Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHOD OF ELECTROCHEMICAL DETECTION OF SOMATIC CELL
MUTATIONS
Related Applications
This application claims priority from U.S. Pat. Application No. 60/424,656
entitled
UNIVERSAL TAG ASSAY filed November 6, 2002. This application also claims
priority
from, and is a continuation-in-part application of U.S. Pat. Application
Serial No.
10/424,542 entitled "UNIVERSAL TAG ASSAY," filed April 24, 2003. The subject
matter of the aforementioned applications is hereby incorporated by reference
in its entirety.
Background of the Invention
Field of the Invention
The present invention relates to the detection of genetic mutations in somatic
cells
and methods of screening patients for cancer or precancer.
Description of the Related Art
Somatic cells are definitionally distinguished from germ cells as the former
are the
cells which make up an individual's body while the latter are those cells
which can
participate in sexual reproduction. Both types of cells can experience genetic
mutations
under a variety of circumstances. Mutations in somatic cells are typically not
passed to an
individual's offspring; they are, however, often passed within an individual
to the daughter
cells of the mutated somatic cell through mitosis. The frequency with which
somatic cells
reproduce is generally related to the type of cell and to various
abnormalities caused by
genetic mutations in the cell. The propagation of somatic cell mutations is a
principal
mechanism behind most cancers.
If a mutation in a somatic cell increases the rate of its reproduction in an
uncontrolled manner, then the number of daughter cells may increase rapidly in
that area.
When this occurs, the daughter cells often divide before reaching their mature
state. This
can result in an ever increasing number of cells that have no beneficial
function to the body,
yet absorb body nutrition at an increasing rate. Tissue of this type may be
referred to as a
tumor. If the cells remain in their place of origin and do not directly invade
surrounding
tissues, the tumor is said to be "benign." If the tumor invades neighboring
tissue and
causes distant secondary growths (called metastasis), it may be termed
"malignant."
The adverse health consequences of a tumor depend on the tissue in which it is
located, how rapidly it grows, and how quickly it is detected and treated.
Numerous
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environmental factors can contribute to somatic cell mutations, though
mutations can arise
spontaneously as well. Mutations owing to either origin may ultimately produce
tumors.
Various techniques for detecting mutations are known in the art. For example,
techniques for detecting colorectal cancer are disclosed in U.S. Pat. Nos.
5,741,650
(Lapidus, et al.); 5,834,181 (Shuber); 5,849,483 (Shuber); 5,952,178 (Lapidus
et al.);
6,268,136 (Shuber et al.); 6,303,304 (Shuber et al.); 6,428,964 (Shuber); all
of which are
hereby incorporated by reference in their entirety.
Present techniques for studying somatic cell mutations typically allow
identification
of an affected genetic region to within 10 to 1000 base pairs, while the
precise position and
nature of the nucleotide change remain elusive. Presently, there is no
reliable, inexpensive
method for rapidly locating and characterizing genetic mutations occurring in
somatic cells.
Such a method could facilitate earlier and more effective treatments for
patients
having, or at risk of developing, mutation-related disorders, including
various cancers.
Accordingly, what is needed in the art is a method that is capable of
identifying the location
and nature of nucleotide changes that occur within somatic cells.
Summary of the Invention
One aspect of the invention is a method for detecting a target polynucleotide,
including the steps of synthetically producing an enlarged target
polynucleotide;
hybridizing the target polynucleotide to a probe polynucleotide in a detection
zone; and
detecting the amount of polynucleotide in the detection zone to ascertain
whether target
polynucleotide has hybridized in the detection zone. In some cases, target
polynucleotide is
enlarged by attaching one or more polynucleotide strands to the target.
Further the target
polynucleotide can be enlarged by attachment of a plurality of polynucleotide
strands,
producing a branched structure. In some embodiments, the target polynucleotide
is
hybridized to more than one probe polynucleotide in the detection zone.
A further aspect of the invention is a method for detecting a nucleic acid
analyte,
including: generating an elongated reporter nucleic acid if the nucleic acid
analyte is
present; capturing the reporter nucleic acid with an innnobilized probe that
is substantially
shorter than the reporter nucleic acid; and generating a signal that is a
function of the size of
the captured reporter nucleic acid to indicate the presence or absence of the
nucleic acid
analyte.
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Brief Description of the Drawings
FIG. lA depicts short strand duplex melting curves.
FIG. 1B depicts long strand duplex melting curves.
FIG. 1C depicts melting curves in which an elongated target strand is
hybridized to
multiple short strand probes.
FIG. 2A illustrates on-chip amplification using head-to-tail polymerization.
FIG. 2B illustrates on-chip amplification using rolling circle amplification.
FIG. 2C illustrates on-chip amplification using a branch technique in
conjunction
with rolling circle amplification.
FIG. 3 shows a voltammagram which illustrates the signal enhancing effect of
on-
chip amplification.
Detailed Description of the Preferred Embodiments
The present invention is generally related to the detection of somatic cell
mutations.
Preferred embodiments include the isolation of oligonucleotides from
biological samples
and an analysis of various oligonucleotide sequences for complementarity using
an
electrochemical hybridization assay. Accordingly, the knowledge that an
oligonucleotide of
unknown sequence is complementary to an oligonucleotide of known sequence can
be used
to identify the unknown sequence. Similarly, comparing to an oligonucleotide
of unknown
sequence to an oligonucleotide known to be healthy or "wild" can be used to
characterize
the unknown sequence as either wild or mutated.
Various techniques for isolating oligonucleotides and conducting hybridization
assays are described in copending U.S. Pat. Application Serial No. 60/424656,
filed
November 6, 2002; U.S. Pat. Application Serial No. 10/424,542 entitled
"UNIVERSAL
TAG ASSAY," filed April 24, 2003; U.S. Pat. Application Serial No. 101429,291
entitled
"ELECTROCHEMICAL METHOD TO MEASURE DNA ATTACHMENT TO AN
ELECTRODE SURFACE IN THE PRESENCE OF MOLECULAR OXYGEN," filed May
2, 2003; all of which are hereby incorporated by reference in their entirety.
Preferred embodiments of the present invention include the detection of
polynucleotide hybridization in a detection zone. Particularly preferred
embodiments
feature the use of a ruthenium complex in conducting an electrochemical assay.
Preferably,
such an electrochemical assay detects nucleic acid hybridization using the
general technique
of Steele et al. (1998, Anal. Chem. 70:4670-4677), hereby expressly
incorporated by
reference in its entirety.
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Typically, in carrying out this technique, a plurality of nucleic acid probes
which are
complementary to a sequence of interest are used. In certain preferred
embodiments,
probes range in length from about 10 to 25 base pairs, with a length of about
17 base pairs
being most preferred. Preferably, the probe strands are positioned within a
detection zone.
In particularly preferred embodiments, the detection zone includes a surface,
such as an
electrode, in contact with a liquid medium, wherein the probe strands are
immobilized on
the surface such they are also in contact with the liquid medium. Preferably,
the surface is a
gold or carbon electrode that is coated with a protein layer such as avidin or
streptavidin to
facilitate the attachment of the nucleic acid probe strands to the electrode.
This protein
layer should be porous, such that it allows ions to pass from the liquid
medium to the
electrode and vice versa. When attaching a probe strand to an avidin layer, it
is preferable
to first bind the probe strand covalently to a biotin complex and then allow
the biotin to
attach to the avidin. Alternatively, probe strands can be attached directly to
the surface, for
example by using a thiol linkage to covalently bind nucleic acid to a gold
electrode.
Carbon electrodes or electrodes of any other suitable conductor can also be
used.
In further carrying out this technique, a target strand (a nucleic acid sample
to be
interrogated relative to the probe) can be contacted with the probe in any
suitable manner
known to those skilled in the art. For example, a plurality of target strands
can be
introduced to the liquid medium described above and allowed to intermingle
with the
immobilized probes. Preferably, the number of target strands exceeds the
number of probe
strands in order to maximize the opportunity of each probe strand to interact
with target
strands and participate in hybridization. If a target strand is complementary
to a probe
strand, hybridization can take place. Techniques for adjusting the stringency
of
hybridization and techniques for detecting hybridization are also discussed
herein.
Further, embodiments of the present invention can include any combination of
the
following steps: extracting a biological sample from a patient, purifying a
nucleic acid from
a biological sample, amplifying a nucleic acid, isolating a nucleic acid in
single stranded
form, cyclizing a nucleic acid, elongating a nucleic acid, controlling
hybridization
stringency, amplifying the nucleic acid on a chip, and detecting
hybridization. Accordingly,
preferred embodiments for each of these steps are discussed in the following
sections.
Tn the present disclosure, references to extracting an oligonucleotide from a
patient
typically refer to obtaining a sequence that will form the basis of a target
strand. However,
in many embodiments, the same techniques, or those which are similar, will
also be
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appropriate for obtaining a sequence that will form the basis of a probe
strand. Those of
skill in the art will recognize that various biological and/or artificial
sources of
oligonucleotides are available and will be able to decide which are most
suitable for
creating probes or targets depending on the particular goals of the assay to
be conducted.
Extracting a Biological Sample
In accordance with the present invention, a variety of methods for extracting
nucleic
acid from various biological samples from a patient can be used. Biological
samples that
are useful in the present invention can include any sample from a patient in
which a nucleic
acid is present. Such samples can be prepared from a any tissue, cell, or body
fluid.
Examples of biological cell sources include blood cells, colon cells, buccal
cells,
cervicovaginal cells, epithelial cells from urine, fetal cells or cells
present in tissue obtained
by biopsy. Exemplary tissues or body fluids include sputum, pancreatic fluid,
bile, lymph,
plasma, urine, cerebrospinal fluid, seminal fluid, saliva, breast nipple
aspirate, pus,
amniotic fluid and stool. Useful biological samples can also include isolated
nucleic acid
from a patient. Nucleic acid can be isolated from any tissue, cell, or body
fluid using any of
numerous methods that are standard in the art.
In some preferred embodiments, a stool sample is taken from a patient as part
of a
method of screening for colorectal cancer. In particular, methods of
extracting biological
samples from stool are described in U.S. Pat. No. 5,741,650 (Lapidus et al.),
herein
incorporated by reference. Lapidus et al. teach sectioning a stool sample to
extract cells
and cellular debris that may be indicative of cancer or precancer. Such a
method can be
used to obtain biological material containing a nucleic acid for further use
in accordance
with the present invention.
Purifyin~ Nucleic Acid Frorn a Sample
Once a sample has been extracted from a patient, a variety of techniques can
be used
to purify nucleic acid. Suitable nucleic acids can include DNA and RNA. The
particular
nucleic acid purification method will typically depend on the source of the
patient sample.
Techniques for purifying nucleic acid are known in the art and can include the
use of
homogenization, centrifugation, extraction with various solvents,
chromatography,
electrophoresis, and other known techniques.
In some preferred embodiments, the biological sample is a stool sample and
nucleic
acid from colorectal tissue is isolated and purified from stool cross sections
according to
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methods disclosed in U.S. Pat. No. 6,406,857 (Shuber et al.), hereby expressly
incorporated
by reference in its entirety.
Amplification of Nucleic Acid
Various techniques which are known in the art can be used to amplify a nucleic
acid
when practicing the present invention. RCA is one technique that can be used,
through
PCR is preferred. It is particularly advantageous to use a "digital PCR"
technique. Digital
PCR refers to a PCR method in which a liquid sample containing nucleic acids
of interest is
so thoroughly diluted and partitioned that each partition contains at most one
nucleic acid
molecule. Accordingly, if subsequent PCR amplification on a partition is
successful, all of
the resulting strands will be derived from one strand. Hence all of the PCR
products for a
given partition will be identical. Because the partitions themselves are
unlikely to be
identical to all the other partitions, it will often be advantageous to study
those partitions
found to contain nucleic acids in separate assays to determine which warrant
further
attention.
Digital PCR is discussed in greater detail in Vogelstein et al. "Digital PCR,"
Proc.
Natl. Acad. Sci. USA, Vol. 96, pp. 9236-41, August 1999; which is hereby
incorporated by
reference in its entirety.
Isolating Single Stranded Nucleic Acid
Various techniques are known in the art for producing or isolating single
stranded
nucleic acid from samples containing double stranded nucleic acid.
In one preferred method, single stranded nucleic acid is isolated using a
streptavidin-coated bead. In performing this technique, an amplification
product is
denatured to generate single-stranded products, wherein at least one strand
contains an
addressable ligand at one terminus. In some preferred embodiments, a
biotinylated single-
stranded PCR product having a copy of the nucleotide sequence of interest is
incubated
with streptavidin-coated beads, under conditions such that the biotinylated
PCR product is
attached to a bead, forming a bead-target sequence complex.
In other preferred embodiments, one strand of a double stranded nucleic acid
is
removed, for example, by selective exonuclease digestion. The remaining single
stranded
nucleic acid can further be used in accordance with the present invention.
Cyclizing the Nucleic Acid and Performin~RCA
In performing an assay in accordance with the present invention, it is
possible to
cyclize and elongate the target nucleic acid prior to hybridization.
"Cyclization" generally
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refers to the process of creating a polynucleotide circle (preferably
containing a particular
sequence), while "elongation" generally refers to the process of increasing
the length of a
polynucleotide. In preferred embodiments, elongation includes a rolling circle
amplification (RCA) step with an appropriate polynucleotide circle and is used
to create a
long strand of target nucleic acid.
In particular, cyclization and elongation can be used to generate one or more
long
target strands in which a sequence being interrogated is repeated several
times. Effectively,
many copies of a small target strand are linked end to end to generate a large
target strand.
Although cyclization/elongation can be used to add as little as one
repetition, it is generally
preferred that multiple repetitions be added, for example, approximately 10,
50, 100, 250,
500; 750, or 1000 repetitions or more may be attached. Circle size is also
adjustable
according to the requirements of the assay. Preferred circle sizes are in the
range of about
40 to about 1000 base pairs, with about 800 base pairs being most preferred.
Notably, the
number of repetitions selected can depend on the length of the circle being
used.
Specifically, it will generally be preferable to use more repetitions with
smaller circles and
fewer repetitions with larger circles so that the strands produced will be
appropriately
manageable and functional according to the demands of the assay.
Generally, any one of the many repetitions of the sequence on a large strand
would
be able to hybridize to a probe just as if that sequence were alone on a
standard short target
strand. Further, just one large target strand can generally hybridize to
multiple probes (by
coiling back toward the electrode surface and allowing another identical
region of the long
strand to attach to another complementary probe).
Elongation and the use of long target strands has various advantages.
Particularly
favorable advantages are related to stringency. "Stringency" refers to a
measurement of the
ease with which various hybridization events can occur. For example, two
strands that are
perfectly complementary generally form a more stable hybrid than two strands
that are not.
Various stringency factors (such as temperature, pH, or the presence of a
species able to
denature various hybridized strands) can be adjusted such that in a single
environment, the
perfectly complementary pair would stay together while the imperfect pair
would fall apart.
Ideal conditions are generally those which strilce a balance between
minimizing the number
of hybridizations between noncomplementary strands (false positives) and
minimizing the
number of probes which remain unhybridized despite the presence of eligible
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complementary target strands (false negatives). Other various techniques for
controlling
stringency are also discussed in the next section.
Elongation is one technique that is useful in improving the effectiveness of
temperature as a stringency factor. A perfect hybrid is typically more stable
than an
imperfect hybrid and will outlast the imperfect hybrid when the temperature is
increased.
However, dehybridization in either case is not a single event when dealing
with populations
of molecules. Instead, more and more molecules give up the hydrogen bonds that
hold
opposing base pairs together over a range of temperature. Perfect hybrids
outlast imperfect
hybrids, but it is often very difficult, if not impossible, to find a single
temperature at which
there are no imperfect hybrids while perfect hybrids abound.
It has been discovered that longer nucleic acid molecules exhibit a less
gradual
transition between their hybridized and unhybridized states when the
temperature is
. changed. This is to say that the melt curve for a given population of
molecules is steeper
and more decisive when the nucleic acid strand is longer. However, the
distance between
the curves of perfect and imperfect hybrids of equivalent length tend to crowd
in a smaller
temperature range, frustrating the initial attempt to create,a stringency
environment that will
distinguish between them.
FIGS. lA and 1B illustrate this phenomenon. FIG. lA shows the melt curves of
matched and mismatched short strand duplexes. FIG. 1B shows the melt curves
for longer
strands. FIG. lA has a large ~ Tm, but the gradual melt of the duplexes makes
it difficult
to select and maintain a temperature range that has a maximum specificity
ratio. FIG. 1B
has steeper and more decisive melting curves, but the 0 Tm is very small,
again making it
difficult to select and maintain a temperature range that allows maximum
specificity.
It has further been discovered that the use of elongated target strands which
can
hybridize to multiple probes enable a larger stringency range. In other words,
the melt
curves are steeper (than those of the short molecules) and that the distance
between the
melting temperatures of perfect and imperfect strands are farther apart (than
those of the
long molecules). This type of hybridization is depicted in FIG. 1C. As shown,
the melt
curves are steep and the ~ Tm is large. This combination facilitates improved
specificity in
the assay because of the large temperature range in which matched duplexes
generally exist
and mismatched duplexes do not.
Accordingly, some embodiments of the present invention include cyclization and
elongation steps to produce a target strand of increased length. Preferably, a
sequence is
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repeated several times on the target strand such that one target strand can
participate in
hybridization with more than one immobilized probe. As this can be used to
create a larger
temperature window in which perfect hybrids remain and imperfect hybrids fall
apart, it is
advantageous to adjust the temperature of the assay environment to minimize
false
positives as well as false negatives.
In some embodiments, it is advantageous to perform an assay in which
hybridization is evaluated at two or more different temperatures. For example,
where a
duplex polynucleotide with a single base mismatch has a melting temperature
Tml and a
duplex polynucleotide with no base mismatch has a higher melting temperature
T",a, it is
possible to first detect whether the duplex exists at a temperature below Tml,
then increase
the temperature of the assay environment above Tmr to detect whether the
duplex exists at a
temperature between Tml and T,,.,z. In this case, the results of such an assay
could indicate
whether a single base mismatch exists in the duplex being interrogated. In
some cases, a
determination of the temperature at which a duplex falls apart can be used to
evaluate the
quantity, type, and/or location of mismatches, if any. Various techniques for
detecting
hybridization are discussed irzf~a.
Those of skill in the art will appreciate that other techniques to elongate
nucleic
acids, including for example, head-to-tail polymerization, can also be used to
achieve
favorable results with regard to temperature stringency.
, In some embodiments, it is advantageous to use "padlock probe" and/or
"addressed
amplicon" techniques when generating a target strand that can be hybridized to
a probe
strand. These techniques are discussed in greater detail in U.S. Pat.
Application Serial No.
10/424,542 entitled "UNIVERSAL TAG ASSAY," filed April 24, 2003, herein
expressly
incorporated by reference in its entirety. Some embodiments of the present
invention
include providing a polynucleotide sample and then performing an assay to
determine
whether it contains a sequence of interest. In some such assays, a nucleic
acid circle is
prepared in connection with the polynucleotide sample that contains both a
portion of a
sequence complementary to the sequence being interrogated and an "address
sequence."
The address sequence is typically an arbitrary sequence of nucleotides that
will also appear
on a probe strand. The circle can be amplified by RCA to produce a long target
that
contains several repetitions of the complement to the address sequence. When
the target
strand is allowed to interact with a probe containing the address sequence,
the two can
hybridize. Detection of hybridization can be used as an indication of the
presence of the
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sequence being interrogated in the original sample. It will be appreciated
that assays of this
type can detect the presence of various sequences as well as the presence of
single
nucleotide polymorphisms (SNPs). When detecting SNPs, for example, it can be
advantageous to use different cyclizable strands containing each of the
possible nucleotides
at the suspected SNP location. Each cyclizable strand should also have a
unique address
sequence. The one cyclizable strand that fits correctly with the sample can
then cyclize and
undergo amplification. Then, by determining which address sequence corresponds
to a
probe-target hybrid, the identity of the nucleotide at the SNP location can be
determined.
Controllin~Hybridization Stringency
In performing a hybridization step, it is preferable to introduce single
stranded
targets derived as described above to the liquid medium such that they may
hybridize with
probes immobilized on an electrode. Preferably, the number of target strands
used in an
assay will exceed the number of probe strands in order to maximize the
opportunity of each
probe strand to interact with target strands and participate in hybridization.
If a target
strand is complementary to a probe strand, hybridization can take place when
the two come
into contact. However, in some cases, even strands which are not truly
complementary may
come together and stay together as an imperfect hybrid. Whether or not various
hybridization events occur can be influenced by various stringency factors
such as
temperature, pH, or the presence of a species able to denature various
hybridized strands.
Increasing the quantity of target strands is one technique that can be useful
in minimizing
the number of probes that should hybridize to targets, but do not (false
negatives).
Preferred techniques for controlling stringency include setting and
maintaining the
temperature and pH of the liquid medium environment. More preferred techniques
also
incorporate introducing one or more chemical species as stringency agents that
can
minimize the number of false positives and/or false negatives. Agents that can
be used for
this purpose include quaternary ammonium compounds such as tetramethylammonium
chloride (TMAC).
TMAC is particularly useful in minimizing false positives. This species
generally
acts through a non-specific salt effect to reduce hydrogen-bonding energies
between G-C
base pairs. At the same time, it binds specifically to A-T pairs and increases
the thermal
stability of these bonds. These opposing influences have the effect of
reducing the
difference in bonding energy between the triple-hydrogen bonded G-C based pair
and the
double-bonded A-T pair. One consequence is that the melting temperature of
nucleic acid
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hybrids formed in the presence of TMAC is solely a function of the length of
the hybrid. A
second consequence is an increase in the slope of the melting curve for each
probe.
Together, these effects allow the stringency of hybridization to be increased
to the point that
single-base differences can be resolved, and non-specific hybridization
minimized. Various
techniques for using a stringency agent such as TMAC are discussed in IJ.S.
Pat. No.
5,849,483 (Shuber), herein incorporated by reference.
Further, specific control of stringency factors can be useful in assays which
seep to
identify mutations occurring at the end of an oligonucleotide fragment. For
example, the
mutator cluster region of the APC gene, wherein mutations are highly
correlated with colon
cancer, is approximately 800 base pairs in length. If a probe oligomer is
approximately 17
base pairs in length, it will typically require approximately 44 oligomers to
blanket the
entire 800 base pair strand. Mutations at the end of a fragment are often
difficult to detect,
so it can be beneficial to use a second series of oligonucleotides that also
blanket that 800
base pair straald, but are offset such that the middle of the second series of
oligonucleotides
corresponds to the ends of the adjacent first series of oligonucleotides.
Allowing for a gap
of three base pairs between adjacent probe sequences, it will typically
require 80 oligomers
to test 800 base pairs for mutations. Various high volume techniques for
testing a mutator
cluster region can be used. In a preferred embodiment, standard multiwell
plates having 96
wells and 20 electrodes per well can be used to test a particular region;
assuming four wells
are used to determine which one of the four bases appears at a particular
point in the
sequence, each 96 well plate can test the properties of 24 different
molecules.
Further, temperature dependence can be adjusted by varying the length of
individual
oligonucleotides since longer sequences tend to be more stable.
Oligonucleotides that are
to be used in an assay need not all be the same length.
Amplifyin the Hybridized Nucleic Acid
In practicing the present invention, it will sometimes be advantageous to
augment
the signal created by the target strand that indicates hybridization has
occurred. One
method for doing this is to elongate the target strand after it has hybridized
to the probe.
This technique may be referred to as "on-chip" amplification. Two methods for
on-chip
amplification are particularly preferred.
The first preferred method of on-chip amplification is depicted in FIG. 2A.
Here,
either the 3' or 5' end of the hybridized PCR product can be targeted for a
head-to-tail
polymerization that builds up the amount of DNA on the electrodes. Typically,
three
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different oligonucleotides (not counting the immobilized probe and the target
strands) will
be used as shown here: the first oligomer is complementary to the 3' end of
the hybridized
PCR product (targeting the complement of the primer sequence), and contains a
sequence A
at its 5' end; the second oligomer has a sequence 5'-A*B-3', where A* is
complementary to
5. A; the third oligonucleotide has sequence 5'-AB*-3'. As depicted in FIG.
2A, these
oligomers can form a polymeric product as shown. The head-to-tail
polymerization can
continue until the strand reaches a desired length. Generally, when performing
head-to-tail
polymerization, the ultimate length of the polynucleotide is limited in part
by a competing
cyclization reaction of the head-to-tail oligomers. A higher concentration of
head-to-tail
oligomers in the liquid medium will generally produce longer linear polymers
attached to
the electrode, however.
The second preferred method of on-chip amplification is depicted in FIG. 2B.
This
method uses rolling circle amplification. Preferably, a preformed circle
(approximately 40
to 300 nucleotides) that has a region complementary to the 3' end of the bound
PCR
product is hybridized to the PCR product as shown. A processive DNA polymerase
can
then be added so that RCA results, elongating the bound PCR product.
Preferably, the PCR
product is elongated by approximately 10 to 100 copies of the circle.
A further technique for on-chip amplification is depicted in FIG. 2C. This
technique may be used in conjunction with other on-chip amplification methods
and is
commonly referred to as "branch" amplification. Here, additional
polynucleotides that are
capable of hybridizing with the target strand in a region beyond the probe-
target
hybridization region can be added to the liquid medium and allowed to
hybridize with the
bound target to further increase the amount of bound polynucleotide material
when probe-
target hybridization occurs. Preferably, these branch polynucleotide strands
are further
amplified, for example by RCA as depicted in FIG. 2C. Further, when a branch
amplification technique is used, it can be advantageous to attach branches on
top of
branches, a technique known as hyperbranching. Additional discussion of
branching and
hyperbranching techniques can be found, for example, in: Urdea, Biotechnology
12:926
(1994); Horn et al., Nucleic Acids Res. 25(23):4835-4841 (1997); Lizardi et
al., Nature
Genetics 19, 225-232 (1998); Kingsmore et al. (U.S. Pat. No. 6,291,187);
Lizardi et al.
(PCT application WO 97/19193); all of which are hereby incorporated by
reference.
After performing an on-chip amplification, the increased amount of DNA can
generate a larger and more detectable signal. This can be advantageous for
assay purposes
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WO 2004/099755 PCT/US2004/013222
since both the probe and the target typically produce some detectable signal.
If the signal of
the target is enhanced, the contrast between hybridized and unhybridized
probes will be
more profound. In some embodiments, however, nucleic acid analogs can be used
as
probes which do not contribute to the overall signal; such designs are
discussed in the
following section. Even when such nucleic acid analogs are used as probes,
target
elongation can still be desirable.
Preferably, nucleic acid hybridization is tested electrochemically using a
transition
metal complex. More preferably, hybridization is detected by measuring the
reduction of a
ruthenium complex as described below.
Detecting Hybridization
Various techniques can be used to determine whether hybridization has
occurred.
As indicated above, preferred embodiments of the present invention feature the
use of a
transition metal complex. In particular, a ruthenium complex can be used as a
counterion
to conduct an electrochemical assay using the general technique of Steele et
al. (199, Anal.
Chefn. 70:4670-4677), herein incorporated by reference.
Counterions, such as Ru(NH3)63+ or Ru(NH3)Spy3+, can be introduced to the
liquid
medium surrounding the immobilized oligonucleotides. Typically, Ru(NH3)Spy3+
is
preferred because its reduction to a divalent ion does not occur at the same
electrical
potential as the reduction of molecular oxygen.
Once introduced, the counterions will tend to cloud around the negatively
charged
backbones of the various nucleic acid strands. Generally, the counterions will
accumulate
electrostatically around the phosphate groups of the nucleic acids whether
they are single or
double stranded. However, because a probe and target together physically
constitute a
larger amount of nucleic acid than the probe alone, the hybridized nucleic
acid will
typically have more counterions surrounding it. In general, the target can be
much longer
than the probe, typically 2 to 100 times, in which case the counterion
accumulation will be
dominated by single stranded regions of the target.
Alternatively, it can be possible to increase the signal contrast between
single
stranded and double stranded nucleic acid by limiting the electrical signal
from the probe
strands. In particular, this can be done by limiting the electrical attraction
between the
probe strand and the counterions which participate in electron transfer. For
example, if the
probe strands are constructed such that they do not contain a negatively
charged backbone,
then they will not attract counterions. Accordingly, more of the detectable
signal will be
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WO 2004/099755 PCT/US2004/013222
due to counterions associated with the target strands. In cases where
hybridization has not
occurred, the detectable signal will be measurably lower since the target
strands are not
present to participate in counterion attraction.
Probe strands without a negatively charged backbone can include peptide
nucleic
acids (PNAs), phosphotriesters, methylphosphonates. These nucleic acid analogs
are
known in the art.
In particular, PNAs are discussed in: Nielsen, "DNA analogues with
nonphosphodiester backbones," Anriu Rep Biophys Biomol Struct, 1995;24:167-83;
Nielsen
et al., "An introduction to peptide nucleic acid," Currlssues Mol Biol,
1999;1(1-2):89-104;
Ray et al., "Peptide nucleic acid (PNA): its medical and biotechnical
applications and
promise for the future," FASEB J., 2000 Jun;l4(9):1041-60; all of which are
hereby
expressly incorporated by reference in their entirety.
Phophotriesters are discussed in: Sung et al., "Synthesis of the human insulin
gene.
Part TI. Further improvements in the modified phosphotriester method and the
synthesis of
seventeen deoxyribooligonucleotide fragments constituting human insulin chains
B and
mini-CDNA," Nucleic Acids Res, 1979 Dec 20;7(8):2199-212; van Boom et al.,
"Synthesis
of oligonucleotides with sequences identical with or analogous to the 3'-end
of 16S
ribosomal RNA of Escherichia coli: preparation of m-6-2-A-C-C-U-C-C and A-C-C-
U-C-
m-4-2C via phosphotriester intermediates," Nucleic Acids Res, 1977
Mar;4(3):747-59;
Marcus-Sekura et al., "Comparative inhibition of chloramphenicol
acetyltransferase gene
expression by antisense oligonucleotide analogues having alkyl
phosphotriester,
methylphosphonate and phosphorothioate linkages," Nucleic Acids Res, 1987 Jul
24;15(14):5749-63; all of which are hereby expressly incorporated by reference
in their
entirety.
Methylphosphonates are discussed in: U.S. Pat. No. 4,469,863 (Ts'o et al.);
Lin et
al., "Use of EDTA derivatization to characterize interactions between
oligodeoxyribonucleoside methylphophonates and nucleic acids," Biochemistfy,
1989, Feb
7;28(3):1054-61; Vyazovkina et al., "Synthesis of specific diastereomers of a
DNA
methylphosphonate heptamer, d(CpCpApApApCpA), and stability of base pairing
with the
normal DNA octamer d(TPGPTPTPTPGPGPC)," Nucleic Acids Res, 1994 Jun
25;22(12):2404-9; Le Bec et al., "Stereospecific Grignard-Activated Solid
Phase Synthesis
of DNA Methylphosphonate Dimers," J Ofg Chem, 1996 Jan 26;61(2):510-513;
Vyazovkina et al., "Synthesis of specific diastereomers of a DNA
methylphosphonate
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CA 02524265 2005-10-31
WO 2004/099755 PCT/US2004/013222
heptamer, d(CpCpApApApCpA), and stability of base pairing with the normal DNA
octamer d(TPGPTPTPTPGPGPC)," Nucleic Acids Res, 1994 Jun 25;22(12):2404-9;
Kibler-Herzog et al., "Duplex stabilities of phosphorothioate,
methylphosphonate, and
RNA analogs of two DNA 14-mers," Nucleic Acids Res, 1991 Jun 11;19(11):2979-
86;
Disney et al., "Targeting a Pneumocystis carinii group I intron with
methylphosphonate
oligonucleotides: backbone charge is not required for binding or reactivity,"
Biochemistry,
2000 Jun 13;39(23):6991-7000; Ferguson et al., "Application of free-energy
decomposition
to determine the relative stability of R and S oligodeoxyribonucleotide
methylphosphonates," Antisense Res Dev, 1991 Fall;l(3):243-54; Thiviyanathan
et al.,
"Structure of hybrid backbone methylphosphonate DNA heteroduplexes: effect of
R and S
stereochemistry," Biochemistry, 2002 Jan 22;41(3):827-38; Reynolds et al.,
"Synthesis and
thermodynamics of oligonucleotides containing chirally pure R(P)
methylphosphonate
linkages," Nucleic Acids Res, 1996 Nov 15;24(22):4584-91; Hardwidge et al.,
"Charge
neutralization and DNA bending by the Escherichia coli catabolite activator
protein,"
Nucleic Acids Res, 2002 May 1;30(9):1879-85; Okonogi et al., "Phosphate
backbone
neutralization increases duplex DNA flexibility: A model for protein binding,"
PNAS
U.SA., 2002 Apr 2;99(7):4156-60; all of which are hereby incorporated by
reference.
In general, an appropriate nucleic acid analog probe will not contribute, or
will
contribute less substantially, to the attraction of counterions compared to a
probe made of
natural DNA. Meanwhile, the target strand will ordinarily feature a natural
phosphate
backbone having negatively charged groups which attract positive ions and make
the strand
detectable.
Alternatively, a probe may be constructed that contains both charged nucleic
acids
and uncharged nucleic acid analogs. Similarly, pure DNA probes can be used
alongside
probes containing uncharged analogs in an assay. However, precision in
distinguishing
between single stranded and double stranded will generally increase according
to the
electrical charge contrast between the probe and the target strands. Hence,
the exclusive
use of probes made entirely of an uncharged DNA analog will generally allow
the greatest
signal contrast between hybridized and non-hybridized molecules on the chip.
In general,
probe strands containing methylphosphonates are preferred when nucleic acid
analogs are
desired.
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Ru(NH3)Spy3+ is a preferred counterion, though any other suitable transition
metal
complexes that bind nucleic acid electrostatically and whose reduction or
oxidation is
electrochemically detectable in an appropriate voltage regime can be used.
Various techniques for measuring the amount of counterions can be used. In
some
preferred embodiments, amperometry is used to detect an electrochemical
reaction at the
electrode. Generally, an electrical potential will be applied to the
electrode. As the
counterions undergo an electrochemical reaction, for example, the reduction of
a trivalent
ion to divalent at the electrode surface, a measurable current is generated.
The amount of
current corresponds to the amount of counterions present which in turn
corresponds to the
amount of negatively-charged phosphate groups on nucleic acids. Accordingly,
measuring
the current allows a quantitation of phosphate groups and can allow the
operator to
distinguish hybridized nucleic acid from unhybridized nucleic acid and
determine whether
the target being interrogated is complementary to the probe (and contains the
sequence of
interest).
Some embodiments of the present invention allow detection of nucleic acid
mutations with improved accuracy and precision. In some embodiments, for
example, a
mutation can be detected at a level of about 1 part in 102 (which means one
mutant version
of a gene in a sample per 100 total versions of the gene in the sample) or
less, about 1 part
in 103 or less, about 1 part in 104 or less, about 1 part in 105 or less, or
about 1 part in 106 or
less.
Although electrochemical measurement is a preferred technique for
hybridization
detection, those of skill in the art will appreciate that many other
techniques can also be
appropriate in practicing the present invention. For example, a detectable
label can be
attached to or otherwise associated with certain polynucleotides in the
detection zone.
Accordingly, such a label can then be detected as an indication of whether
hybridization has
occurred. Such labels are well known in the art and can include, for example,
chemical
moieties, dyes, radioactive probes, quantum dots, and nanoparticles.
Techniques for
detection of various labels can include, for example, chemical detection,
radioactivity
detection, UV and/or visible spectroscopy, fluorescence, and the like.
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Examples
Example 1: Augmentin~~an Electrical Signal Usin On-chip~lification
The following procedure was performed to determine the effectiveness of on-
chip
amplification for enhancing an electrical signal. The results are discussed
with reference to
FIG. 3.
To prepare a nucleic acid film on the surface of an electrode, 1.5 ~.l of a
solution
consisting of biotinylated capture probe and NeutrAvidin was deposited to the
surface of a
carbon electrode and allowed to air dry.
The carbon electrode was transferred to a solution containing 5 ~.M of
Ru(NH3)6C13
in 10 mM Tris + 10 mM NaCI. The electrochemical response was detected and
recorded
using Osteryoung Square Wave Voltammetry (OSWV). Tlus measurement corresponds
to
the quantity of nucleic acid immobilized on the electrode before RCA is
performed. The
resulting current is represented by the smaller curve (which peaks at
approximately 0.100
p,A) as depicted in FIG. 3.
The immobilized capture probe was then hybridized with a circularized DNA as
follows. The electrode was rinsed with Tris buffer solution. Then, 10 ~,l of
solution
containing circularized DNA in 10 mM Hepes + 1 M LiCI was applied to the
surface of the
carbon electrode. The electrode was maintained at 60 °C for 5 minutes
and then cooled to
room temperature and maintained at the room temperature for 30 minutes.
RCA was then performed as follows. The electrode was rinsed with the Tris
buffer.
A mixture of RCA working solution containing phi29 polymerase and dNTPs in
tris buffer
was applied. RCA was allowed to proceed at 37 °C for 1 hour.
The electrode was rinsed with tris buffer and transferred to a solution
containing 5
~M of Ru(NH3)6C13 in 10 mM Tris + 10 mM NaCI. The electrochemical response was
again detected and recorded using OSWV. The resulting current is represented
by the
larger curve (which peaks at approximately 1.800 ~,A) as depicted in FIG. 3.
The change from the smaller curve to the larger curve corresponds to the
increased
number of ruthenium complexes which associate with the more numerous P03-
moieties on
a larger amount of DNA.
Example 2: Screening a Patient for Colorectal Cancer
A patient at risk for colorectal cancer can be screened for cancer or
precancer as
follows:
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1. An oligonucleotide sequence indicative of colorectal cancer is identified;
this
sequence is approximately 20 base pairs in length and is known as the
"sequence of
interest."
2. A probe strand having a length of approximately 20 base pairs is produced
having a sequence that is complementary to the sequence of interest.
3. A patient suspected of having colorectal cancer or suspected of later
developing
colorectal cancer is identified.
4. A stool sample voided from the patient is collected and sectioned to
extract cells
and cellular debris containing nucleic acids from the epithelial cells of the
patient's
colorectal tract.
5. DNA is extracted and isolated from the cells and cellular debris using
methods
disclosed in U.S. Pat. Nos. 5,741,650 (Lapidus et al.) and 6,406,857 (Shuber
et al.).
6. DNA molecules isolated from the stool sample are amplified using digital
PCR.
7. Single stranded PCR products are isolated.using a streptavidin-coated bead.
8. The isolated, single stranded nucleic acid is cyclized and subj ect to RCA
to
produce elongated target strands.
9. A plurality of probe strands containing a biotin complex are immobilized on
a
gold electrode coated with streptavidin. A liquid medium is placed in contact
with the
electrode surface and with the immobilized probe strands.
10. A plurality of target strands are introduced to the liquid medium such
that they
are allowed to interact with the probe strands. Hybridization stringency is
controlled by
adjusting temperature, pH, and the quantity of TMAC in the liquid medium.
Hybridization
stringency is set such that perfectly complementary sequences hybridize but
that all others
do not.
11. On-chip amplification is conducted using head-to-tail polymerization to
increase the length of any hybridized target on the electrode. This
amplification adds
approximately 10,000 base pairs to each bound target strand.
12. Ru(NH3)Spy~+ ions in a liquid are added to the liquid medium as
counterions
able to form an electrostatic cloud around nucleic acids.
13. An electrical potential is applied to the electrode on which the nucleic
acid
probes are immobilized.
14. As Ru(NH3)Spy3+ ions are reduced from trivalent to divalent at the
electrode
surface, current is measured at the electrode. The amount of current is
recorded and used to
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determine whether the immobilized probes have hybridized to target strands or
remain
unhybridized.
15. The hybridization status of the probes to the targets is used to evaluate
the
health of the patient with regard to colorectal cancer and to decide whether
to administer
further healthcare services to the patient, including, for example,
counseling, additional
testing, administration of pharmaceutical agents, surgery, etc.
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