FokI recognizes a specific 5-base site
(5'-GGATG-3'), but it cleaves the double stranded nucleic acid at a
position nine bases downstream of the recognition site provided
that these nine bases form perfectly matched double-stranded
sequence (FIG. 11). Other examples of such restriction
endonucleases include, without limitation, AlwI, MnlI, CspCI, AjuI,
AloI, PpiI, PsrI, and AarI. Probe nucleic acid designed for use
with a recognition restriction endonuclease that has separate
recognition and cleavage sites can be used to detect any SNP of
interest, including those that do not change a known restriction
site with respect to, for example, corresponding wild-type
sequences. In such cases, probe nucleic acid can be designed to
contain a double stranded portion between 10 and 100 bp in length
(e.g., 10 and 75, 10 and 50, 10 and 40, 10 and 30, 20 and 100, 30
and 100, 15 and 75, 15 and 50, 15 and 40, 20 and 50, or 20 and 40
bp in length) that has the recognition site of a recognition
restriction endonuclease that has separate recognition and cleavage
sites (e.g., FokI) adjacent to a single stranded portion 10 and 100
nucleotides in length (e.g., 10 and 75, 10 and 50, 10 and 40, 10
and 30, 20 and 100, 30 and 100, 15 and 75, 15 and 50, 15 and 40, 20
and 50, or 20 and 40 nucleotides in length) that is designed to
have a sequence complementary to a desired target nucleic acid
(e.g., a wild-type target nucleic acid or a target nucleic acid
containing a SNP) such that hybridization of the desired target
nucleic acid creates the cleavage site of the recognition
restriction endonuclease. Probe nucleic acid containing any
hybridized nucleic acid can be subjected to blunting and ligation
reactions. For example, T4 DNA polymerase (or a blunting kit
containing this enzyme) can be used to remove free single-stranded
ends of nucleic acid hybridized to probe nucleic acid. T4 DNA
polymerase can convert DNA with single-stranded 5' or 3' overhangs
to 5' phosphorylated, blunt-ended DNA for efficient blunt-end
ligation. A DNA ligase (e.g., E. coli DNA ligase) can be used
subsequently (or simultaneously with T4 DNA polymerase) to ligate
the blunted hybridized nucleic acid to the adjacent strand of probe
nucleic acid (see, e.g., FIG. 12).
[0067] If the desired target nucleic acid is present in a sample
being tested, hybridizes to the single stranded portion of probe
nucleic acid to form the cleavage site of the recognition
restriction endonuclease that has separate recognition and cleavage
sites, and is ligated to the adjacent strand of probe nucleic acid,
then the recognition restriction endonuclease can cleave the probe
nucleic acid:target nucleic acid hybrid (see, e.g., FIG. 12). Such
cleavage can be detected using the methods and materials provided
herein. For example, the portion of cleaved probe nucleic acid
containing the amplifying restriction endonuclease can be allowed
to cleave reporter nucleic acid as described herein.
[0068] With reference to FIG. 12, probe nucleic acid can be
designed to have a double-stranded DNA section between 10 and 100
bp in length (e.g., 10 and 75, 10 and 50, 10 and 40, 10 and 30, 20
and 100, 30 and 100, 15 and 75, 15 and 50, 15 and 40, 20 and 50, or
20 and 40 bp in length) carrying the FokI recognition site (GGATG)
at the free end and a single-stranded DNA section 10 and 100
nucleotides in length (e.g., 10 and 75, 10 and 50, 10 and 40, 10
and 30, 20 and 100, 30 and 100, 15 and 75, 15 and 50, 15 and 40, 20
and 50, or 20 and 40 nucleotides in length) that is complementary
to a target nucleic acid DNA fragment (in this example, a wild-type
nucleic acid sequence of interest). The single-stranded sequence of
the probe nucleic acid is designed such that a potential SNP
location is positioned at the potential FokI cleavage site, which
is exactly nine nucleotides away from the FokI recognition site.
The probe nucleic acid is allowed to hybridize to nucleic acid
present in the sample being tested, and any free single-stranded
ends of hybridized nucleic acid from the sample being tested are
removed using T4 DNA polymerase (or a blunting kit containing this
enzyme). A DNA ligase (e.g., E. coli DNA ligase) is used to ligate
any blunted hybridized nucleic acid from the sample being tested to
the adjacent available end of a strand of the probe nucleic acid.
If the desired target nucleic acid is present in the sample, a
target nucleic acid:probe nucleic acid hybrid is formed such that
the hybrid contains both double-stranded FokI recognition and
cleavage sites. At this point, FokI can cleave the target nucleic
acid:probe nucleic acid hybrid, which can be detected as described
herein. If any blunted hybridized nucleic acid from the sample
being tested contains a SNP such that a mismatch exists with the
probe nucleic acid at the cleavage site, then such probe nucleic
acid are not cleaved.
[0069] In some cases, an assay or kit provided herein can have one
probe nucleic acid designed to detect target nucleic acid having an
un-mutated sequence (e.g., a wild-type sequence) and another probe
nucleic acid designed to detect target nucleic acid having a
mutated version of the sequence (e.g., a sequence containing a
SNP). Comparison of signals for mutated versus un-mutated target
nucleic acids can provide information about the homozygosity and
heterozygosity of the corresponding genotype in terms of the allele
of interest.
[0070] In general, probe nucleic acid can be designed to have a
single-stranded section that is designed to hybridize with desired
target nucleic acid and to form a single recognition restriction
endonuclease cut site upon target nucleic acid hybridization. In
some cases, probe nucleic acid can be designed to have a
single-stranded section that is designed to hybridize with desired
target nucleic acid and to form more than one (e.g., two, three,
four, five, six, seven, eight, nine, ten, or more) recognition
restriction endonuclease cut site upon target nucleic acid
hybridization. When more than one recognition restriction
endonuclease cut site is used, the multiple recognition restriction
endonuclease cut sites can be cut sites for the same restriction
endonuclease or cut sites for different restriction endonucleases.
For example, probe nucleic acid can be designed to have a
single-stranded section that is designed to hybridize with desired
target nucleic acid and to form one recognition restriction
endonuclease cut site for an EcoRI recognition restriction
endonuclease and one recognition restriction endonuclease cut site
for an XbaI recognition restriction endonuclease upon target
nucleic acid hybridization. In such cases, each recognition
restriction endonuclease can be used individually or in combination
(e.g., as a mixture) to cleave probe nucleic acid that hybridized
to target nucleic acid and formed the corresponding recognition
restriction endonuclease cut site via such hybridization.
[0071] Probe nucleic acid can be designed such that any target
nucleic acid containing a genetic or epigenetic element can be
detected. Examples of target nucleic acid that can be detected
using the methods and materials provided herein include, without
limitation, genomic DNA, RNA, cDNA, methylated DNA, and
combinations thereof. In some cases such as those involving
assessing a biological sample for a genetic or epigenetic element
in RNA, the target nucleic acid can be an RNA or a cDNA generated
from an RNA. When detecting an RNA target nucleic acid, restriction
endonucleases having the ability to cleave a recognition
restriction endonuclease cut site that is created between a DNA
section of the probe nucleic acid and the RNA target nucleic acid
can be used as recognition restriction endonucleases. Examples of
such restriction endonucleases include, without limitation, HhaI,
AluI, TaqI, HaeIII, EcoRI, HindII, SalI, and MspI restriction
endonucleases. When detecting methylated target nucleic acid (e.g.,
a methylated DNA), restriction endonucleases having the ability to
cleave a recognition restriction endonuclease cut site that
includes a methylated nucleotide to be assessed can be used as
recognition restriction endonucleases. Examples of restriction
endonucleases having the ability to recognize methylated
nucleotides include, without limitation, DpnI, GlaI, HpaII, MspI,
AciI, HhaI, and SssI restriction endonucleases. In such cases, a
control can include detecting the same target nucleic acid without
the methylated nucleotide. In some cases, a combination of
methylation insensitive and methylation sensitive restriction
endonucleases can be used to assess a sample for methylated target
nucleic acid. For example, similar generation of cleavage products
using both methylation insensitive and methylation sensitive
restriction endonucleases designed for the same site can indicate
that the target nucleic acid lacks methylation at that site, while
an increased level of cleavage products using a methylation
insensitive restriction endonuclease as compared to the level
generated using a methylation sensitive restriction endonuclease
designed for the same site can indicate that the target nucleic
acid is methylated at that site (see, e.g., FIG. 8).
[0072] Any appropriate pair of methylation sensitive and
methylation insensitive isoschizomers can be used as described
herein. Examples of such recognition restriction endonuclease pairs
include, without limitation, the MspI/HpaII pair that cut CCGG
sites, with HpaII being sensitive to methylation of the second C
(blocked by CCmGG) and the EcoRII/BstN1 pair that cut CCNGG sites,
with BstN1 being methylation-sensitive. There are more than 300
methylation sensitive restriction endonucleases known that can be
used as described herein, and about 30 of them have methylation
insensitive isoschizomers (McClelland et al., Nucleic Acids Res.,
22:3640-3659 (1994)).
[0073] In some cases, the presence, absence, or amount of target
methylated DNA can be determined using methylation sensitive
recognition restriction endonucleases as opposed to a pair of
methylation sensitive and methylation insensitive isoschizomers
(see, e.g., FIG. 9). For example, target DNA can be contacted with
the same probe nucleic acid in two different compartments or wells
(e.g., compartment 1 and compartment 2). A first recognition
restriction endonuclease (e.g., Rr1), which cleaves a cut site
(e.g., cut site A) that does not contain any C nucleotides and thus
can not be methylated, can be added to compartment 1. Upon
hybridization with the target DNA, all probe-target hybrids are
cleaved by Rr1, and the signal from compartment 1 corresponds to
the total amount of the target DNA in the sample. A second
recognition restriction endonuclease (e.g., Rr2), which is
methylation-sensitive and can only cleave a different site (e.g.,
site B) if it is unmethylated, can be added to compartment 2. Upon
hybridization with the target DNA, probe-target hybrids are cleaved
by Rr2 only if site B is unmethylated, and the signal from
compartment 2 corresponds only to the unmethylated target DNA
within the sample. Signal detection for both compartments can be
carried out as described herein. The resultant signal from
compartment 1 can correspond to the total amount of target DNA,
while the compartment 2 signal can correspond only to the amount of
unmethylated target DNA. Thus, the amount of methylated target can
be calculated by subtracting the compartment 2 signal from the
compartment 1 signal.
[0074] The nucleotide sequence of target nucleic acid to be
detected can be obtained from, for example, common nucleic acid
sequence databases such as GenBank.RTM. (e.g., the SNP database of
GenBank.RTM.). A portion of target nucleic acid sequence can be
selected using a computer-based program. For example, a
computer-based program can be used to detect restriction
endonuclease cut sites within a portion of target nucleic acid
(e.g., at the location of a genetic or epigenetic element whether
the genetic or epigenetic element is a single nucleotide element or
a larger nucleotide sequence element such as a 5, 10, 15, 20, 50,
100, or more nucleotide insertion). Such information can be used to
design probe nucleic acid such that the single-stranded section
creates at least one recognition restriction endonuclease cut site
upon hybridization of the target nucleic acid. In some cases,
bioinformatics computer-based programs and tools can be used to
assist in the design of probe nucleic acid. For example, computer
programs (e.g., BLAST.RTM. and alignment programs) and computer
databases (e.g., GenBank.RTM.) can be used to indentify nucleic
acid sequences of a particular organism's genome (e.g., sequences
from particular genes, coding sequences, promotors, enhancers, or
untranslated regions). In addition, computer programs such as CLC
Workbench or Vector NTI (Invitrogen) can be used to identify the
location of restriction endonuclease cut sites within a particular
nucleic acid sequence. In some cases, sequence analysis computer
programs can be used to identify sequences with limited or an
absence of repeats, a presence of high sequence complexity of a
potential recognition restriction endonuclease cut site, and/or
limited or an absence of hairpin structures. Identification of such
sequences can help reduce the risk of probe self-hybridization and
potentially unintended cutting by a recognition endonuclease.
[0075] Any appropriate method can be used to obtain the nucleic
acid component of the probe nucleic acid. For example, common
molecular cloning and chemical nucleic acid synthesis techniques
can be used to obtain the nucleic acid component of the probe
nucleic acid. In some cases, the nucleic acid component of the
probe nucleic acid can be synthesized using commercially available
automated oligonucleotide synthesizers such as those available from
Applied Biosystems (Foster City, Calif.). In some cases, probe
nucleic acids can be synthesized de novo using any of a number of
procedures widely available in the art. Examples of such methods of
synthesis include, without limitation, the P-cyanoethyl
phosphoramidite method (Beaucage et al., Tet. Let., 22:1859-1862
(1981)) and the nucleoside H-phosphonate method (Garegg et al.,
Tet. Let., 27:4051-4054 (1986); Froehler et al., Nucl. Acid Res.,
14:5399-5407 (1986); Garegg et al., Tet. Let., 27:4055-4058 (1986);
and Gaffney et al., Tet. Let., 29:2619-2622 (1988)). These methods
can be performed by a variety of commercially-available automated
oligonucleotide synthesizers. In some cases, recombinant nucleic
acid techniques such as PCR and those that include using
restriction enzyme digestion and ligation of existing nucleic acid
sequences (e.g., genomic DNA or cDNA) can be used to obtain the
nucleic acid component of the probe nucleic acid.
[0076] Probe nucleic acid described herein can be attached to a
solid support. Examples of solid supports include, without
limitation, a well of a microtiter plate (e.g., a 96-well
microtiter plate or ELISA plate), beads (e.g., magnetic, glass,
plastic, or gold-coated beads), slides (e.g., glass or gold-coated
slides), micro- or nano-particles (e.g., carbon nanotubes),
platinum solid supports, palladium solid supports, and a surface of
a chamber or channel within a microfluidic device. In some cases, a
solid support can be a silicon oxide-based solid support, a plastic
polymer-based solid support (e.g., a nylon, nitrocellulose, or
polyvinylidene fluoride-based solid support), or a biopolymer-based
(e.g., a cross-linked dextran or cellulose-based solid support)
solid support. Probe nucleic acid can be directly or indirectly
attached to a solid support. For example, biotin can be a component
of the probe nucleic acid, and the probe nucleic acid containing
biotin can be indirectly attached to a solid support that is coated
with streptavidin via a biotin-streptavidin interaction. In some
cases, probe nucleic acid can be attached to a solid support via a
covalent or non-covalent interaction. For example, probe nucleic
acid can be covalently attached to magnetic beads as described
elsewhere (Albretsen et al., Anal. Biochem., 189(1):40-50
(1990)).
[0077] Probe nucleic acid can be designed to contain any type of
restriction endonuclease as an amplifying restriction endonuclease.
In general, an amplifying restriction endonuclease of the probe
nucleic acid is typically a different restriction endonuclease than
the restriction endonuclease that is used as a recognition
restriction endonuclease. For example, when an EcoRI restriction
endonuclease is used as a recognition restriction endonuclease, a
restriction endonuclease other than an EcoRI restriction
endonuclease (e.g., a HindIII restriction endonuclease) is used as
an amplifying restriction endonuclease. Examples of restriction
endonucleases that can be used as amplifying restriction
endonucleases include, without limitation, EcoRI, EcoRII, BamHI,
HindIII, TaqI, NotI, HinfI, Sau3A, PovII, SmaI, HaeIII, HgaI, AluI,
EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI, SphI, StuI, XbaI,
AarI, BanII, BseGI, BspPI, CfrI, EcoNI, Hsp92II, NlaIV, RsaI, TaiI,
AasI, BbsI, BseLI, BspTI, ClaI, EcoO109I, I-PpoI, NmuCI, RsrII,
TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI, KasI, Acc65I, BbvCI, BseMI,
BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI, AccB7I, BbvI, BseMII,
BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI, BceAI, BseNI,
BsrGI, CspI, Esp3I, KspAI, NsiI, SapI, and TauI restriction
endonucleases. Any number of molecules of the same amplifying
restriction endonuclease can be attached to one probe nucleic acid
molecule. For example, a single probe nucleic acid molecule can
contain one, two, three, four, five, or more EcoRI amplifying
restriction endonuclease molecules. In some cases, a single probe
nucleic acid molecule can contain two or more (e.g., two, three,
four, five, or more) different types of amplifying restriction
endonucleases. For example, a single probe nucleic acid molecule
can contain three EcoRI amplifying restriction endonuclease
molecules and two BanII amplifying restriction endonuclease
molecules.
[0078] Any appropriate method can be used to attach an amplifying
restriction endonuclease to a nucleic acid component of the probe
nucleic acid. In some cases, an amplifying restriction endonuclease
can be attached by an ionic or covalent attachment. For example,
covalent bonds such as amide bonds, disulfide bonds, and thioether
bonds, or bonds formed by crosslinking agents can be used. In some
cases, a non-covalent linkage can be used. The attachment can be a
direct attachment or an indirect attachment. For example, a linker
can be used to attach an amplifying restriction endonuclease to a
nucleic acid component of the probe nucleic acid. In some cases,
nucleic acid can include a thiol modification, and a restriction
endonuclease can be conjugated to the thiol-containing nucleic acid
based on succinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) using
techniques similar to those described elsewhere (Dill et al.,
Biosensors and Bioelectronics, 20:736-742 (2004)). In some cases, a
biotinylated nucleic acid and a streptavidin-containing restriction
endonuclease can be attached to one another via a
biotin-streptavidin interaction. A restriction endonuclease can be
conjugated with streptavidin using, for example, sulfosuccinimidyl
6-(3'-[2-pyridyldithio]-propionamido)hexanoate. An amplifying
restriction endonuclease can be attached at any location of a
nucleic acid component of the probe nucleic acid. For example, an
amplifying restriction endonuclease can be attached at an end
(e.g., a 5' end or 3' end) of a nucleic acid component, in the
middle of a nucleic acid component, or at any position along the
length of a nucleic acid component.
[0079] Signal expansion nucleic acid (e.g., first signal expansion
nucleic acid and second signal expansion nucleic acid) and reporter
nucleic acid described herein typically include at least one
double-stranded DNA section that includes an amplifying restriction
endonuclease cut site (e.g., an initial amplifying restriction
endonuclease cut site, a secondary amplifying restriction
endonuclease cut site, or a tertiary amplifying restriction
endonuclease cut site). The other portions of the signal expansion
nucleic acid or reporter nucleic acid can include DNA, RNA, or
other molecules. For example, reporter nucleic acid can include
biotin such that the reporter nucleic acid can be attached to a
streptavidin-coated solid support. In some cases, one or both
strands of the double-stranded section of the signal expansion
nucleic acid or the reporter nucleic acid that contains an
amplifying restriction endonuclease cut site can be RNA or a
nucleic acid analog (e.g., a peptide nucleic acid (PNA)) provided
that such a double-stranded section is capable of being cleaved by
the amplifying restriction endonuclease. Examples of restriction
endonucleases that can be used as amplifying restriction
endonucleases to cleave a DNA:RNA hybrid section of signal
expansion nucleic acid or reporter nucleic acid include, without
limitation, HhaI, AluI, TaqI, HaeIII, EcoRI, HindII, SalI, and MspI
restriction endonucleases.
[0080] Signal expansion nucleic acid or reporter nucleic acid
described herein can be any length provided that the
double-stranded section that contains the amplifying restriction
endonuclease cut site is capable of being cleaved by the amplifying
restriction endonuclease. In general, the double-stranded section
of signal expansion nucleic acid or reporter nucleic acid can be
between about 10 and about 500 or more nucleotides (e.g., between
about 10 and about 400 nucleotides, between about 10 and about 300
nucleotides, between about 10 and about 200 nucleotides, between
about 10 and about 100 nucleotides, between about 10 and about 50
nucleotides, between about 10 and about 25 nucleotides, between
about 20 and about 500 nucleotides, between about 30 and about 500
nucleotides, between about 40 and about 500 nucleotides, between
about 50 and about 500 nucleotides, between about 15 and about 50
nucleotides, between about 15 and about 25 nucleotides, between
about 20 and about 50 nucleotides, or between about 18 and about 25
nucleotides, between about 20 and about 60 nucleotides, between
about 25 and about 55 nucleotides, between about 30 and about 50
nucleotides, between about 35 and about 45 nucleotides, or between
about 38 and about 42 nucleotides) in length. In some cases, the
double-stranded section of signal expansion nucleic acid or
reporter nucleic acid can be between 5 and 50 nucleotides in
length. The amplifying restriction endonuclease cut site of the
signal expansion nucleic acid or the reporter nucleic acid can be
located at any position alone the double-stranded section. For
example, the amplifying restriction endonuclease cut site can be
towards the 5' end, towards the '3 end, or near the center of the
double-stranded section of the signal expansion nucleic acid or the
reporter nucleic acid. In general, the overall length of signal
expansion nucleic acid or reporter nucleic acid described herein
can be between about 10 and about 2500 or more nucleotides (e.g.,
between about 10 and about 2000 nucleotides, between about 10 and
about 1000 nucleotides, between about 10 and about 500 nucleotides,
between about 10 and about 400 nucleotides, between about 10 and
about 300 nucleotides, between about 10 and about 200 nucleotides,
between about 10 and about 100 nucleotides, between about 10 and
about 50 nucleotides, between about 10 and about 25 nucleotides,
between about 20 and about 500 nucleotides, between about 30 and
about 500 nucleotides, between about 40 and about 500 nucleotides,
between about 50 and about 500 nucleotides, between about 75 and
about 500 nucleotides, between about 100 and about 500 nucleotides,
between about 150 and about 500 nucleotides, between about 15 and
about 50 nucleotides, between about 15 and about 25 nucleotides,
between about 20 and about 50 nucleotides, between about 18 and
about 25 nucleotides, between about 20 and about 60 nucleotides,
between about 25 and about 55 nucleotides, between about 30 and
about 50 nucleotides, between about 35 and about 45 nucleotides, or
between about 38 and about 42 nucleotides) in length.
[0081] The amplifying restriction endonuclease cut site of signal
expansion nucleic acid or reporter nucleic acid described herein
can be a cut site of any type of restriction endonuclease. In
addition, any type of restriction endonuclease can be used as an
amplifying restriction endonuclease to cleave signal expansion
nucleic acid or reporter nucleic acid. Examples of restriction
endonucleases that can be used as amplifying restriction
endonucleases include, without limitation, EcoRI, EcoRII, BamHI,
HindIII, TaqI, NotI, HinfI, Sau3A, PovII, SmaI, HaeIII, HgaI, AluI,
EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI, SphI, StuI, XbaI,
AarI, BanII, BseGI, BspPI, CfrI, EcoNI, Hsp92II, NlaIV, RsaI, TaiI,
AasI, BbsI, BseJI, BspTI, ClaI, EcoO109I, I-PpoI, NmuCI, RsrII,
TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI, KasI, Acc65I, BbvCI, BseMI,
BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI, AccB7I, BbvI, BseMII,
BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI, BceAI, BseNI,
BsrGI, CspI, Esp3I, KspAI, NsiI, SapI, and TauI restriction
endonucleases.
[0082] In general, signal expansion nucleic acid or reporter
nucleic acid can be designed to have a double-stranded section that
contains a single amplifying restriction endonuclease cut site. In
some cases, signal expansion nucleic acid or reporter nucleic acid
provided herein can be designed to have a double-stranded section
that contains more than one (e.g., two, three, four, five, six,
seven, eight, nine, ten, or more) amplifying restriction
endonuclease cut site. When more than one amplifying restriction
endonuclease cut site is used, the multiple amplifying restriction
endonuclease cut sites can be cut sites for the same restriction
endonuclease or cut sites for different restriction endonucleases.
For example, reporter nucleic acid can be designed to have a
double-stranded section that contains one initial amplifying
restriction endonuclease cut site for an EcoRI initial amplifying
restriction endonuclease and one secondary amplifying restriction
endonuclease cut site for an XbaI secondary amplifying restriction
endonuclease.
[0083] Any appropriate method can be used to obtain the nucleic
acid component of signal expansion nucleic acid or reporter nucleic
acid. For example, common molecular cloning and chemical nucleic
acid synthesis techniques can be used to obtain the nucleic acid
component of signal expansion nucleic acid or reporter nucleic
acid. In some cases, the nucleic acid component of signal expansion
nucleic acid or reporter nucleic acid can be synthesized using
commercially available automated oligonucleotide synthesizers such
as those available from Applied Biosystems (Foster City, Calif.).
In some cases, signal expansion nucleic acid or reporter nucleic
acid can be synthesized de novo using any of a number of procedures
widely available in the art. Examples of such methods of synthesis
include, without limitation, the .beta.-cyanoethyl phosphoramidite
method (Beaucage et al., Tet. Let., 22:1859-1862 (1981)) and the
nucleoside H-phosphonate method (Garegg et al., Tet. Let.,
27:4051-4054 (1986); Froehler et al., Nucl. Acid Res., 14:5399-5407
(1986); Garegg et al., Tet. Let., 27:4055-4058 (1986); and Gaffney
et al., Tet. Let., 29:2619-2622 (1988)). These methods can be
performed by a variety of commercially-available automated
oligonucleotide synthesizers. In some cases, recombinant nucleic
acid techniques such as PCR and those that include using
restriction enzyme digestion and ligation of existing nucleic acid
sequences (e.g., genomic DNA or cDNA) can be used to obtain the
nucleic acid component of signal expansion nucleic acid or reporter
nucleic acid.
[0084] Signal expansion nucleic acid or reporter nucleic acid
described herein can be attached to a solid support. Examples of
solid supports include, without limitation, a well of a microtiter
plate (e.g., a 96-well microtiter plate or ELISA plate), beads
(e.g., magnetic, glass, plastic, or gold-coated beads), slides
(e.g., glass or gold-coated slides), micro- or nano-particles
(e.g., carbon nanotubes), platinum solid supports, palladium solid
supports, and a surface of a chamber or channel within a
microfluidic device. In some cases, a solid support can be a
silicon oxide-based solid support, a plastic polymer-based solid
support (e.g., a nylon, nitrocellulose, or polyvinylidene
fluoride-based solid support) or a biopolymer-based (e.g., a
cross-linked dextran or cellulose-based solid support) solid
support.
[0085] Signal expansion nucleic acid or reporter nucleic acid can
be directly or indirectly attached to a solid support. For example,
biotin can be a component of signal expansion nucleic acid or
reporter nucleic acid, and the signal expansion nucleic acid or the
reporter nucleic acid containing biotin can be indirectly attached
to a solid support that is coated with streptavidin via a
biotin-streptavidin interaction. In some cases, signal expansion
nucleic acid or reporter nucleic acid can be attached to a solid
support via a covalent or non-covalent interaction. For example,
signal expansion nucleic acid or reporter nucleic acid can be
covalently attached to magnetic beads as described elsewhere
(Albretsen et al., Anal. Biochem., 189(1):40-50 (1990)).
[0086] Signal expansion nucleic acid can be designed to contain any
type of restriction endonuclease as an amplifying restriction
endonuclease (e.g., an initial amplifying restriction endonuclease,
a secondary amplifying restriction endonuclease, or a tertiary
amplifying restriction endonuclease). In general, an amplifying
restriction endonuclease of signal expansion nucleic acid is
typically a different restriction endonuclease than the restriction
endonuclease that is used as a recognition restriction
endonuclease. For example, when an EcoRI restriction endonuclease
is used as a recognition restriction endonuclease, a restriction
endonuclease other than an EcoRI restriction endonuclease (e.g., a
HeaIII restriction endonuclease) is used as an amplifying
restriction endonuclease. Examples of restriction endonucleases
that can be used as amplifying restriction endonucleases include,
without limitation, EcoRI, EcoRII, BamHI, HindIII, TaqI, NotI,
HinfI, Sau3A, PovII, SmaI, HaeIII, HgaI, AluI, EcoRV, EcoP15I,
KpnI, PstI, SacI, SalI, ScaI, SphI, StuI, XbaI, AarI, BanII, BseGI,
BspPI, CfrI, EcoNI, Hsp92II, NlaIV, RsaI, TaiI, AasI, BbsI, BseLI,
BspTI, ClaI, EcoO109I, I-PpoI, NmuCI, RsrII, TaqaI, AatII, BbuI,
BseLI, BsrBI, CpoI, KasI, Acc65I, BbvCI, BseMI, BsrDI, Csp45I,
Kpn2I, NruI, SacII, TasI, AccB7I, BbvI, BseMII, BsrFI, Csp6I, EheI,
KpnI, NsbI, SalI, TatI, AccI, BceAI, BseNI, BsrGI, CspI, Esp3I,
KspAI, NsiI, SapI, and TauI restriction endonucleases. Any number
of molecules of the same amplifying restriction endonuclease can be
attached to one signal expansion nucleic acid molecule. For
example, a single signal expansion nucleic acid molecule can
contain one, two, three, four, five, or more EcoRI amplifying
restriction endonuclease molecules. In some cases, a single signal
expansion nucleic acid molecule can contain two or more (e.g., two,
three, four, five, or more) different types of amplifying
restriction endonucleases. For example, a single signal expansion
nucleic acid molecule can contain three BanII amplifying
restriction endonuclease molecules and two SacII amplifying
restriction endonuclease molecules.
[0087] Reporter nucleic acid can be designed to contain a label to
aid in the detection of cleaved reporter nucleic acid. In some
cases, signal expansion nucleic acid can be designed to contain a
label. In such cases, signal expansion nucleic acid containing a
label can be used in addition to reporter nucleic acid or in place
of reporter nucleic acid to detect target nucleic acid. Examples of
labels that can be a component of reporter nucleic acid or signal
expansion nucleic acid include, without limitation, fluorescent
labels (with or without the use of quenchers), dyes, antibodies,
radioactive material, enzymes (e.g., horse radish peroxidase,
alkaline phosphatase, laccase, galactosidase, or luciferase), redox
labels (e.g., ferrocene redox labels), metallic particles (e.g.,
gold nanoparticles), and green fluorescent protein-based labels. In
some cases, for a redox label, such as ferrocene, the detector can
be an electrode for amperometric assay of redox molecules. For
example, if the redox label is present in a reduced form of
ferrocene, then the electrode at high electrode potential can
provide an oxidation of the reduced form of ferrocene, thereby
converting it to an oxidized form of ferrocene. The generated
current can be proportional to the concentration of ferrocene label
in the solution.
[0088] In one embodiment, reporter nucleic acid or signal expansion
nucleic acid can contain a fluorescent label and a quencher such
that cleaved reporter nucleic acid provides a fluorescent signal
and uncleaved reporter nucleic acid does not provide a fluorescent
signal. In some cases, the reporter nucleic acid or signal
expansion nucleic acid can contain a label (e.g., a fluorescent
label or an enzyme such as horse radish peroxidase) and can be
attached to a solid support (e.g., a well of a microtiter plate).
For example, the reporter nucleic acid or signal expansion nucleic
acid can be attached to a solid support such that cleavage at the
amplifying restriction endonuclease cut site by the amplifying
restriction endonuclease releases a portion of the reporter nucleic
acid or the signal expansion nucleic acid that contains the label.
The resulting reaction mixture can be collected and assessed for
the presence, absence, or amount of released portions of the
reporter nucleic acid or signal expansion nucleic acid using the
label. For example, the released portions of the reporter nucleic
acid or the signal expansion nucleic acid, if present, can be
transferred from one well of a microtiter plate (e.g., a 96-well
plate) that contained the reporter nucleic acid or the signal
expansion nucleic acid to another well of a microtiter plate, where
the transferred material can be assessed for a signal from the
label. Any number of molecules of a label can be attached to one
reporter nucleic acid molecule or one signal expansion nucleic acid
molecule. For example, a reporter nucleic acid molecule or a single
signal expansion nucleic acid molecule can contain one, two, three,
four, five, or more fluorescent molecules.
[0089] Any appropriate method can be used to attach a label to a
nucleic acid component of reporter nucleic acid or signal expansion
nucleic acid. In some cases, a label can be attached by an ionic or
covalent attachment. For example, covalent bonds such as amide
bonds, disulfide bonds, and thioether bonds, or bonds formed by
crosslinking agents can be used. In some cases, a non-covalent
linkage can be used. The attachment can be a direct attachment or
an indirect attachment. For example, a linker can be used to attach
a label to a nucleic acid component of reporter nucleic acid or
signal expansion nucleic acid. In some cases, nucleic acid can
include a thiol modification, and a label can be conjugated to the
thiol-containing nucleic acid based on succinimidyl
4-[N-maleimidomethyl]cyclo-hexane-1-carboxylate (SMCC) using
techniques similar to those described elsewhere (Dill et al.,
Biosensors and Bioelectronics, 20:736-742 (2004)). In some cases, a
biotinylated nucleic acid and a streptavidin-containing label can
be attached to one another via a biotin-streptavidin interaction. A
label can be conjugated with streptavidin using, for example,
sulfosuccinimidyl 6-(3'-[2-pyridyldithio]-propionamido)hexanoate. A
label can be attached at any location of a nucleic acid component
of reporter nucleic acid or signal expansion nucleic acid. For
example, a label can be attached at an end (e.g., a 5' end or 3'
end) of a nucleic acid component, in the middle of a nucleic acid
component, or at any position along the length of a nucleic acid
component of reporter nucleic acid or signal expansion nucleic
acid.
[0090] As described herein, the methods and materials provided
herein can be used to detect target nucleic acid containing a
genetic or epigenetic element in any type of sample (e.g., a
biological sample). For example, a blood sample or cheek swab
sample can be collected from a mammal and assessed for target
nucleic acid to determine if the mammal has one or more genetic or
epigenetic elements of interest. Once obtained, a sample to be
assessed can be processed to obtain nucleic acid. For example, a
nucleic acid extraction can be performed on a blood sample to
obtain a sample that is enriched for nucleic acid. In some cases, a
sample can be heated or treated with a cell lysis agent to release
nucleic acid from cells present in the sample.
[0091] As described herein, a sample (e.g., a biological sample)
can be assessed for the presence, absence, or amount of target
nucleic acid (e.g., target nucleic acid containing a genetic or
epigenetic element) using an enzymatic amplification cascade of
restriction endonucleases described herein without using a nucleic
acid amplification technique (e.g., a PCR-based nucleic acid
technique). Assessing samples (e.g., biological samples) for the
presence, absence, or amount of target nucleic acid using an
enzymatic amplification cascade of restriction endonucleases
described herein without using a nucleic acid amplification
technique can allow patients as well as medical, laboratory, or
veterinarian personnel (e.g., clinicians, physicians, physician's
assistants, laboratory technicians, research scientists, and
veterinarians) to test for one or more genetic or epigenetic
elements without the need for potentially expensive thermal cycling
devices and potentially time consuming thermal cycling techniques.
In some cases, the methods and materials provided herein can be
used in combination with a PCR-based nucleic acid technique. For
example, a PCR-based nucleic acid technique can be performed to
amplify nucleic acid (e.g., a target nucleic acid containing a
genetic or epigenetic element) present within a biological sample,
and the resulting amplification material can be assessed using an
enzymatic amplification cascade of restriction endonucleases
described herein to detect the presence, absence, or amount of a
particular nucleic acid (e.g., a target nucleic acid a genetic or
epigenetic element). In some cases, a limited PCR-based nucleic
acid technique can be performed to amplify a target nucleic acid to
a point where the amount of amplified target nucleic acid is
increased only slightly over the amount of target nucleic acid
originally present within the biological sample. For example, a two
to twelve cycle PCR technique (e.g., a 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, or 12 cycle PCR technique) can be performed to slightly
increase the amount of amplified target nucleic acid as compared to
the amount of unamplified target nucleic acid originally present
within the biological sample. Such limited PCR-based nucleic acid
techniques, when used in combination with an enzymatic
amplification cascade of restriction endonucleases described
herein, can allow medical, laboratory, or veterinarian personnel to
test organisms with a potentially increased level of sensitivity
and/or specificity without the potentially lengthy time involved in
thermal cycling techniques that include a greater number of cycles.
This increased level of sensitivity and/or specificity can be over
the high level of sensitivity and specificity of a comparable
testing procedure that includes an enzymatic amplification cascade
of restriction endonucleases described herein without the limited
PCR-based nucleic acid technique. In some cases, the PCR-based
nucleic acid technique can be performed to amplify a target nucleic
acid to a point where the amount of amplified target nucleic acid
is easily detectable (e.g., visually detectable using gel
electrophoresis and ethidium bromide staining). For example, a 15
or more cycle PCR technique (e.g., a 20 cycle PCR technique) can be
performed to produce at least ng amounts (e.g., greater than 1 ng,
10 ng, 100 ng, 1 .mu.g, 10 .mu.g, or more) of amplified nucleic
acid. Such PCR-based nucleic acid techniques, when used in
combination with an enzymatic amplification cascade of restriction
endonucleases described herein, can allow medical, laboratory, or
veterinarian personnel to test organisms with a potentially
increased level of sensitivity and/or specificity. This increased
level of sensitivity and/or specificity can be over the high level
of sensitivity and specificity of a comparable testing procedure
that includes an enzymatic amplification cascade of restriction
endonucleases described herein without the PCR-based nucleic acid
technique.
[0092] In some cases, a sample (e.g. a biological sample) can be
obtained and subjected to a culturing technique. For example, a
cell sample can be obtained and cultured with medium (e.g.,
enrichment medium) to enrich the sample such that the number of
cells present in the sample can increase. Examples of enrichment
media include, without limitation, Dulbecco's Modified Eagle Medium
(DMEM), Minimum Essential Medium (MEM), Iscove's Modified
Dulbecco's Media (IMDM), and AIM V.RTM. Medium. In some cases, the
culture medium can contain a nutrient (e.g. serum such as fetal
calf serum), ingredient, or drug that prevents certain cells from
dividing while allowing other cells to divide. In some cases, the
culturing technique can include incubating a sample at an
appropriate temperature (e.g. between 15.degree. C. and 45.degree.
C., between 20.degree. C. and 45.degree. C., between 25.degree. C.
and 45.degree. C., between 30.degree. C. and 45.degree. C., between
30.degree. C. and 40.degree. C., between 35.degree. C. and
45.degree. C., or between 35.degree. C. and 40.degree. C.) for an
appropriate period of time (e.g., between about 0.5 hours and 48
hours, between about 0.5 hours and 36 hours, between about 0.5
hours and 24 hours, between about 0.5 hours and 12 hours, between
about 0.5 hours and 8 hours, between about 0.5 hours and 6 hours,
between about 0.5 hours and 5 hours, between about 0.5 hours and 4
hours, between about 0.5 hours and 3 hours, between about 0.5 hours
and 2 hours, between about 1 hour and 4 hours, or between about 2
hours and 4 hours). For example, a sample can be obtained and
cultured in tissue culture medium at 37.degree. C. for 24-48 hours.
Examples of tissue culture techniques that can be used as described
herein include, without limitation, those described elsewhere
(Animal Cell Culture: A Practical Approach, 3rd edition, J.
Masters, ed., Oxford University Press, 2000, 336 pp).
[0093] In some cases, a sample, obtained and subjected to a
culturing technique or not, can be processed, for example, to
remove non-nucleic acid material, to disrupt cell membranes to
release nucleic acid, and/or to collect or extract nucleic acid,
such that nucleic acid of the sample, if present within the sample,
is available for hybridization to probe nucleic acid. For example,
a blood or cheek swab sample can be treated with a lysis buffer and
subjected to nucleic acid extraction such that a major component of
the sample is nucleic acid. In some cases, a sample can be
homogenized and treated to disrupt cells that are present in the
sample. For example, a blood sample can be subjected to high speed
mechanical homogenization with glass/silica/zirconium/stainless
steel beads, can be subjected to high temperature (e.g., boiling or
autoclaving), can be subjected to chemical lysis with detergents
and/or surfactants (e.g., sodium dodecyl sulfate,
cetyltrimethylammonium bromide, or sodium lauroyl sarcosin), can be
subjected to one or more freeze-thaw cycles using, e.g., liquid
nitrogen or dry ice, can be subjected to sonication, or can be
subjected to combinations thereof. The resulting sample can be
subjected to a standard nucleic acid extraction technique such as
those described elsewhere (e.g., Sambrook and Russell, (2001)
Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring
Harbor Press) or a nucleic acid extraction technique that includes
the use of magnetic beads or selective DNA-binding membranes (see,
e.g., QIAGEN DNeasy.RTM. Blood & Tissue Kit, or Mo Bio
PowerFood.TM. Microbial DNA Isolation Kit). For example, the blood
sample can be contacted with magnetic beads that bind nucleic acid,
the beads can be removed, and bound nucleic acid can be eluted into
an appropriate buffer to form a processed sample for further
analysis using the methods and materials provide herein. Such a
process can be carried out using a variety of kits including,
without limitation, Qiagen BioSprint 96 One-For-All Vet Kit (a
rapid and economical automated purification of viral nucleic acid
and/or bacterial nucleic acid from samples based on magnetic beads)
and Chemicell geneMAG-PCR cleanup kit. In some cases, a sample
(e.g., a blood sample or body fluid sample) can be subjected to DNA
isolation using Qiagen QIAcard FTA Spots or Qiagen QIAamp UltraSens
Virus Kits.
[0094] In some cases, a sample can be processed in a manner
designed to fragment any nucleic acid present within the sample.
For example, genomic or large pieces of nucleic acid present within
a sample can be subjected to a sonication technique, nebulization
technique, and/or restriction digestion with a restriction
endonuclease such as DpnII or CviJI to generate nucleic acid
fragments. Such fragmentation can be performed using restriction
endonucleases that are different from those used as recognition or
amplifying restriction endonucleases to assess the sample as
described herein.
[0095] In some cases, the sample can be treated such that any
double-stranded nucleic acid present within the sample is
separated. For example, a biological sample can be heated and then
snap-cooled or can be subjected to chemical (e.g., sodium
hydroxide) denaturation. In some cases, when the sample is
subjected to a PCR-based technique, certain primer or reaction
modifications can be used to generate preferentially
single-stranded product. For example, unidirectional DNA polymerase
reactions can be performed with a single specific primer. In some
cases, the strands of nucleic acid can be separated, and the strand
of interest can be enrichment using specific biotinylated primers
and streptavidin-conjugated magnetic beads. In some cases,
selective digestion of one of the strands can be accomplished using
lambda exonucleases.
[0096] As described herein, a sample (e.g., a biological sample)
can be subjected to a nucleic acid amplification technique. For
example, a tissue sample containing extracted nucleic acid can be
subjected to a quick PCR-based amplification of one or more
specific targets (e.g., 1 hour, end-point PCR) or to a whole genome
amplification technique (e.g., Qiagen REPLI-g Screening Kit for
high-throughput manual or automated whole genome
amplification).
[0097] Once obtained, a sample to be assessed, whether subjected to
a PCR-based nucleic acid technique or not, can be contacted with a
probe nucleic acid as described herein. This contacting step can be
carried out for any period of time and at any temperature that
allows target nucleic acid to hybridize with probe nucleic acid.
For example, this step can be performed between 10 seconds and 24
hours (e.g., between 30 seconds and 12 hours, between 30 seconds
and 8 hours, between 30 seconds and 4 hours, between 30 seconds and
2 hours, between 30 seconds and 1 hour, between 1 minute and 24
hours, between 1 minute and 12 hours, between 1 minute and 8 hours,
between 1 minute and 4 hours, between 1 minute and 2 hours, between
1 minute and 1 hour, between 5 minutes and 1 hour, between 10
minutes and 1 hour, between 15 minutes and 1 hour, or between 30
minutes and 1 hour). The initial temperature can be between
15.degree. C. and 100.degree. C. (e.g., between 23.degree. C. and
98.degree. C., between 23.degree. C. and 90.degree. C., between
23.degree. C. and 85.degree. C., between 23.degree. C. and
75.degree. C., between 23.degree. C. and 65.degree. C., between
23.degree. C. and 55.degree. C., between 23.degree. C. and
45.degree. C., between 23.degree. C. and 35.degree. C., between
30.degree. C. and 95.degree. C., between 30.degree. C. and
85.degree. C., between 30.degree. C. and 75.degree. C., between
30.degree. C. and 65.degree. C., between 30.degree. C. and
55.degree. C., between 30.degree. C. and 45.degree. C., between
20.degree. C. and 40.degree. C., between 20.degree. C. and
30.degree. C., and between 25.degree. C. and 35.degree. C.). The
temperature during this contacting step can remain constant or can
be increased or decreased. For example, the initial temperature can
be between about 40.degree. C. and about 85.degree. C., and then
the temperature can be allowed to decrease to room temperature over
a period of about 30 seconds to about 30 minutes (e.g., between
about 30 seconds and about 15 minutes, between about 30 seconds and
about 10 minutes, between about 1 minute and about 30 minutes,
between about 1 minute and about 15 minutes, or between about 1
minute and about 5 minutes).
[0098] Contact of the sample (e.g., a biological sample to be
tested) with probe nucleic acid can occur in the presence of the
recognition restriction endonucleases, or a separate step of adding
the recognition restriction endonucleases to the reaction can be
performed. The recognition restriction endonuclease step can be
carried out for any period of time and at any temperature that
allows the recognition restriction endonuclease to cleave
recognition restriction endonuclease cut sites formed by the
hybridization of target nucleic acid to the probe nucleic acid. For
example, this step can be performed between one second and 24 hours
(e.g., between one second and 30 minutes, between one second and
one hour, between five seconds and one hour, between 30 seconds and
24 hours, between 30 seconds and 12 hours, between 30 seconds and 8
hours, between 30 seconds and 4 hours, between 30 seconds and 2
hours, between 30 seconds and 1 hour, between 1 minute and 24
hours, between 1 minute and 12 hours, between 1 minute and 8 hours,
between 1 minute and 4 hours, between 1 minute and 2 hours, between
1 minute and 1 hour, between 5 minutes and 1 hour, between 10
minutes and 1 hour, between 15 minutes and 1 hour, or between 30
minutes and 1 hour). The temperature can be between 15.degree. C.
and 75.degree. C. (e.g., between 15.degree. C. and 75.degree. C.,
between 15.degree. C. and 65.degree. C., between 15.degree. C. and
55.degree. C., between 15.degree. C. and 45.degree. C., between
15.degree. C. and 35.degree. C., between 15.degree. C. and
30.degree. C., between 23.degree. C. and 55.degree. C., between
23.degree. C. and 45.degree. C., between 30.degree. C. and
65.degree. C., between 30.degree. C. and 55.degree. C., between
30.degree. C. and 45.degree. C., between 30.degree. C. and
40.degree. C., between 35.degree. C. and 40.degree. C., and between
36.degree. C. and 38.degree. C.). Any appropriate concentration of
recognition restriction endonuclease can be used. For example,
between about 0.001 units and 1000 units (e.g., between about 0.001
units and 750 units, between about 0.001 units and 500 units,
between about 0.001 units and 250 units, between about 0.001 units
and 200 units, between about 0.001 units and 150 units, between
about 0.001 units and 100 units, between about 0.001 units and 50
units, between about 0.001 units and 25 units, between about 0.001
units and 10 units, between about 0.001 units and 1 unit, between
about 0.001 units and 0.1 units, between about 0.01 units and 1000
units, between about 0.1 units and 1000 units, between about 1 unit
and 1000 units, between about 10 units and 1000 units, between
about 50 units and 1000 units, between about 0.5 units and 100
units, or between about 1 unit and 100 units) of restriction
endonuclease can be used. Other restriction endonuclease reaction
conditions such as salt conditions can be used according to the
manufacturer's instructions.
[0099] When one step of a method provided herein is completed, the
resulting reaction product containing cleaved nucleic acid can be
used in the next step. For example, cleaved nucleic acid of a
reaction product can be removed from uncleaved nucleic acid and
used in the next step of the method. For example, when probe
nucleic acid is attached to a solid support, the released portions
of probe nucleic acid that contain an amplifying restriction
endonuclease can be collected and placed in contact with reporter
nucleic acid or signal expansion nucleic acid as described herein.
The resulting reaction products of a particular step can be
manually or automatically (e.g., robotically) transferred to a
location containing nucleic acid for the next step (e.g., reporter
nucleic acid or signal expansion nucleic acid), which nucleic acid
can be attached or not attached to a solid support. In some cases,
one reaction of a method described herein can be carried out at one
location (e.g., a chamber) of a microfluidic device or blister
package device, and the reaction products that are generated can be
moved to another location (e.g., another chamber) that contains
nucleic acid for the next step (e.g., reporter nucleic acid or
signal expansion nucleic acid) via a channel. In some cases,
cleaved nucleic acid of a reaction product can be used in the next
step of the method by removing the uncleaved nucleic acid from the
reaction product. For example, when magnetic beads are used as a
solid support, a magnetic force can be used to remove the magnetic
beads and any attached uncleaved nucleic acid from the reaction
product. In some cases, two or more reactions of a method provided
herein can be carried out at one location (e.g., a single well of a
microtiter plate or a single chamber of a microfluidic device). For
example, a single compartment can have one region that contains
immobilized probe nucleic acid and another region that contains
immobilized reporter nucleic acid provided that the amplifying
restriction endonuclease of the immobilized probe nucleic acid is
not capable of cleaving the amplifying restriction endonuclease cut
site of the reporter nucleic acid unless target nucleic acid
hybridizes to the probe nucleic acid and the recognition
restriction endonuclease cleaves the probe nucleic acid, thereby
releasing a portion of the probe nucleic acid that contains the
amplifying restriction endonuclease so that it is capable of
cleaving the reporter nucleic acid. In another example, a single
compartment can have one region that contains immobilized probe
nucleic acid, other regions that contain immobilized signal
expansion nucleic acid (e.g., one region that contains a first
signal expansion nucleic acid and another region that contains a
second signal expansion nucleic acid), and another region that
contains immobilized reporter nucleic acid provided that the
amplifying restriction endonucleases of immobilized probe nucleic
acid and signal expansion nucleic acid are not capable of cleaving
their intended amplifying restriction endonuclease cut sites until
they are released as described herein. Such single compartments can
be made using partitions or sub-compartments within the single
compartment. For example, a sample to be tested can be placed into
a single well of a microtiter plate that contains probe nucleic
acid, recognition restriction endonucleases, first and second
signal expansion nucleic acid, and reporter nucleic acid such that
cleaved reporter nucleic acid and/or signal expansion nucleic acid
is produced as described herein when target nucleic acid is present
in the sample being tested and little or no cleaved reporter
nucleic acid and/or signal expansion nucleic acid is produced when
target nucleic acid is not present in the sample being tested.
[0100] Any appropriate method can be used to detect cleaved
reporter nucleic acid and/or signal expansion nucleic acid to
determine the presence, absence, or amount of target nucleic acid
in a sample, which can indicate the presence, absence, or amount of
a target genetic or epigenetic element. For example, size
separation techniques can be used to assess reaction products for
cleaved reporter nucleic acid and/or signal expansion nucleic acid.
Examples of such size separation techniques include, without
limitation, gel electrophoresis and capillary electrophoresis
techniques. In some cases, a melt curve analysis can be performed
to assess reaction products for cleaved reporter nucleic acid
and/or signal expansion nucleic acid. As described herein, a label
can be used to aid in the detection of cleaved nucleic acid (e.g.,
reporter nucleic acid and/or signal expansion nucleic acid).
Examples of labels that can be used include, without limitation,
fluorescent labels (with or without the use of quenchers), dyes,
antibodies, radioactive material, enzymes (e.g., horse radish
peroxidase, alkaline phosphatase, laccase, galactosidase, or
luciferase), redox labels (e.g., ferrocene redox labels), metallic
particles (e.g., gold nanoparticles), and green fluorescent protein
based labels. For example, the release of fluorescently labeled
portions of reporter nucleic acid and/or signal expansion nucleic
acid from a solid support can be assessed using common fluorescent
label detectors. In some cases, cleaved reporter nucleic acid
and/or signal expansion nucleic acid can be detected
electrochemically. For electrochemical detection, the reporter
nucleic acid and/or signal expansion nucleic acid can include a
ferrocene redox label. Reporter nucleic acid and/or signal
expansion nucleic acid containing ferrocene can be obtained by
coupling ferrocene carboxylic acid with an amino-modified
oligonucleotide using the carbodiimide reaction in the presence of
an excess of ferrocene carboxylic acid. In one embodiment, for a
redox label, such as ferrocene, the detector can be an electrode
for amperometric assay of redox molecules. For example, if the
redox label is present in a reduced form of ferrocene, then the
electrode at high electrode potential can provide an oxidation of
the reduced form of ferrocene, thereby converting it to an oxidized
form of ferrocene. The generated current can be proportional to the
concentration of ferrocene label in the solution.
[0101] The methods and materials provided herein can be used to
assess one or more samples for target nucleic acid in real-time.
For example, a fluorescent label/quencher system or an
electrochemical redox label system can be used to detect cleavage
of reporter nucleic acid and/or signal expansion nucleic acid in
real time.
[0102] The methods and materials provided herein can be used to
assess one or more samples (e.g., two, three, four, five, six,
seven, eight, nine, ten, 20, 50, 100, 500, 1000, or more) for a
single type of target nucleic acid. For example, 100s of tissue
samples (e.g., tissue biopsy samples) can be assessed for target
nucleic acid containing a particular genetic or epigenetic element.
In some case, the methods and materials provided herein can be used
in a multiplex manner to assess one or more samples for more than
one (e.g., two, three, four, five, six, seven, eight, nine, ten,
20, 50, 100, 500, 1000, or more) type of target nucleic acid. For
example, target nucleic acid for ten different sequences (e.g., ten
different SNP sequences) can be used to design ten different probe
nucleic acid molecules. In these cases, each probe nucleic acid can
be used in a separate series of reactions within the same device
(e.g., microtiter plate or microfluidic device), and the same label
can be used for the reporter nucleic acid for each probe nucleic
acid. In addition, in some cases, the same amplifying restriction
endonuclease can be used for each probe nucleic acid, and the same
reporter nucleic acid can be used for each reaction series. In some
cases, when multiple different probe nucleic acid molecules are
used in the same reaction series, a different reporter nucleic acid
having different labels can be used to correspond to each probe
nucleic acid such that the detected signals can indicate which of
the ten target nucleic acids are being detected.
[0103] This document also provides kits for performing the methods
described herein. For example, a kit provided herein can include
probe nucleic acid with or without being attached to a solid
support and/or reporter nucleic acid with or without being attached
to a solid support. In some cases, such a kit can include a
recognition restriction endonuclease, first signal expansion
nucleic acid, second signal expansion nucleic acid, or a
combination thereof In some cases, a kit can be configured into a
microfluidic device that allows for the movement of probe nucleic
acid, first signal expansion nucleic acid, second signal expansion
nucleic acid, reporter nucleic acid, or recognition restriction
endonucleases (or any combination thereof) as well as a cleaved
portion of any such nucleic acid in a manner that allows a
detection method provided herein to be carried out with or without
the nucleic acid being attached to a solid support. For example, a
kit provided herein can be a microfluidic device capable of
receiving a sample and contacting that sample with probe nucleic
acid. The probe nucleic acid can be designed to include a length of
nucleotides followed by the sequence complementary to the target
nucleic acid, which can create a recognition restriction
endonuclease cut site, followed by an amplifying restriction
endonuclease. The distance from the recognition restriction
endonuclease cut site to the amplifying restriction endonuclease
can be relatively short (e.g., 100, 50, 25, 10, or less
nucleotides), while the distance from the recognition restriction
endonuclease cut site to the beginning of the length of nucleotides
can be relatively long (e.g., 50, 100, 150, 200, 500, 1000, 2000,
or more). In such cases, cleavage of the probe nucleic acid at the
recognition restriction endonuclease cut site can result in a
relatively small portion that contains the amplifying restriction
endonuclease and is capable of traveling faster than the larger
uncleaved probe nucleic acid. This difference can allow the cleaved
portion containing the amplifying restriction endonuclease to reach
an area of the microfluidic device containing signal expansion
nucleic acid or reporter nucleic acid so that the next reaction can
be carried out without the presence of uncleaved probe nucleic
acid. In some cases, after the smaller portion containing the
amplifying restriction endonuclease enters the area containing
signal expansion nucleic acid or reporter nucleic acid, a valve can
be used to prevent the larger uncleaved probe nucleic acid from
entering. In some cases, a filter can be used to limit the ability
of larger uncleaved probe nucleic acid from proceeding to the next
reaction location. Similar approaches can be used during other
steps of a method provided herein to separate cleaved nucleic acid
from uncleaved nucleic acid.
[0104] In some cases, a kit provided herein can be a portable or
self-contained device, packet, vessel, or container that can be
used, for example, in point of care applications. For example, such
a kit can be configured to allow a patient or physician's assistant
to insert a sample for analysis. In some cases, a kit can be
designed for use in a home setting or any other setting. Once
inserted, the sample can be heated (e.g., heated to about 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, or more
.degree. C.) and/or cooled by a heating or cooling mechanism
located within the kit. For example, an exothermic or endothermic
chemical reaction can be initiated within the kit to increase,
decrease, or maintain the temperature. Such exothermic or
endothermic chemical reactions can be carried out within the kit
without being in fluid communication with the reactions of the
target nucleic acid detection method. An iron oxidation reaction is
an example of an exothermic chemical reaction that can be used to
heat a kit provided herein. An endothermic chemical reaction that
can be used to cool a kit provided herein can be a reaction that
includes the use of ammonium chloride and water, potassium chloride
and water, or sodium carbonate and ethanoic acid. In general, when
detecting DNA target nucleic acid, the kit can be designed to
generate, if needed, enough heat to denature double stranded DNA
present within the sample. The kit also can be designed to generate
appropriate heating and cooling temperatures to carry out each step
of a detection method provided herein. In some cases, a kit
provided herein can include a temperature indicator (e.g., color
indicator or thermometer) to allows a user to assess
temperature.
[0105] In some cases, a kit can be designed to provide a user with
a "yes" or "no" indication about the presence of target nucleic
acid within a tested sample. For example, a label having the
ability to generate a change in pH can be used, and a visual
indicator (e.g., a pH-based color indicator) can be used to inform
the user of the presence of target nucleic acid based on a change
in pH.
[0106] In some cases, a point of care or home use device can be
designed to carry out the reactions described herein. For example,
point of care or home use device can be designed to include a
series of adjacent chambers. In a relatively simple configuration,
for example, a first "sample" chamber can be configured for sample
insertion, and can contain reagents (e.g., in dry or liquid form)
to effect generation of single stranded nucleic acid fragments. A
second "recognition" chamber can be configured to receive single
stranded nucleic acid fragments from the first chamber, and can
contain probe nucleic acid and recognition restriction endonuclease
(e.g., in dry or liquid form). A third "amplification" chamber can
be configured to receive cleaved portions of probe nucleic acid
from the second chamber, and can contain reporter nucleic acid
(e.g., in dry or liquid form). A fourth "detection" chamber can be
configured to receive cleaved portions of marker nucleic acid from
the third chamber, and can contain a reagent (e.g., in dry or
liquid form) that serves as an indicator of whether or not target
nucleic acid was present in the sample. It is noted that one or
more additional "signal expansion" chambers can be present between
the "recognition" chamber and the "amplification" chamber.
[0107] In some cases, a point of care or home use device can be
configured such the chambers are separated from each other by
membranes that can provide controlled passage of reaction
materials. For example, chambers can be separated by membranes that
are subject to degradation by particular reagents or solutions. In
such cases, a reaction can be confined to a particular chamber
until the membrane separating it from the adjacent chamber
degrades, permitting passage of reaction components there
between.
[0108] In some cases, a point of care or home use device can be
adapted for automatic transfer of the reaction mixture between
chambers. For example, insertion of a sample into the first chamber
can trigger a reaction or provide a reagent that gradually degrades
the membrane separating the first chamber from the second chamber.
Movement of all or a portion of the reaction mixture into the
second chamber can in turn provide a reagent or trigger a reaction
that gradually degrades the membrane separating the second chamber
from the third chamber. For example, if the sample reaction mixture
in the first chamber is an aqueous solution, the reagents in the
second chamber are dry, and the membrane in the second chamber is
degraded by water, movement of the aqueous reaction mixture into
the second chamber can trigger degradation of the membrane
therein.
[0109] In some cases, a point of care or home use device can be
adapted for automatic controlled flow transfer of reaction mixture
between chambers. For example, insertion of a sample into the first
chamber can trigger a reaction or provide a reagent that allows
controlled flow movement of the sample through absorption media.
Movement of all or a portion of the reaction mixture into the
second chamber can in turn provide a reagent or trigger a reaction
that allows controlled flow movement of the sample through
absorption media to a third chamber. In such cases, a reaction can
be confined to a particular chamber until the media separating it
from the adjacent chamber absorbs and permits passage of reaction
components there between.
[0110] In some cases, a point of care or home use device can be
adapted for automatic controlled flow transfer of reaction mixture
between chambers. For example, insertion of a sample into the first
chamber can trigger a reaction or provide a reagent that allows
controlled capillary flow movement of the sample through
micro-fluidic channels. Movement of all or a portion of the
reaction mixture into the second chamber can in turn provide a
reagent or trigger a reaction that allows controlled flow movement
of the sample through micro-fluidic channels to a third chamber. In
such cases, a reaction can be confined to a particular chamber
until the microfluidic channel permits passage of reaction
components there between.
[0111] In some cases, a point of care or home use device can be
adapted for automatic controlled flow transfer of reaction mixture
without chambers. For example, insertion of a sample into the
device can trigger a reaction or provide a reagent that allows
controlled capillary flow movement of the sample through
microfluidic channels. Movement of all or a portion of the reaction
mixture in the microfluidic channel can trigger a reaction that
allows reagents to enter the reaction mixture in a continuous
flow-through manner with no specific chamber for a reaction. In
such cases, a reaction does not need to be confined to a particular
section of the microfluidic channel.
[0112] In some cases, transfer of a reaction mixture from one
chamber to the next can be controlled by a user. An exemplary
user-controlled, pen-style point of care or home use device is
depicted in FIG. 7. Device 300 can include sample collector 310 and
reaction unit 320. Sample collector 310 can have cap 312 with screw
threads 314, shaft 316, and swabber 318. Swabber 318 can be smooth
or rough, and in some cases can have bristles (e.g., smooth or
rough bristles) or a matted texture to facilitate sample collection
from, for example, the inside cheek, throat, or skin of an
individual to be tested.
[0113] Reaction unit 320 can include tube 322, open end 324
reversibly closed by safety cap 326, and closed end 328. Open end
324 can have internal screw threads, and cap 326 can have external
screw threads 329. Screw threads 329 of safety cap 326, as well as
screw threads 314 of sample collector cap 312, can be adapted to
mate with the internal screw threads at open end 324, such that
either safety cap 326 or sample collector 310 can be screwed into
open end 324.
[0114] Tube 322 can contain several chambers, such as lysing and
isolation chamber 330, recognition and amplification chamber 360,
and detection chamber 390. As described herein, the chambers can be
separated from one another to prevent premature mixing of reaction
components. Tube 322 and the chambers contained therein can be made
from, for example, clear plastic (e.g., polycarbonate, acrylic,
nylon, or PVC). Tube 322 also can contain first and second safety
bands 340 and 370, and first and second spring returns 350 and
380.
[0115] Lysing and isolation chamber 330 can be positioned proximal
to open end 324. Lysing and isolation chamber 330 can have proximal
end 332, distal end 334, proximal membrane 336, distal membrane
337, and reaction completion indicator 338. Proximal membrane 336
can be located adjacent to proximal end 332, and distal membrane
337 can be located adjacent to distal end 334. Membranes 336 and
337 can be made from, for example, synthetic rubber, natural latex
rubber, or silicone. Chamber 330 can contain reagents for lysing
cells as well as reagents for cleaving and denaturing cellular
nucleic acids. Reaction completion indicator 338 can be, for
example, a built in timer or stop watch, a built in pH indicator, a
built in color change reagent, or a conductivity probe, and can
indicate when cell lysis and nucleic acid sample generation are
sufficient to proceed to the next step.
[0116] First safety band 340 can be positioned distal to lysing and
isolation chamber 330 within tube 322, and first spring return 350
can be positioned distal to first safety band 340. First safety
band 340 can be, for example, connected to a tab or strap, and can
be moved or removed from reaction unit 320 by pulling on the tab or
strap. First spring return 350 can be made from a shape memory
material that can be compressed and then automatically return to or
toward its original configuration.
[0117] The safety band 340 can be attached to the tube as a secured
ring that can be, for example, over molded as a soft rubber
component or inserted as a spring like split ring component. The
safety band 340 can lock the position of the lysing and isolation
tube chamber 340, preventing linear sliding of the lysing and
isolation chamber 330 to that of the recognition and amplification
chamber 360. Upon removal of safety band 340, the user can actuate
linear movement of the entire device 300 by holding the proximal
end firm and pressing the distal closed end 328 such that both
distal chambers recognition and amplification 360 and detection
chamber 390 are moved toward the lysing and isolation chamber 330.
The needle and sample collector 362 can pierce membrane 337 and
enter the lysing and isolation chamber 330. The user can release a
firm hold on the assembly and spring return 350 can draw the sample
into recognition and amplification chamber 360. After completion of
the reaction, the user can remove safety band 370, and the user can
actuate linear movement of the assembly by holding the recognition
and amplification chamber 360 firm and pressing the distal closed
end 328 such that the detection chamber 390 moves toward the
recognition and amplification chamber 360. The needle and sample
collector 392 can pierce membrane 366. The user can release the
firm hold on the assembly, and spring return 380 can draw the
sample into detection chamber 390.
[0118] Recognition and amplification chamber 360 can be positioned
distal to first spring return 350. Chamber 360 can have proximal
end 361, which in turn can have piercing needle and sample
collector 362, distal end 364, membrane 366, and reaction
completion indicator 368. Recognition and amplification chamber 360
can contain, for example, probe nucleic acid and reporter nucleic
acid and restriction endonucleases for use in enzymatic
amplification cascades as described herein. Piercing needle and
sample collector 362 can have a pointed, beveled, or barbed tip. In
addition, the interior of piercing needle and sample collector 362
can be in fluid communication with the interior of recognition and
amplification chamber 360, such that a nucleic acid test sample can
be collected from lysing and isolation chamber 330 and transferred
to recognition and amplification chamber 360 via collector 362.
Membrane 364 can be located adjacent to distal end 364, and can be
made from, for example, synthetic rubber, natural latex rubber, or
silicone. Reaction completion indicator 368 can be, for example, a
built in timer or stop watch, a built in pH indicator, a built in
color change reagent, or a conductivity probe, and can indicate
when cell lysis and nucleic acid sample generation are sufficient
to proceed to the next step.
[0119] Second safety band 370 can be positioned distal to
recognition and amplification chamber 360 within tube 322, and
second spring return 380 can be positioned distal to second safety
band 370. Second safety band 370 can be, for example, connected to
a tab or strap, and can be moved or removed from reaction unit 320
by pulling on the tab or strap. Second spring return 380 can be
made from a shape memory material (e.g., spring steel, plastic, or
rubber) that can be compressed and then automatically return to or
toward its original configuration.
[0120] Detection chamber 390 can be positioned distal to second
spring return 380, adjacent to closed end 328 of tube 322.
Detection chamber 390 can have proximal end 391, which in turn can
have piercing needle and sample collector 392, and distal end 394.
Piercing needle and sample collector 392 can have a pointed,
beveled, or barbed tip. In addition, the interior of piercing
needle and sample collector 392 can be in fluid communication with
the interior of detection chamber 390, such that a reaction sample
can be collected from recognition and amplification chamber 360 and
transferred to detection chamber 390 via collector 392. Detection
chamber 390 can contain a substrate for an enzyme marker such as a
substrate for horseradish peroxidase (HRP) (e.g., ABTS, TMB, OPD)
or alkaline phosphatase (AP) (e.g., PNNP).
[0121] Sample collector 310 and reaction unit 320 can be packaged
together and sold as a kit. In use, the sample collector 310 can be
removed from the package, and a swab can be obtained from, for
example, a subject's body. Cap 326 can be removed from open end 324
of tube 322, and sample collector 310 can be screwed into open end
324 such that all or a portion of swabber 318 extends through
proximal membrane 336 and into the interior of lysing and isolation
chamber 330. The sample can be mixed (e.g., by shaking), and the
lysing and nucleic acid preparation can proceed for a particular
length of time, or until reaction completion indicator 338
indicates that the user can proceed to the next reaction step.
[0122] When the nucleic acid sample is ready, the user can remove
first safety band 350 from reaction unit 320, and can actuate
reaction unit 320 such that piercing needle and sample collector
362 moves proximally to penetrate distal membrane 337 of lysing and
isolation chamber 330, collects a sample from chamber 330, and, by
virtue of first spring return 350, moves distally to its original
position. The sample can again be mixed, and the recognition and
amplification steps can proceed for a particular length of time, or
until reaction completion indicator 368 indicates that the user can
proceed to the next reaction step.
[0123] When the reaction sample is ready, the user can remove
second safety band 380 from reaction unit 320, and can actuate
reaction unit 320 such that piercing needle and sample collector
392 moves proximally to penetrate membrane 366 of recognition and
amplification chamber 360, collects a sample from chamber 360, and,
by virtue of second spring return 380, moves distally to its
original position. The sample can again be mixed, and marker
released during the amplification step can be detected (e.g.,
colorimetrically or fluorescently). In some cases, the outer
surface of tube 322 can have a color code printed thereon, so a
user can compare the color of detection chamber 390 with the color
code to determine whether or not the tested sample contains target
nucleic acid.
[0124] Device 300 can have any suitable dimensions. For example,
the size of device 300 can approximate that of a pen or a marker,
which can make it particularly convenient to transport. In some
cases, device 300 can have a diameter at its widest point of about
0.25 to about 2 cm (e.g., 0.25, 0.3, 0.4, 0.5, 0.6, 0.75, 0.8, 0.9,
1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 cm), and a
length of about 5 cm to about 200 cm (e.g., 5, 10, 15, 20, 25, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, or 200 cm).
[0125] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Formation and Cleavage of Target-Probe Hybrids
[0126] An oligonucleotide probe (5'-thiol-GGT AGT GCG AAA TGC CAT
TGC TAG TTG TTT-biotin-3'; SEQ ID NO:2) that was modified with a
thiol group at the 5' end and a biotin molecule at the 3' end was
conjugated to horseradish peroxidase (HRP). Conjugation was
performed using the SMCC reagent according to a technique modified
from Dill et al. (Biosensors and Bioelectronics, 20:736-742
(2004)). The HRP conjugate solution was incubated with a
streptavidin-coated ELISA plate to immobilize the
HRP-oligonucleotide probe to the surface via a biotin-streptavidin
interaction. The ELISA plate was then incubated with different
concentrations of a target oligonucleotide (5'-AAA CAA CTA GCA ATG
GCA TTT-3'; SEQ ID NO:3). The target oligonucleotide sequence was
reverse-complementary to the probe sequence to form a
double-stranded hybrid molecule. After washing, the plate was
incubated in a solution containing the restriction endonuclease
BfaI. BfaI specifically recognizes the sequence 5'-CTAG-3' and
cleaves the double-stranded, target-probe hybrids to release the
HRP-oligonucleotide into the reaction solution. After a two-hour
incubation at 37.degree. C., the reaction solution was transferred
to a new ELISA plate. The cleaved HRP-oligonucleotide was contacted
to 3,3',5,5'-tetramethyl benzidine (TMB) to form a colored reaction
product.
[0127] When the restriction endonuclease BfaI was added in excess
to the reaction mixture, a clear direct dependence between the
amount of released HRP-probe and the concentration of
oligonucleotide target was observed (FIG. 6A). The detectable
target concentration was approximately 1 nM. This detection limit
was obtained by direct measurement without any secondary signal
amplification. The addition of a restriction endonuclease signal
amplification cascade as described herein can further improve the
detection limit by several orders of magnitude.
[0128] When the HRP-oligonucleotide probes were pre-incubated with
an excess of target oligonucleotide (500 nM), the amount of cleaved
HRP-oligonucleotide probe was limited by the amount of recognition
restriction endonuclease BfaI (FIG. 6B). Taken together, these data
demonstrate that recognition restriction endonucleases can be used
to initiate the restriction endonuclease cascades described
herein.
Example 2
Detecting Target Nucleic Acid Using Probe Nucleic Acid and Reporter
Nucleic Acid
[0129] A target nucleic acid is selected. Once selected, target
nucleic acid is analyzed using a common genetic database such as
GenBank.RTM. and/or a computer-based sequence analysis program to
identify a portion of the target nucleic acid that contains a cut
site for a restriction endonuclease. Probe nucleic acid is designed
to be complementary to at least a portion of target nucleic acid
that contains a cut site. Once designed and obtained by standard
oligonucleotide synthesis methods, probe nucleic acid is conjugated
to an amplifying restriction endonuclease and immobilized to the
surface of a first well of a microtiter plate. A sample to be
tested is incubated in the first well. If target nucleic acid is
present in the sample, at least a portion of the target nucleic
acid hybridizes to the probe nucleic acid, and thereby forms a
recognition restriction endonuclease cut site. The recognition
restriction endonuclease is added to the first well having the
sample and probe nucleic acid. The microtiter plate is incubated at
37.degree. C. for an appropriate length of time for the cleavage
reaction to proceed.
[0130] Upon cleavage of probe nucleic acid by the recognition
restriction endonuclease, the reaction solution containing the
released portion of the probe nucleic acid is transferred into a
second well. The second well contains reporter nucleic acid that is
immobilized to the surface and contains at least one
double-stranded portion having an amplifying restriction
endonuclease cut site. Reporter nucleic acid also has a fluorescent
label. Upon transfer to the second chamber, the amplifying
restriction endonuclease bound to the released portion of the probe
nucleic acid contacts the reporter nucleic acid. The amplifying
restriction endonuclease cleaves reporter nucleic acid at the
double-stranded amplifying restriction endonuclease cut site to
form at least two portions. The liberated portion of the reporter
nucleic acid having the fluorescent label is moved to a third
microtiter plate well, and a standard fluorescent reader is used to
measure any fluorescent signal.
[0131] A standard curve of known amounts of target nucleic acid is
used to quantify the amount of target nucleic acid in the tested
sample.
Example 3
Detecting Target Nucleic Acid Using Probe Nucleic Acid, First
Signal Expansion Nucleic Acid, Second Signal Expansion Nucleic
Acid, and Reporter Nucleic Acid
[0132] Once selected, target nucleic acid is analyzed using a
common genetic database such as GenBank.RTM. and/or a
computer-based sequence analysis program to identify a portion of
target nucleic acid that contains a cut site for a restriction
endonuclease. Probe nucleic acid is designed based on the desired
target nucleic acid as described herein. Standard oligonucleotide
synthesis methods are used to make the probe nucleic acid, which is
then conjugated to an initial amplifying restriction endonuclease
and immobilized to the surface of a first well of a microtiter
plate. A sample to be tested for the target nucleic acid is
incubated in the first well. If target nucleic acid is present in
the sample, at least a portion of target nucleic acid hybridizes to
probe nucleic acid and thereby forms a recognition restriction
endonuclease cut site. Recognition restriction endonuclease is
added to the first well having the sample and probe nucleic acid.
The microtiter plate is incubated at 37.degree. C. for an
appropriate length of time for the cleavage reaction to
proceed.
[0133] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by the recognition restriction endonuclease, the reaction
solution containing the free portion of probe nucleic acid is
transferred to another well that includes first signal expansion
nucleic acid and second signal expansion nucleic acid. The first
signal expansion nucleic acid and second signal expansion nucleic
acid creates a positive feedback loop that causes an exponential
acceleration of release of initial amplifying restriction enzymes.
The reaction product from this well is transferred to another well
containing reporter nucleic acid, and cleavage of the reporter
nucleic acid is used to determine the presence, absence, or amount
of target nucleic acid in the sample. A standard curve of known
amounts of target nucleic acid is used to quantify the amount of
target nucleic acid in the tested sample.
Example 4
Detecting Methylated Cyclin D2 Promoter in Circulating Blood of
Breast Cancer Patients
[0134] The presence or absence methylated cyclin D2 promoter in
circulating blood of breast cancer patients can be indicative of
breast cancer lesions (Rykova et al., Ann. N.Y. Acad. Sci.,
1137:232-235 (2008)). The presence or absence of methylated cyclin
D2 promoter is determined in total circulating DNA (cirDNA) from
the blood that can include cell-free and cell-surface-bound DNA
fractions isolated as described elsewhere (e.g., Sunami et al.,
Methods Mol. Biol., 507:349-356 (2009)). The presence or absence
methylated cyclin D2 promoter is detected using an enzymatic
amplification cascade. The gene for cyclin D2 (CCDN2, G1/S-specific
cyclin-D2; Ensembl ID: ENSG00000118971) is located in the forward
strand of human Chromosome 12 at position 4,382,902-4,414,521. Its
promoter is composed of at least 7 fragments, and the longest 700
bp fragment (sequence ID ENSR00000172023; chromosome 12 positions
4386022-4386721) was analyzed using the Ensembl ("http" colon,
slash, slash "uswest" dot "ensemble" dot "org" slash "index" dot
"html") genetic database and CLC DNA Workbench software to identify
a portion of target sequence with a cut site for the MspI/HpaII
restriction endonucleases, which cleave at the 4 bp nucleotide
sequence 5'-CCGG-3'. A 40 nt probe nucleic acid
(5'-GTTTATTGGGGTGCTTTACCCCGGCTGTACACAGAAAGCC-3' (SEQ ID NO:4)) was
designed to be complementary to nucleotides 520 to 559 of the
selected target nucleic acid (chromosome 12 positions
4386542-4386581).
[0135] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid is conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of two wells of a microtiter plate. A sample of circulating DNA to
be tested is obtained from patient blood or body fluids, and added
to the two wells. If the cyclin D2 promoter sequence is present in
circulating DNA, it will bind to the probe in both wells thereby
forming a CCGG restriction site for the MspI recognition
restriction endonuclease, which is methylation insensitive, and the
HpaII recognition restriction endonuclease, which is methylation
sensitive. MspI and HpaII are either added or present in the first
and second wells, respectively, and they are allowed to cleave any
formed recognition restriction endonuclease cut sites by incubating
the microtiter plate at 37.degree. C. for an appropriate length of
time (e.g., 1 minute to 2 hours) for the cleavage reaction to
proceed.
[0136] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by MspI and HpaII, the reaction solutions in the first and
second wells are transferred to third and fourth wells,
respectively, both containing reporter nucleic acid that is
immobilized to the surface and that has at least one
double-stranded portion having an amplifying restriction
endonuclease NcoI cut site. The reporter nucleic acid can be a
double-stranded nucleic acid having a first strand (e.g.,
5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3' (SEQ ID NO:5)) and a
second strand (e.g., 5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3'
(SEQ ID NO:6)). The reporter nucleic acid also has a fluorescent
label. In some cases, first signal expansion nucleic acid and
second signal expansion nucleic acid are used prior to the reporter
nucleic acid step to increase the level of target nucleic acid
detection. The first signal expansion nucleic acid and second
signal expansion nucleic acid can include labels, in which case
they can be used together with reporter nucleic acid or in place of
reporter nucleic acid.
[0137] After transferring the reaction mixture to the third and
fourth wells, the amplifying restriction endonucleases of the
released portions of probe nucleic acid contact reporter nucleic
acid, and the microtiter plate is incubated at an appropriate
temperature (e.g., at 37.degree. C.) for an appropriate length of
time (e.g., 1 minute to 2 hours) for the cleavage reaction to
proceed. The amplifying restriction endonucleases cleave reporter
nucleic acid at the double-stranded amplifying restriction
endonuclease cut site to form at least two portions. The reaction
solutions of the third and fourth wells are transferred to fifth
and six wells, respectively, for fluorescence detection using a
fluorescent microtiter plate reader. The fluorescent signal in the
fifth well (corresponding to the MspI recognition restriction
endonuclease-treated well) is indicative of total amount of the
cyclin D1 promoter in the circulating blood. The fluorescent signal
in the sixth well (corresponding to the HpaII recognition
restriction endonuclease-treated well) is indicative of the amount
of unmethylated cyclin D1 promoter in the circulating blood. If the
latter signal is smaller than the former signal then at least a
part of the corresponding cyclin D2 promoter DNA is methylated. The
proportion of methylated promoter can be calculated as a difference
between the signal in the fifth well minus the signal in the sixth
well.
Example 5
Detecting Methylated RASSF1A Promoter in Circulating Blood of
Hepatocellular Carcinoma and Lung Cancer Patients
[0138] The presence or absence methylated RASSF1A promoter in
circulating blood of hepatocellular carcinoma and lung cancer
patients is indicative of tumor growth and disease progression (Di
Gioia et al., BMC Cancer, 6:89 (2006); Fischer et al., Lung Cancer,
56:115-123 (2007); and Allen Chan et al., Clin. Chem., 10:1373
(2008)). The presence or absence of methylated RASSF1A promoter is
determined in total circulating DNA (cirDNA) from the blood that
can include cell-free and cell-surface-bound DNA fractions isolated
as described elsewhere (e.g., Sunami et al., Methods Mol. Biol.,
507:349-356 (2009)).
[0139] The presence or absence methylated RASSF1A promoter is
detected using an enzymatic amplification cascade. The gene for
RASSF1A (Ras association domain-containing protein 1; Ensembl ID:
ENSG00000068028) is located in the reverse strand of Chromosome 3
at positions 50,367,217-50,378,411. Its promoter is composed of at
least three fragments, and the longest 1,702 bp fragment (sequence
ID ENSR00000059407, Chromosome 3:50369843-50371544) was analyzed
using the Ensembl (http://uswest.ensembl.org/index.html) genetic
database and CLC DNA Workbench software to identify a portion of
target sequence with cut sites for two restriction endonucleases,
PstI and SmaI. PstI cleaves at the 6 bp nucleotide sequence
5'-CTGCAG-3', and a 40 nt probe nucleic acid
(5'-AGTCCGAGTCCTCTTGGCTGCAGTAGCCACTGCTCGTCGT-3' (SEQ ID NO:7)) was
designed to be complementary to nucleotides 503 to 542 of the
selected target nucleic acid (Chromosome 3:50370346-50370385). SmaI
cleaves at the 6 bp nucleotide sequence 5'-CCCGGG-3', and a 40 nt
probe nucleic acid (5'-GTGTCAGTGTGCGCGTGCGCCCGGGCCAGAGCCGCGCCGC-3'
(SEQ ID NO:8)) was designed to be complementary to nucleotides 746
to 785 of the selected target nucleic acid (Chromosome
3:50370589-50370628). The PstI cut site is not methylated, and the
SmaI cut site is a CpG island that can be methylated in cancer
cells, and its methylation blocks cleavage by SmaI.
[0140] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acids are conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of two wells of a microtiter plate. A sample of circulating DNA to
be tested is obtained from patient blood or body fluids, and added
to the two wells. If the RASSF1A promoter sequence is present in
circulating DNA, it will bind to the probe nucleic acid in both
wells thereby forming restriction sites for PstI and for SmaI in
the first and second wells, respectively. PstI and SmaI are either
added or present in the first and second wells, respectively, and
they are allowed to cleave any formed recognition restriction
endonuclease cut sites by incubating the microtiter plate at
37.degree. C. for an appropriate length of time (e.g., 1 minute to
2 hours) for the cleavage reaction to proceed.
[0141] After cleavage of the probe nucleic acid:target nucleic acid
hybrids by PstI or SmaI, the reaction solutions in the first and
second wells are transferred to third and fourth wells,
respectively, both containing reporter nucleic acid that is
immobilized to the surface and that has at least one
double-stranded portion having an amplifying restriction
endonuclease NcoI cut site. The reporter nucleic acid can be a
double-stranded nucleic acid having a first strand (e.g.,
5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3' (SEQ ID NO:5)) and a
second strand (e.g., 5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3'
(SEQ ID NO:6)). The reporter nucleic acid also has a fluorescent
label. In some cases, first signal expansion nucleic acid and
second signal expansion nucleic acid are used prior to the reporter
nucleic acid step to increase the level of target nucleic acid
detection. The first signal expansion nucleic acid and second
signal expansion nucleic acid can include labels, in which case
they can be used together with reporter nucleic acid or in place of
reporter nucleic acid.
[0142] After transferring the reaction mixture to the third and
fourth chambers, the amplifying restriction endonucleases of the
released portions of probe nucleic acid contact reporter nucleic
acid, and the microtiter plate is incubated at an appropriate
temperature (e.g., at 37.degree. C.) for an appropriate length of
time (e.g., 1 minute to 2 hours) for the cleavage reaction to
proceed. The amplifying restriction endonucleases cleave reporter
nucleic acid at the double-stranded amplifying restriction
endonuclease cut site to form at least two portions. The reaction
solutions of the third and fourth wells are transferred to fifth
and six wells, respectively, for fluorescence detection using a
fluorescent microtiter plate reader. The fluorescent signal in the
fifth well (corresponding to the PstI recognition restriction
endonuclease-treated well) is indicative of total amount of the
RASSF1A promoter in the circulating blood. The fluorescent signal
in the sixth well (corresponding to the SmaI recognition
restriction endonuclease-treated well) is indicative of the amount
of unmethylated RASSF1A promoter in the circulating blood. If the
latter signal is smaller than the former signal then at least a
part of the corresponding RASSF1A promoter DNA is methylated. The
proportion of methylated promoter can be calculated as the
difference between the signal in the fifth well minus the signal in
the sixth well.
Example 6
Assessing Alleles of the Thiopurine S-Methyltransferase Gene Based
on a Sequence that Creates/Destroys a Restriction Endonuclease
Site
[0143] Thiopurine S-methyltransferase (EC 2.1.1.67) enzyme (TPMT)
is a drug-metabolizing enzyme that catalyzes the S-methylation of
thiopurine drugs such as 6-mecaptopurine and azathioprine that are
used to treat childhood leukemia, autoimmune diseases, and
transplant recipients (Wang et al., Proc. Natl. Acad. Sci. USA,
102(26):9394-9399 (2005)). These drugs can have potentially
life-threatening drug-induced toxicity, depending on the levels of
the TPMT enzyme in patient's tissues. Large individual variations
in levels of TPMT activity are regulated primarily by common
genetic polymorphisms with several most common alleles. Some of
these alleles can result in a virtual lack of TPMT enzyme activity,
and correspondingly, patients homozygous for these alleles can
suffer severe, life-threatening toxicity when treated with standard
doses of thiopurines.
[0144] The known SNP variants of the TPMT gene (Ensemble gene ID
ENSG00000137364; positioned on Chromosome 6: 18,128,542-18,155,305
reverse strand) were analyzed using the Ensembl ("http" colon,
slash, slash, "uswest" dot "ensemble" dot "org" slash "index" dot
"html") genetic database and CLC DNA Workbench to determine whether
these SNPs create or destroy restriction sites. One of these SNPs
(Ensembl ID rs72552739; chromosome 6 position 18143901, C to A
substitution causing premature stop codon and truncated 98-amino
acid truncated polypeptide) was selected since this C/A
substitution destroyed an ApoI restriction site 5'-AAATTC-3'
present in un-mutated DNA (chromosome 6 positions
18143896-18143901). Another restriction site for BfuI, 5'-GTATCC-3'
was found to be present in both mutated and un-mutated target DNA
(chromosome 6 positions 18143904-18143909). Two probes (P1 and P2)
were designed to be complementary to nucleotides 18143882-18143922
of the selected target nucleic acid
(5'-TTCTGCTCTGTAAAAAATTcTTGTATCCCAAGTTCACTGAT-3' (P1; SEQ ID NO:9)
and 5'-TTCTGCTCTGTAAAAAATTaTTGTATCCCAAGTTCACTGAT-3' (P2; SEQ ID
NO:10)), one corresponding to the un-mutated (P1), and another to
mutated DNA (P2), respectively (the SNP positions are shown with
lowercase letters).
[0145] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid P1 is conjugated to an
amplifying restriction endonuclease (NcoI) and immobilized to the
surface of two wells of a microtiter plate. A sample of genomic DNA
to be tested is obtained from a patient's blood (with or without
PCR amplification and single-stranded target preparation), and
added to the two wells. If the target nucleic acid for P1 is
present in the genomic DNA, it will bind to P1 in both wells
thereby forming restriction sites for recognition restrictases ApoI
and BfuI. ApoI is added to the first well, and BfuI is added to the
second well, and they are allowed to cleave any formed recognition
restriction endonuclease cut sites by incubating the microtiter
plate at 37.degree. C. for an appropriate length of time (e.g., 1
minute to 2 hours) for the cleavage reaction to proceed.
[0146] After cleavage of the P1:target nucleic acid hybrids by ApoI
or BfuI, the reaction solutions in the first and second wells are
transferred to third and fourth wells, respectively, both
containing reporter nucleic acid that is immobilized to the surface
and that has at least one double-stranded portion having an
amplifying restriction endonuclease NcoI cut site. The reporter
nucleic acid can be a double-stranded nucleic acid having a first
strand (e.g., 5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3' (SEQ ID
NO:5)) and a second strand (e.g.,
5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3' (SEQ ID NO:6)). The
reporter nucleic acid also has a fluorescent label. In some cases,
first signal expansion nucleic acid and second signal expansion
nucleic acid are used prior to the reporter nucleic acid step to
increase the level of target nucleic acid detection. The first
signal expansion nucleic acid and second signal expansion nucleic
acid can include labels, in which case they can be used together
with reporter nucleic acid or in place of reporter nucleic
acid.
[0147] After transferring the reaction mixture to the third and
fourth chambers, the amplifying restriction endonucleases of the
released portions of P1 contact reporter nucleic acid, and the
microtiter plate is incubated at an appropriate temperature (e.g.,
at 37.degree. C.) for an appropriate length of time (e.g., 1 minute
to 2 hours) for the cleavage reaction to proceed. The amplifying
restriction endonucleases cleave reporter nucleic acid at the
double-stranded amplifying restriction endonuclease cut site to
form at least two portions. The reaction solutions of the third and
fourth wells are transferred to fifth and six wells, respectively,
for fluorescence detection using a fluorescent microtiter plate
reader. The fluorescent signal in the fifth well is indicative of
the amount of un-mutated ApoI cleaved TPMT allelic variant in the
sample. The TPMT allelic variant containing the C/A SNP, if
present, is not cleaved and doesn't contribute to the signal in the
fifth well. The fluorescent signal in the sixth wells is indicative
of total amount of the target TPMT nucleic acid in the sample, both
mutated and un-mutated. Thus, the allelic composition of the
patient's genotype in terms of the corresponding SNP can be
evaluated from the ratio of un-mutated TPMT allele to total amount
of the TPMT nucleic acid (signal in the fifth well versus signal in
the sixth well). A ratio of approximately 0.5 is indicative of
heterozygosity. A ratio of approximately 1 is indicative of
homozygosity of the un-mutated allele, and a ratio close to zero is
indicative of homozygosity of the mutated allele.
Example 7
Assessing Alleles of the Thiopurine S-Methyltransferase Gene Based
on a Sequence that does not Appear to Create/Destroy a Restriction
Endonuclease Site
[0148] An allele of the TPMT gene (Ensemble gene ID
ENSG00000137364; positioned on Chromosome 6: 18,128,542-18,155,305
reverse strand) was selected to design an enzymatic amplification
cascade of restriction endonucleases using a recognition
restriction endonuclease that has separate recognition and cleavage
sites (FokI) since the SNP of this allele does not appear to create
or destroy a restriction site. The SNP (Ensembl ID rs1800460;
chromosome 6 position 18139228, C to T substitution causing
non-synonymous substitution in the codon 154) is one of the most
common variant alleles (Wang et al., Proc. Natl. Acad. Sci. USA,
102(26):9394-9399 (2005)). The corresponding nucleic acid sequence
was analyzed using the Ensembl ("http" colon, slash, slash,
"uswest" dot "ensemble" dot "org" slash "index" dot "html") genetic
database and CLC DNA Workbench to select the SNP site (position
18139228) and flanking sequences, 8 nucleotides upstream (5'
direction, 18139220-18139227), and 31 nucleotides downstream (3'
direction, 18139229-18139259). These sequences are used to design
the single stranded parts of two probe nucleic acids (P1 and P2),
one corresponding to the non-mutated genetic element (P1,
5'-ATAGAGGAcCATTAGTTGCCATCAATCCAGGTGATCGCAA-3'; (SEQ ID NO:11)),
and one corresponding to the mutated genetic element (P2,
5'-ATAGAGGAtCATTAGTTGCCATCAATCCAGGTGATCGCAA-3' (SEQ ID NO:12)). The
common double-stranded part of the P1 and P2 probe nucleic acids
contained a 15-bp spacer followed by the FokI recognition site:
5'-CATTGCGCGCCTAGTGGATG-3' (SEQ ID NO:13) as shown in FIG. 13.
[0149] Once designed and obtained by standard oligonucleotide
synthesis methods, the probe nucleic acids are conjugated to an
amplifying restriction endonuclease (NcoI) and immobilized to the
surface of two wells of a microtiter plate. The first and second
wells thus contain P1 for the un-mutated DNA and P2 for the mutated
DNA, respectively. A sample of genomic DNA to be tested is obtained
from a patient's blood (with or without PCR amplification and
single-stranded target preparation), and added to the two wells. If
the target nucleic acid is present in the genomic DNA, it can
hybridize to the probe nucleic acid in the wells and create a FokI
cleavage site with its corresponding probe nucleic acid. The
protruding ends of target nucleic acid are removed using blunting
by adding T4 DNA polymerase, and then the blunted nucleic acid is
ligated to the adjacent available strand of the probe nucleic acid
using E. coli DNA ligase by incubating the microtiter plate at 20
to 37.degree. C. for an appropriate length of time (e.g., 1 minute
to 2 hours) for the enzymatic reactions to proceed. FokI is added
to both first and second wells, and is allowed to cleave any formed
cleavage sites by incubating the microtiter plate at 37.degree. C.
for an appropriate length of time (e.g., 1 minute to 2 hours) for
the cleavage reaction to proceed. FokI is only able to cleave the
perfect match between the probe nucleic acid and target nucleic
acid, and not a single nucleotide mismatch at the cleavage site.
After cleavage of the probe nucleic acid:target nucleic acid
hybrids by FokI, the reaction solutions in the first and second
wells are transferred to third and fourth wells, respectively, both
containing reporter nucleic acid that is immobilized to the surface
and that has at least one double-stranded portion having an
amplifying restriction endonuclease NcoI cut site. The reporter
nucleic acid can be a double-stranded nucleic acid having a first
strand (e.g., 5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3' (SEQ ID
NO:5)) and a second strand (e.g.,
5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3' (SEQ ID NO:6)). The
reporter nucleic acid also has a fluorescent label. In some cases,
first signal expansion nucleic acid and second signal expansion
nucleic acid are used prior to the reporter nucleic acid step to
increase the level of target nucleic acid detection. The first
signal expansion nucleic acid and second signal expansion nucleic
acid can include labels, in which case they can be used together
with reporter nucleic acid or in place of reporter nucleic
acid.
[0150] After transferring the reaction mixture to the third and
fourth chambers, the amplifying restriction endonucleases of the
released portions of probe nucleic acid contact reporter nucleic
acid, and the microtiter plate is incubated at an appropriate
temperature (e.g., at 37.degree. C.) for an appropriate length of
time (e.g., 1 minute to 2 hours) for the cleavage reaction to
proceed. The amplifying restriction endonucleases cleave reporter
nucleic acid at the double-stranded amplifying restriction
endonuclease cut site to form at least two portions. The reaction
solutions of the third and fourth wells are transferred to fifth
and six wells, respectively, for fluorescence detection using a
fluorescent microtiter plate reader. The fluorescent signal in the
fifth wells is indicative of the amount of the un-mutated TPMT
target nucleic acid in the sample. The fluorescent signal in the
sixth well is indicative of the amount of mutated TPMT target
nucleic acid in the sample. Thus, the allelic composition of the
patient's genotype in terms of the corresponding SNP can be
evaluated from the ratio of un-mutated TPMT allele to mutated TPMT
allele (signal in the fifth well versus signal in the sixth well).
The ratio of approximately 1 is indicative of heterozygosity. If
the signal in the fifth well greatly exceeding the one in the sixth
well, then the results are indicative of homozygosity for the
un-mutated allele, and the opposite is indicative of homozygosity
for the mutated allele.
Other Embodiments
[0151] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
13140DNAArtificial Sequencenucleic acid containing cleavage
sitemisc_feature6-20 and 26-40n = any nucleic acid 1ggatgnnnnn
nnnnnnnnnn cctacnnnnn nnnnnnnnnn 40230DNAArtificial Sequencethiol
and biotin containing nucleic acid 2ggtagtgcga aatgccattg
ctagttgttt 30321DNAArtificial Sequencepartial complement of SEQ ID
NO2 3aaacaactag caatggcatt t 21440DNAHomo sapiens 4gtttattggg
gtgctttacc ccggctgtac acagaaagcc 40536DNAArtificial Sequencenucleic
acid containing cleavage site 5cattgctagt tgtttccatg gggtagtgcg
aaatgc 36636DNAArtificial Sequencepartial complement of SEQ ID NO5
6gcatttcgca ctaccccatg gaaacaacta gcaatg 36740DNAHomo sapiens
7agtccgagtc ctcttggctg cagtagccac tgctcgtcgt 40840DNAHomo sapiens
8gtgtcagtgt gcgcgtgcgc ccgggccaga gccgcgccgc 40941DNAHomo sapiens
9ttctgctctg taaaaaattc ttgtatccca agttcactga t 411041DNAHomo
sapiens 10ttctgctctg taaaaaatta ttgtatccca agttcactga t
411140DNAHomo sapiens 11atagaggacc attagttgcc atcaatccag gtgatcgcaa
401240DNAHomo sapiens 12atagaggatc attagttgcc atcaatccag gtgatcgcaa
401320DNAArtificial Sequencespacer containing FokI restriction site
13cattgcgcgc ctagtggatg 20
* * * * *
References