U.S. patent application number 13/905882 was filed with the patent office on 2013-11-14 for high resolution dna detection methods and devices.
The applicant listed for this patent is Integrated Nano-Technologies, LLC. Invention is credited to Dennis Michael Connolly.
Application Number | 20130303404 13/905882 |
Document ID | / |
Family ID | 22433882 |
Filed Date | 2013-11-14 |
United States Patent
Application |
20130303404 |
Kind Code |
A1 |
Connolly; Dennis Michael |
November 14, 2013 |
HIGH RESOLUTION DNA DETECTION METHODS AND DEVICES
Abstract
The present invention provides methods and devices for detecting
a target nucleic acid molecule. A set of oligonucleotide probes
integrated into an electric circuit, where the oligonucleotide
probes are positioned such that they cannot come into contact with
one another, are contacted with a sample. If the sample contains a
target nucleic acid molecule, one which has sequences complimentary
to both probes, the target nucleic acid molecule can bridge the gap
between the probes. The resulting bridge can then carry electrical
current between the two probes, indicating the presence of the
target nucleic acid molecule.
Inventors: |
Connolly; Dennis Michael;
(Rochester, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Integrated Nano-Technologies, LLC |
Rochester |
NY |
US |
|
|
Family ID: |
22433882 |
Appl. No.: |
13/905882 |
Filed: |
May 30, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11210071 |
Aug 23, 2005 |
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13905882 |
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10347781 |
Jan 17, 2003 |
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11210071 |
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09918140 |
Jul 30, 2001 |
6593090 |
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10347781 |
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09545010 |
Apr 7, 2000 |
6399303 |
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09918140 |
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60128149 |
Apr 7, 1999 |
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Current U.S.
Class: |
506/16 |
Current CPC
Class: |
C12Q 1/689 20130101;
C12Q 1/6816 20130101; B01J 2219/00529 20130101; C12Q 1/6825
20130101; H01L 51/0093 20130101; Y10S 436/806 20130101; C12Q 1/6825
20130101; B01J 2219/00722 20130101; B01J 2219/00653 20130101; C12Q
1/6825 20130101; Y10S 436/807 20130101; C12Q 1/70 20130101; B82Y
10/00 20130101; C12Q 2565/607 20130101; G01N 2001/021 20130101;
C12Q 2523/313 20130101; C12Q 2565/607 20130101; C12Q 2565/543
20130101; C12Q 2523/313 20130101; C12Q 1/6883 20130101; C12Q 1/6816
20130101; C40B 40/06 20130101 |
Class at
Publication: |
506/16 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12Q 1/70 20060101 C12Q001/70 |
Claims
1. A device for detecting the presence of a target nucleic acid
molecule, comprising: two electronic leads, where an end of a first
lead is located near an end of second lead but where the leads are
not in contact, one or more sets of two oligonucleotide probes
attached to the electronic leads, where the oligonucleotide probes
are positioned such that the probes can not come into contact with
one another and such that a target nucleic acid molecule, which has
two sequences, a first sequence complimentary to a first probe
attached to the first lead and a second sequence complimentary to a
second probe attached to the second lead, can bind to both probes
concurrently completing an electrical circuit, when the target
nucleic acid molecule is present, a fluidic channel for introducing
a reagent to coat the target nucleic acid molecule with a metal
conductor, an electric potential for generating a current flow
through the electrical circuit, a computer for detecting an
electrical current and correlating the presence or absence of an
electrical current to the presence or absence of the target nucleic
acid molecule; wherein a bridged probe having an electrical current
flowing through the two electrical leads and a target nucleic acid
molecule denotes the presence of the target nucleic acid molecule;
and an unbridged probe lacks an electrical current denoting the
absence of the target nucleic acid molecule.
2. The device according to claim 1, further comprising: a chamber
for treating a sample to release nucleic acid molecules from a
sample.
3. The device according to claim 1, wherein the device contains
proteins for processing the sample.
4. The device according to claim 3, wherein the protein is selected
from the group consisting of a ligase, protease, restriction
endonuclease, nuclease, or nucleic acid binding protein.
5. The device according to claim 3, wherein the protein is a
thermostable protein.
6. The device according to claim 1, wherein the nucleic acid
molecule is DNA.
7. The device according to claim 1, wherein the nucleic acid
molecule is RNA.
8. The device according to claim 1, wherein the metal conductor is
silver.
9. The device according to claim 1, wherein the metal conductor is
gold.
10. The device according to claim 1, further comprising: a chamber
containing nucleases for contacting the target nucleic acid after
binding with the probes.
11. The device according to claim 1, further comprising: heating
elements for heating the sample.
12. The device according to claim 1, wherein the probes are
complimentary to sequences from the genetic material of a
pathogenic bacteria.
13. The device according to claim 12, wherein the pathogenic
bacteria is a biowarfare agent.
14. The device according to claim 12, wherein the pathogenic
bacteria is a food borne pathogen.
15. The device according to claim 1, wherein the probes are
complimentary to sequences from the genetic material of a
virus.
16. The device according to claim 1, wherein the probes are
complimentary to sequences from the genetic material of a
human.
17. The device according to claim 16, wherein one or both of the
probes has a sequence which is complimentary to a sequence having a
polymorphism, where the base or bases complimentary to the
polymorphism are located at the end of the probe.
18. A device for detecting the presence of a target nucleic acid
molecule, comprising: two electronic leads, where an end of a first
lead is located near an end of second lead but where the leads are
not in contact, one or more sets of two oligonucleotide probes
attached to the electronic leads, where the oligonucleotide probes
are positioned such that the probes can not come into contact with
one another and such that a target nucleic acid molecule, which has
two sequences, a first sequence complimentary to a first probe
attached to the first lead and a second sequence complimentary to a
second probe attached to the second lead, can bind to both probes
concurrently completing an electrical circuit, when the target
nucleic acid molecule is present, a first microfluidic channels for
contacting the probes with a sample which may have the target
nucleic acid molecule to permit the target nucleic acid molecule,
if present in the sample, to bind to both probes, a second fluidic
channel for introducing a reagent to coat the target nucleic acid
molecule with a metal conductor, an electric potential for
generating a current flow through the electrical circuit, a
computer for detecting an electrical current, the electrical
current indicating the presence of the target nucleic acid molecule
in the sample and no electrical current indicating the lack of the
target nucleic acid molecule in the sample.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
11/210,071 (filed Aug. 23, 2005) which is a continuation of U.S.
Ser. No. 10/347,781 (filed Jan. 17, 2003), which is a divisional of
U.S. Ser. No. 09/918,140 (filed Jul. 30, 2001), now U.S. Pat. No.
6,593,090, which is a continuation of U.S. Ser. No. 09/545,010
(filed Apr. 7, 2000), now U.S. Pat. No. 6,399,303, which claims
benefit of U.S. Ser. No. 60/128,149 (filed Apr. 7, 1999). Each of
these applications are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] DNA identification technology has numerous uses including
identification of pathogenic organisms, genetic testing, and
forensics. Advances have been made to allow for automated screening
of thousands of sequences concurrently. Gene chip technologies
exist where DNA probes are immobilized on a substrate such as a
glass or silicon chip. A sample containing nucleic acid molecules
is applied to the chip and the nucleic acid molecules within the
sample are allowed to hybridize to the probe DNA on the chip.
Fluorescence detection is typically used to identify double
stranded nucleic acid molecule products. The advantage of the
system is the ability to screen hundreds or thousands of sequences
using automated systems.
[0003] Hybridization screening with fluorescence detection is a
powerful technique for detecting nucleic acid sequences. However,
in order to detect target DNA molecules, the target must first be
amplified by PCR to get a reliable signal. The gene chip technology
also requires a system capable of detecting fluorescent or
radioactive materials. Such a system is expensive to use and is not
amenable to a portable system for biological sample detection and
identification. Furthermore, the hybridization reactions take up to
two hours. For many potential uses, such as detecting biological
warfare agents, the gene chip system is simply not effective.
Therefore, there is a need for a system which can rapidly detect
small quantities of a target nucleic acid molecule without relying
on PCR amplification.
[0004] The discussion above is merely provided for general
background information and is not intended to be used as an aid in
determining the scope of the claimed subject matter.
BRIEF DESCRIPTION OF THE INVENTION
[0005] The present invention provides a method for detecting a
target nucleic acid molecule. A device for detecting the presence
of a target nucleic acid molecule is provided having two electronic
leads, where the ends of the leads are located near each other but
are not in contact. One or more sets of two oligonucleotide probes
are attached to the electronic leads. The oligonucleotide probes
are positioned such that the probes can not come into contact with
one another and such that a target nucleic acid molecule, which has
two sequences complimentary to the probes can bind to both probes
concurrently. A sample which may have the target nucleic acid
molecule is contacted with the probes under selective hybridization
conditions. If the target is present it bridges the gap between the
probes. The target nucleic acid molecule may then carry current
between the probes, or be used as a support to form a conductive
wire between the two probes.
[0006] The present invention also provides a device for detecting
the presence of a target nucleic acid molecule. The device has two
electronic leads, where the ends of the leads are located near each
other but are not in contact. One or more sets of two
oligonucleotide probes are attached to the electronic leads. The
oligonucleotide probes are positioned such that the probes cannot
come into contact with one another and such that a target nucleic
acid molecule, which has two sequences complimentary to the probes
can bind to both probes concurrently.
[0007] This brief description of the invention is intended only to
provide a brief overview of subject matter disclosed herein
according to one or more illustrative embodiments, and does not
serve as a guide to interpreting the claims or to define or limit
the scope of the invention, which is defined only by the appended
claims. This brief description is provided to introduce an
illustrative selection of concepts in a simplified form that are
further described below in the detailed description. This brief
description is not intended to identify key features or essential
features of the claimed subject matter, nor is it intended to be
used as an aid in determining the scope of the claimed subject
matter. The claimed subject matter is not limited to
implementations that solve any or all disadvantages noted in the
background.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] So that the manner in which the features of the invention
can be understood, a detailed description of the invention may be
had by reference to certain embodiments, some of which are
illustrated in the accompanying drawings. It is to be noted,
however, that the drawings illustrate only certain embodiments of
this invention and are therefore not to be considered limiting of
its scope, for the scope of the invention encompasses other equally
effective embodiments. The drawings are not necessarily to scale,
emphasis generally being placed upon illustrating the features of
certain embodiments of the invention. In the drawings, like
numerals are used to indicate like parts throughout the various
views. Thus, for further understanding of the invention, reference
can be made to the following detailed description, read in
connection with the drawings in which:
[0009] FIG. 1 graphically depicts the method of the present
invention. Two leads are provided each having a probe which is
complimentary to sequences on a target nucleic acid molecule (FIG.
1A). A target nucleic acid molecule binds to the two probes at the
complimentary sequences (FIG. 1B). The complimentary strand is
filled in (FIG. 1C). Nucleases are used to remove the free ends of
the target nucleic acid molecule (FIG. 1D). Current can be passed
through the double stranded molecule or the target nucleic acid
molecule and probes may be coated with a conductor and then tested
for current flow.
[0010] FIG. 2 is a variation on the method shown in FIG. 1 using a
ligase method to distinguish a single base variation. The variation
is identified by the asterisk. After step D, a ligase is used. Only
those targets which have an exact match at the ends of the probes
will ligate. After ligation, the sample is heated to remove
non-ligated target molecules (FIG. 2E). The structure in FIG. 2E is
stable at higher temperatures, whereas the un-ligated structure in
FIG. 2D would denature under heat treatment.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The present invention provides devices and methods for
rapidly detecting the presence of nucleic acid molecules. The
target nucleic acid molecule either itself, or as a support, is
used to complete a electrical circuit. The presence of the target
nucleic acid molecule is indicated by the ability to conduct an
electrical signal through the circuit. In the case where the target
nucleic acid molecule is not present, the circuit is not be
completed. Thus, the target nucleic acid molecule acts as a switch.
The presence of the nucleic acid molecule provides an on signal for
an electrical circuit, whereas the lack of the target nucleotide is
interpreted as an off signal. Due to the direct detection of the
target nucleic acid molecule, the method allows for extremely
sensitive detection of target molecules connect two wires.
[0012] The detection device is constructed on a support. Examples
of useful substrate materials include, e.g., glass, quartz and
silicon as well as polymeric substrates, e.g. plastics. In the case
of conductive or semi-conductive substrates, it will generally be
desirable to include an insulating layer on the substrate. However,
any solid support which has a non-conductive surface may be used to
construct the device. The support surface need not be flat. In
fact, the support may be on the walls of a chamber in a chip.
[0013] Two leads are provided having ends located close together,
within the spanning distance of a target nucleic acid molecule, but
not contacting one another. Current cannot flow effectively between
the leads without the presence of a target nucleic acid molecule to
bridge the two leads. Two probes specific to the target nucleic
acid molecule are used. The first is attached to one lead, the
second to the other lead. The two probes are specific to sequences
on the target molecule which are separated by sufficient distance
to span the region between the leads. Typically, the gap will be in
micron or fractions of microns in length. However, as chip
manufacturing has improved, it has become possible to shrink the
distance between elements on a chip, requiring shorter lengths of
target nucleic acid molecules.
[0014] The target nucleic acid molecule is passed over the two
leads. If a target molecule has a sequence complimentary to one of
the probes, it can bind to that probe. Once bound to that probe,
the nucleic acid molecule is tethered at that site. The sequence
complimentary to the second probe can then bind to the second
probe. To facilitate such a reaction, the two complimentary
sequences should be chosen such that the length of the molecule in
between can span the distance between the two leads and provide
flexibility for the nucleic acid molecule to move easily, such that
the second complimentary sequence readily binds to the second
probe.
[0015] In a preferred embodiment, the probes are selected to bind
with the target such that they have approximately the same melting
temperature. This can be done by varying the lengths of the
hybridization region. A-T rich regions may have longer target
sequences, whereas G-C rich regions would have shorter target
sequences.
[0016] Hybridization assays on substrate-bound oligonucleotide
arrays involve a hybridization step and a detection step. In the
hybridization step, a hybridization mixture containing the target
and an isostabilizing agent, denaturing agent or renaturation
accelerant is brought into contact with the probes of the array and
incubated at a temperature and for a time appropriate to allow
hybridization between the target and any complementary probes.
Usually, unbound target molecules are then removed from the array
by washing with a wash mixture that does not contain the target,
such as hybridization buffer. This leaves only bound target
molecules. In the detection step, the probes to which the target
has hybridized are identified. In the present method the detection
is carried out by detecting a completed electronic circuit. Since
the nucleotide sequence of the probes at each feature is known,
identifying the locations at which target has bound provides
information about the particular sequences of these probes.
[0017] Including a hybridization optimizing agent in the
hybridization mixture significantly improves signal discrimination
between perfectly matched targets and single-base mismatches. As
used herein, the term "hybridization optimizing agent" refers to a
composition that decreases hybridization between mismatched nucleic
acid molecules, i.e., nucleic acid molecules whose sequences are
not exactly complementary.
[0018] An isostabilizing agent is a composition that reduces the
base-pair composition dependence of DNA thermal melting
transitions. More particularly, the term refers to compounds that,
in proper concentration, result in a differential melting
temperature of no more than about 1.degree. C. for double stranded
DNA oligonucleotides composed of AT or GC, respectively.
Isostabilizing agents preferably are used at a concentration
between 1 M and 10 M, between 2 M and 6 M, between 4 M and 6 M,
between 4 M and 10 M and, optimally, at about 5 M. For example, 5 M
agent in 2.times.SSPE is suitable. Betaines and lower tetraalkyl
ammonium salts are examples of isostabilizing agents. In one
embodiment, the isostabilizing agent is not an alkylammonium
ion.
[0019] Betaine (N,N,N,-trimethylglycine; (Rees et al., Biochem.,
(1993) 32:137-144), which is hereby incorporated by reference) can
eliminate the base pair composition dependence of DNA thermal
stability. Unlike TMAC1, betaine is zwitterionic at neutral pH and
does not alter the polyelectrolyte behavior of nucleic acids while
it does alter the composition-dependent stability of nucleic acids.
Inclusion of betaine at about 5 M can lower the average
hybridization signal, but increases the discrimination between
matched and mismatched probes.
[0020] A denaturing agent is a compositions that lowers the melting
temperature of double stranded nucleic acid molecules by
interfering with hydrogen bonding between bases in a
double-stranded nucleic acid or the hydration of nucleic acid
molecules. Denaturing agents can be included in hybridization
buffers at concentrations of about 1 M to about 6 M and,
preferably, about 3 M to about 5.5 M.
[0021] Denaturing agents include formamide, formaldehyde, DMSO
("dimethylsulfoxide"), tetraethyl acetate, urea, GuSCN, glycerol
and chaotropic salts. As used herein, the term "chaotropic salt"
refers to salts that function to disrupt van der Waal's attractions
between atoms in nucleic acid molecules. Chaotropic salts include,
for example, sodium trifluoroacetate, sodium tricholoroacetate,
sodium perchlorate, guanidine thiocyanate ("GuSCN"), and potassium
thiocyanate.
[0022] A renaturation accelerant is a compound that increases the
speed of renaturation of nucleic acids by at least 100-fold. They
generally have relatively unstructured polymeric domains that
weakly associate with nucleic acid molecules. Accelerants include
heterogenous nuclear ribonucleoprotein ("hnRP") A1 and cationic
detergents such as, preferably, CTAB ("cetyltrimethylammonium
bromide") and DTAB ("dodecyl trimethylammonium bromide"), and,
also, polylysine, spermine, spermidine, single stranded binding
protein ("SSB"), phage T4 gene 32 protein and a mixture of ammonium
acetate and ethanol. Renaturation accelerants can be included in
hybridization mixtures at concentrations of about 1 mu M to about
10 mM and, preferably, 1 mu M to about 1 mM. The CTAB buffers work
well at concentrations as low as 0.1 mM.
[0023] Homologous nucleotide sequences can be detected by
selectively hybridizing to each other. Selectively hybridizing is
used herein to mean hybridization of DNA or RNA probes from one
sequence to the "homologous" sequence under stringent or
non-stringent conditions (Ausubel, et al., Eds., 1989, Current
Protocols in Molecular Biology, Vol. I, Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., New York, at page
2.10.3, which is hereby incorporated by reference). Hybridization
and wash conditions are also exemplified in Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor, N.Y., (1989), which is hereby incorporated by
reference.
[0024] A variety of hybridization buffers are useful for the
hybridization assays of the invention. Addition of small amounts of
ionic detergents (such as N-lauroyl-sarkosine) are useful. LiCl is
preferred to NaCl. Hybridization can be at 20.degree.-65.degree.
C., usually 37.degree. C. to 45.degree. C. for probes of about 14
nucleotides. Additional examples of hybridization conditions are
provided in several sources, including: Sambrook et al., Molecular
Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor,
N.Y.; and Berger and Kimmel, "Guide to Molecular Cloning
Techniques," Methods in Enzymology, (1987), Volume 152, Academic
Press, Inc., San Diego, Calif.; Young and Davis, Proc. Natl. Acad.
Sci. USA, 80:1194 (1983), which are hereby incorporated by
reference.
[0025] In addition to aqueous buffers, non-aqueous buffers may also
be used. In particular non-aqueous buffers which facilitate
hybridization but have low electrical conductivity are
preferred.
[0026] The hybridization mixture is placed in contact with the
array and incubated. Contact can take place in any suitable
container, for example, a dish or a cell specially designed to hold
the probe array and to allow introduction of the fluid into and
removal of it from the cell so as to contact the array. Generally,
incubation will be at temperatures normally used for hybridization
of nucleic acids, for example, between about 20.degree. C. and
about 75.degree. C., e.g., about 25.degree. C., about 30.degree.
C., about 35.degree. C., about 40.degree. C., about 45.degree. C.,
about 50.degree. C., about 55.degree. C., about 60.degree. C. or
about 65.degree. C. For probes longer than about 14 nucleotides,
37.degree. C.-45.degree. C. is preferred. For shorter probes,
55.degree. C.-65.degree. C. is preferred. More specific
hybridization conditions can be calculated using formulas for
determining the melting point of the hybridized region. Preferably,
hybridization is carried out at a temperature at or between ten
degrees below the melting temperature and the melting temperature.
More preferred, the hybridization is carried out at a temperature
at or between five degrees below the melting temperature and the
melting temperature. The target is incubated with the probe array
for a time sufficient to allow the desired level of hybridization
between the target and any complementary probes in the array. After
incubation with the hybridization mixture, the array usually is
washed with the hybridization buffer, which also can include the
hybridization optimizing agent. These agents can be included in the
same range of amounts as for the hybridization step, or they can be
eliminated altogether. Then the array can be examined to identify
the probes to which the target has hybridized.
[0027] The target polynucleotide whose sequence is to be determined
is usually isolated from a tissue sample. If the target is genomic,
the sample may be from any tissue (except exclusively red blood
cells). For example, whole blood, peripheral blood lymphocytes or
PBMC, skin, hair or semen are convenient sources of clinical
samples. These sources are also suitable if the target is RNA.
Blood and other body fluids are also a convenient source for
isolating viral nucleic acids. If the target is mRNA, the sample is
obtained from a tissue in which the mRNA is expressed. If the
polynucleotide in the sample is RNA, it may be reverse transcribed
to DNA, but in this method need not be converted to DNA.
[0028] Various methods exist for attaching the probes to the
electronic circuit. For example, U.S. Pat. Nos. 5,861,242;
5,861,242; 5,856,174; 5,856,101; and 5,837,832, which are hereby
incorporated by reference, disclose a method where light is shone
through a mask to activate functional (for oligonucleotides,
typically an --OH) groups protected with a photo-removable
protecting group on a surface of a solid support. After light
activation, a nucleoside building block, itself protected with a
photo-removable protecting group (at the 5'-OH), is coupled to the
activated areas of the support. The process can be repeated, using
different masks or mask orientations and building blocks, to place
probes on a substrate.
[0029] Alternatively, new methods for the combinatorial chemical
synthesis of peptide, polycarbamate, and oligonucleotide arrays
have recently been reported (see Fodor et al., Science, 251:767-773
(1991); Cho et al., Science, 261:1303-1305 (1993); and Southern et
al., Genomics 13:1008-10017 (1992), which are hereby incorporated
by reference). These arrays, or biological chips (see Fodor et al.,
Nature, 364:555-556 (1993), which is hereby incorporated herein by
reference), harbor specific chemical compounds at precise locations
in a high-density, information rich format, and are a powerful tool
for the study of biological recognition processes.
[0030] Preferably, the probes are attached to the leads through
spatially directed oligonucleotide synthesis. Spatially directed
oligonucleotide synthesis may be carried out by any method of
directing the synthesis of an oligonucleotide to a specific
location on a substrate. Methods for spatially directed
oligonucleotide synthesis include, without limitation,
light-directed oligonucleotide synthesis, microlithography,
application by ink jet, microchannel deposition to specific
locations and sequestration with physical barriers. In general
these methods involve generating active sites, usually by removing
protective groups; and coupling to the active site a nucleotide
which, itself, optionally has a protected active site if further
nucleotide coupling is desired.
[0031] In one embodiment the lead-bound oligonucleotides are
synthesized at specific locations by light-directed oligonucleotide
synthesis which is disclosed in U.S. Pat. No. 5,143,854; PCT
application WO 92/10092; and PCT application WO 90/15070. In a
basic strategy of this process, the surface of a solid support
modified with linkers and photolabile protecting groups is
illuminated through a photolithographic mask, yielding reactive
hydroxyl groups in the illuminated regions. A
3'-O-phosphoramidite-activated deoxynucleoside (protected at the
5'-hydroxyl with a photolabile group) is then presented to the
surface and coupling occurs at sites that were exposed to light.
Following the optional capping of unreacted active sites and
oxidation, the substrate is rinsed and the surface is illuminated
through a second mask, to expose additional hydroxyl groups for
coupling to the linker. A second 5'-protected,
3'-O-phosphoramidite-activated deoxynucleoside (C-X) is presented
to the surface. The selective photodeprotection and coupling cycles
are repeated until the desired set of probes are obtained.
Photolabile groups are then optionally removed and the sequence is,
thereafter, optionally capped. Side chain protective groups, if
present, are also removed. Since photolithography is used, the
process can be miniaturized to specifically target leads in high
densities on the support.
[0032] This general process can be modified. For example, the
nucleotides can be natural nucleotides, chemically modified
nucleotides or nucleotide analogs, as long as they have activated
hydroxyl groups compatible with the linking chemistry. The
protective groups can, themselves, be photolabile. Alternatively,
the protective groups can be labile under certain chemical
conditions, e.g., acid. In this example, the surface of the solid
support can contain a composition that generates acids upon
exposure to light. Thus, exposure of a region of the substrate to
light generates acids in that region that remove the protective
groups in the exposed region. Also, the synthesis method can use
3'-protected 5'-O-phosphoramidite-activated deoxynucleoside. In
this case, the oligonucleotide is synthesized in the 5' to 3'
direction, which results in a free 5' end.
[0033] The general process of removing protective groups by
exposure to light, coupling nucleotides (optionally competent for
further coupling) to the exposed active sites, and optionally
capping unreacted sites is referred to herein as "light-directed
nucleotide coupling."
[0034] The probe molecules can be targeted to the leads through
chemical and electrical methods. The probes may be targeted to the
leads by using a chemical reaction for attaching the probe or
nucleotide to the lead which preferably binds the probe or
nucleotide to the lead rather than the support material.
Alternatively, the probe or nucleotide may be targeted to the lead
by building up a charge on the lead which electrostatically
attracts the probe or nucleotide.
[0035] Nucleases can be used to remove probes which are attached to
the chip or lead in the wrong position. More particularly, a target
nucleic acid molecule may be added to the probes. Targets which
bind at both ends to probes, one end to each lead, will have no
free ends and will be resistant to exonuclease digestion. However,
probes which are positioned so that the target cannot contact both
leads will be bound only one end, leaving the molecule subject to
digestion. Thus, improperly located probes can be removed while
protecting the properly located probes. After the protease is
removed or inactivated the target nucleic acid molecule can be
removed and the device is ready for use.
[0036] Interest has been growing in the fabrication of microfluidic
devices. Typically, advances in the semiconductor manufacturing
arts have been translated to the fabrication of micromechanical
structures, e.g., micropumps, microvalves and the like, and
microfluidic devices including miniature chambers and flow
passages.
[0037] A number of researchers have attempted employ these
microfabrication techniques in the miniaturization of some of the
processes involved in genetic analysis in particular. For example,
published PCT Application No. WO 94/05414, to Northrup and White,
incorporated herein by reference in its entirety for all purposes,
reports an integrated micro-PCR apparatus for collection and
amplification of nucleic acids from a specimen. U.S. Pat. No.
5,304,487 to Wilding et al., and U.S. Pat. No. 5,296,375 to Kricka
et al., discuss devices for collection and analysis of cell
containing samples. Similar techniques can be used to produce chips
which can accept a sample, release the nucleic acid molecules and
then detect the target sequences.
[0038] Microfluidic devices are disclosed in U.S. Pat. No.
6,046,056, which is hereby incorporated by reference. The devices
includes a series of channels fabricated into the surface of the
substrate. At least one of these channels will typically have very
small cross sectional dimensions, e.g., in the range of from about
0.1 micrometer to about 500 micrometers. Preferably the
cross-sectional dimensions of the channels will be in the range of
from about 0.1 to about 200 micrometers and more preferably in the
range of from about 0.1 to about 100 micrometers. In particularly
preferred aspects, each of the channels will have at least one
cross-sectional dimension in the range of from about 0.1
micrometers to about 100 micrometers. Although generally shown as
straight channels, it will be appreciated that in order to maximize
the use of space on a substrate, serpentine, saw tooth or other
channel geometries, to incorporate effectively longer channels in
shorter distances.
[0039] Manufacturing of these microscale elements into the surface
of the substrates may generally be carried out by any number of
microfabrication techniques that are well known in the art. For
example, lithographic techniques may be employed in fabricating,
e.g., glass, quartz or silicon substrates, using methods well known
in the semi-conductor manufacturing industries such as
photolithographic etching, plasma etching or wet chemical etching.
Alternatively, micromachining methods such as laser drilling,
micromilling and the like may be employed.
[0040] Similarly, for polymeric substrates, well known
manufacturing techniques may also be used. These techniques include
injection molding or stamp molding methods where large numbers of
substrates may be produced using, e.g., rolling stamps to produce
large sheets of microscale substrates or polymer microcasting
techniques where the substrate is polymerized within a
micromachined mold.
[0041] The devices will typically include an additional planar
element which overlays the channeled substrate enclosing and
fluidly sealing the various channels to form conduits. Attaching
the planar cover element may be achieved by a variety of means,
including, e.g., thermal bonding, adhesives or, in the case of
certain substrates, e.g., glass, or semi-rigid and non-rigid
polymeric substrates, a natural adhesion between the two
components. The planar cover element may additionally be provided
with access ports and/or reservoirs for introducing the various
fluid elements needed for a particular screen.
[0042] The device may also include reservoirs disposed and fluidly
connected at the ends of the channels. A sample channel is used to
introduce the test compounds into the device. The introduction of a
number of individual, discrete volumes of compounds into the sample
may be carried out by a number of methods. For example,
micropipettors may be used to introduce the test compounds into the
device. In preferred aspects, an electropipettor may be used which
is fluidly connected to sample channel. Generally, an
electropipettor utilizes electroosmotic fluid direction, to
alternately sample a number of test compounds, or subject
materials, and spacer compounds. The pipettor then delivers
individual, physically isolated samples into the sample channel for
subsequent manipulation within the device.
[0043] Alternatively, the sample channel may be individually
fluidly connected to a plurality of separate reservoirs via
separate channels. The separate reservoirs each contain a reactant
compound, such as proteins or detergents, with additional
reservoirs being provided for appropriate spacer compounds. The
test compounds, reactant compounds, and/or spacer compounds are
then transported from the various reservoirs into the sample
channels using appropriate fluid direction schemes.
[0044] The sample collection portion of a device of the present
invention, whether or not on a micro scale, generally provides for
the identification of the sample, while preventing contamination of
the sample by external elements, or contamination of the
environment by the sample. Generally, this is carried out by
introducing a sample for analysis, e.g., preamplified sample,
tissue, blood, saliva, etc., directly into a sample collection
chamber within the device. Typically, the prevention of
cross-contamination of the sample may be accomplished by directly
injecting the sample into the sample collection chamber through a
sealable opening, e.g., an injection valve, or a septum. Generally,
sealable valves are preferred to reduce any potential threat of
leakage during or after sample injection. Alternatively, the device
may be provided with a hypodermic needle integrated within the
device and connected to the sample collection chamber, for direct
acquisition of the sample into the sample chamber. This can
substantially reduce the opportunity for contamination of the
sample.
[0045] In addition to the foregoing, the sample collection portion
of the device may also include reagents and/or treatments for
neutralization of infectious agents, stabilization of the specimen
or sample, pH adjustments, and the like. Stabilization and pH
adjustment treatments may include, e.g., introduction of heparin to
prevent clotting of blood samples, addition of buffering agents,
addition of protease or nuclease inhibitors, preservatives and the
like. Such reagents may generally be stored within the sample
collection chamber of the device or may be stored within a
separately accessible chamber, wherein the reagents may be added to
or mixed with the sample upon introduction of the sample into the
device. These reagents may be incorporated within the device in
either liquid or lyophilized form, depending upon the nature and
stability of the particular reagent used.
[0046] For those embodiments where whole cells, viruses or other
tissue samples are being analyzed, it will typically be necessary
to extract the nucleic acids from the cells or viruses, prior to
continuing with the various sample preparation operations.
Accordingly, following sample collection, nucleic acids may be
liberated from the collected cells, viral coat, etc., into a crude
extract, followed by additional treatments to prepare the sample
for subsequent operations, e.g., denaturation of contaminating (DNA
binding) proteins, purification, filtration, desalting, and the
like.
[0047] Liberation of nucleic acids from the sample cells or
viruses, and denaturation of DNA binding proteins may generally be
performed by physical or chemical methods. For example, chemical
methods generally employ lysing agents to disrupt the cells and
extract the nucleic acids from the cells, followed by treatment of
the extract with chaotropic salts such as guanidinium
isothiocyanate or urea to denature any contaminating and
potentially interfering proteins. Generally, where chemical
extraction and/or denaturation methods are used, the appropriate
reagents may be incorporated within the extraction chamber, a
separate accessible chamber or externally introduced.
[0048] Alternatively, physical methods may be used to extract the
nucleic acids and denature DNA binding proteins. U.S. Pat. No.
5,304,487, herein incorporated by reference, discusses the use of
physical protrusions within microchannels or sharp edged particles
within a chamber or channel to pierce cell membranes and extract
their contents. More traditional methods of cell extraction may
also be used, e.g., employing a channel with restricted
cross-sectional dimension which causes cell lysis when the sample
is passed through the channel with sufficient flow pressure.
Alternatively, cell extraction and denaturing of contaminating
proteins may be carried out by applying an alternating electrical
current to the sample. More specifically, the sample of cells is
flowed through a microtubular array while an alternating electric
current is applied across the fluid flow. A variety of other
methods may be utilized within the device of the present invention
to effect cell lysis/extraction, including, e.g., subjecting cells
to ultrasonic agitation, or forcing cells through microgeometry
apertures, thereby subjecting the cells to high shear stress
resulting in rupture.
[0049] Following extraction, it will often be desirable to separate
the nucleic acids from other elements of the crude extract, e.g.,
denatured proteins, cell membrane particles, and the like. Removal
of particulate matter is generally accomplished by filtration,
flocculation or the like. A variety of filter types may be readily
incorporated into the device. Further, where chemical denaturing
methods are used, it may be desirable to desalt the sample prior to
proceeding to the next step. Desalting of the sample, and isolation
of the nucleic acid may generally be carried out in a single step,
e.g., by binding the nucleic acids to a solid phase and washing
away the contaminating salts or performing gel filtration
chromatography on the sample. Suitable solid supports for nucleic
acid binding include, e.g., diatomaceous earth, silica, or the
like. Suitable gel exclusion media is also well known in the art
and is commercially available from, e.g., Pharmacia and Sigma
Chemical. This isolation and/or gel filtration/desalting may be
carried out in an additional chamber, or alternatively, the
particular chromatographic media may be incorporated in a channel
or fluid passage leading to a subsequent reaction chamber.
[0050] Alternatively, the interior surfaces of one or more fluid
passages or chambers may themselves be derivatized to provide
functional groups appropriate for the desired purification, e.g.,
charged groups, affinity binding groups and the like.
[0051] In a preferred embodiment of the invention, ligation methods
may be used to specifically identify single base differences in
sequences. Previously, methods of identifying known target
sequences by probe ligation methods have been reported. U.S. Pat.
No. 4,883,750 to N. M. Whiteley et al.; D. Y. Wu et al., Genomics,
4:560 (1989); U. Landegren et al., Science, 241:1077 (1988); and E.
Winn-Deen et al., Clin. Chem., 37:1522 (1991), which are hereby
incorporated by reference. In one approach, known as
oligonucleotide ligation assay ("OLA"), two probes or probe
elements which span a target region of interest are hybridized to
the target region. Where the probe elements basepair with adjacent
target bases, the confronting ends of the probe elements can be
joined by ligation, e.g., by treatment with ligase. The ligated
probe element is then assayed, evidencing the presence of the
target sequence.
[0052] In the present invention, one or both probes may be designed
to specifically recognize a variation in the sequence at the end of
the probe. After the target binds to the probes, the target is
treated with nucleases to remove the ends of the molecules which do
not bind to the probes. The junction is then treated with ligase.
If the complimentary sequence is present at the end of the probe,
the ligase will ligate the target to the probe. The test chamber
can then be heated up to denature non-ligated targets. Detection of
the specific target can then be carried out.
[0053] In one embodiment of the invention, the probe set is
contacted with a target nucleic acid molecule and after
hybridization the nucleic acid molecules are coated with a
conductor, such as a metal, as described in U.S. Patent
Applications Ser. Nos. 60/095,096, 60/099,506, or Ser. No.
09/315,750 which are hereby incorporated by reference. The coated
nucleic acid molecule can then conduct electricity across the gap
between the pair of probes, thus producing a detectable signal
indicative of the presence of a target nucleic acid molecule.
[0054] Braun demonstrated that silver could be deposited along a
DNA molecule. A three-step process is used. First, silver is
selectively localized to the DNA molecule through a Ag+/Na+
ion-exchange (Barton, in Bioinorganic Chemistry (eds Bertini, et
al.) ch. 8 (University Science Books, Mill Valley, 1994, which is
hereby incorporated by reference) and complexes are formed between
the silver and the DNA bases (Spiro (ed.) Nucleic Acid-Metal Ion
Interactions (Wiley Interscience, New York 1980; Marzeilli, et al.,
J. Am. Chem. Soc. 99:2797 (1977); Eichorn (ed.) Inorganic
Biochemistry, Vol. 2, ch 33-34 (Elsevier, Amsterdam, 1973), which
are hereby incorporated by reference). The ion-exchange process may
be monitored by following the quenching of the fluorescence signal
of the labeled DNA. The silver ion-exchanged DNA is then reduced to
form aggregates with bound to the DNA skeleton. The silver
aggregates are further developed using standard procedures, such as
those used in photographic chemistry (Holgate, et al., J.
Histochem. Cytochem. 31:938 (1983); Birell, et al., J. Histochem.
Cytochem. 34:339 (1986), which are hereby incorporated by
reference).
[0055] The nucleic acid molecule itself may have some conductive
properties of its own. These properties may be modified to reduce
any detrimental effects on the function of the electronic circuit
(Meade, et al, U.S. Pat. No. 5,770,369, "Nucleic Acid Mediated
Electron Transfer" (1998), which is hereby incorporated by
reference). Modification of the electrical properties of the
nucleic acid molecule may be made by intercalating compounds
between the bases of the nucleic acid molecule, modifying the
sugar-phosphate backbone, or by cleaving the nucleic acid molecule
after the circuit elements are formed. Cleavage of the nucleic acid
molecule may be accomplished by irradiation, chemical treatment, or
enzymatic degradation. Irradiation using gamma-radiation is
preferred because radiation may penetrate materials coating the
nucleic acid molecule.
[0056] In another aspect of the invention, the electrical
conductivity of nucleic acid molecules is relied upon to transmit
the electrical signal. Hans-Werner Fink and Christian
Schoenenberger reported in Nature (1999), which is hereby
incorporated by reference, that double-stranded DNA conducts
electricity like a semiconductor. This flow of current can be
sufficient to construct a simple switch. The present invention
provides an electronic detector based upon such a nucleic acid
switch, which will indicate whether or not a target nucleic acid
molecule is present within a sample.
[0057] Probes to the target nucleic acid molecule are immobilized
within an electrical circuit. The probes are physically located at
a distance sufficient that they cannot come into contact with one
another. The sample to be tested is contacted with the probes. If a
nucleic acid molecule is present in the sample which has sequences
homologous or complementary to the two probes, the nucleic acid
molecule can bridge the gap between the probes. The detection unit
can then detect an electrical current which can flow through the
nucleic acid molecule. A computer unit can detect the presence of
the nucleic acid molecule as an "on" switch, while an unbridged
probe set would be an "off" switch. The information is processed by
a digital computer which correlates the status of the switch with
the presence of a particular target. The computer can also analyze
the results from several switches specific for the same target, to
determine specificity of binding and target concentration. The
information could be quickly identified to the user by indicating
the presence or absence of the biological material, organism,
mutation, or other target of interest on the nucleic acid
molecule.
[0058] A detection device could comprise numerous different probe
sets which could detect a wide variety of targets. Thus a detection
device could screen for multiple target DNA molecules. For example,
a detection device could have probe sets directed at multiple
pathogenic organisms. In that way, a sample could be screened for
several pathogens simultaneously. Each probe set would be a
separate switch which would indicate the presence or absence of the
complimentary nucleic acid molecule.
[0059] A cell sample can be prepared by either chemical (including
enzymatic) or physical disruption, or a combination thereof. After
lysis the sample can be further processed. For example, the sample
can be treated with RNase to remove any RNA to limit detection to
DNA.
[0060] Prior to or at the point of contact with the probes, the
nucleic acid molecules in the sample are denatured. Denaturation is
preferentially carried out by heat treatment. Denaturation can also
be carried out by varying the ionic concentration of the carrier
solution or by a combination of ionic and heat treatment.
[0061] The present invention also has the advantage of being used
for multiple samples. The probe sets can be recycled by stripping
the target DNAs from the probe sets. In a preferred embodiment the
stripping is accomplished by increasing temperature and/or salt
concentration. The probe set is then ready for analysis of an
additional sample.
[0062] The nucleic acid molecule of the present invention is
preferentially a DNA or RNA molecule. In the present invention,
preferred nucleic acid molecules include RNA and DNA. RNA detection
may allow for more sensitivity since RNA transcripts may be at
higher levels. Also included within the invention are chemically
modified nucleic acid molecules or nucleic acid analogs. Such RNA
or DNA analogs comprise but are not limited to 2'-O-alkyl sugar
modifications, methylphosphonate, phosphorothioate,
phosphorodithioate, formacetal, 3'-thioformacetal, sulfone,
sulfamate, and nitroxide backbone modifications, amides, and
analogs wherein the base moieties have been modified. In addition,
analogs of oligomers may be polymers in which the sugar moiety has
been modified or replaced by another suitable moiety, resulting in
polymers which include, but are not limited to, polyvinyl backbones
(Pitha et al., "Preparation and Properties of Poly
(I-vinylcytosine)," Biochim. Biophys. Acta, 204:381-8 (1970); Pitha
et al., "Poly(1-vinyluracil): The Preparation and Interactions with
Adenosine Derivatives," Biochim. Biophys. Acta, 204:39-48 (1970),
which are hereby incorporated by reference), morpholino backbones
(Summerton, et al., "Morpholino Antisense Oligomers: Design,
Preparation, and Properties," Antisense Nucleic Acid Drug Dev.,
7:187-9 (1997), which is hereby incorporated by reference) and
peptide nucleic acid (PNA) analogs (Stein et al., "A Specificity
Comparison of Four Antisense Types: Morpholino, 2'-O-methyl RNA,
DNA, and Phosphorothioate DNA," J. Antisense Nucleic Acid Drug
Dev., 7:151-7 (1997); Egholm et al., "Peptide Nucleic Acids
(PNA)-Oligonucleotide Analogues with an Achiral Peptide Backbone,"
J. Am. Chem. Soc., 114:1895-1897 (1992); Faruqi et al., "Peptide
Nucleic Acid-Targeted Mutagenesis of a Chromosomal Gene in Mouse
Cells," Proc. Natl. Acad. Sci. USA, 95:1398-403 (1998); Christensen
et al., "Solid-Phase Synthesis of Peptide Nucleic Acids," J. Pept.
Sci., 1:175-83 (1995); Nielsen et al., "Peptide Nucleic Acid (PNA).
A DNA Mimic with a Peptide Backbone," Bioconjug. Chem., 5:3-7
(1994), which are hereby incorporated by reference). In addition
linkages may contain the following exemplary modifications: pendant
moieties, such as, proteins (including, for example, nucleases,
toxins, antibodies, signal peptides and poly-L-lysine);
intercalators (e.g., acridine and psoralen), chelators (e.g.,
metals, radioactive metals, boron and oxidative metals),
alkylators, and other modified linkages (e.g., alpha anomeric
nucleic acids). Such analogs include various combinations of the
above-mentioned modifications involving linkage groups and/or
structural modifications of the sugar or base for the purpose of
improving RNAseH-mediated destruction of the targeted RNA, binding
affinity, nuclease resistance, and or target specificity.
[0063] In one embodiment, the bridging nucleic acid molecule can be
made double stranded by adding a segment of a nucleic acid molecule
which is complimentary to the region of the target nucleic acid
molecule located between the sequences complimentary to the probes.
Ligase can be used to ligate the fragments into one molecule. The
device may be recycled by passing through a restriction
endonuclease to release the bridging nucleic acid molecule.
Alternatively, a polymerase can be used to fill in the
complimentary sequence. In that case, the solution must contain
nucleotides for the synthesis of the complimentary strand.
[0064] Each probe set consists of two probes. Each probe may
consist of one or more copies of the oligonucleotide, where all the
copies for that probe attach to the circuit so that electrical
current can be carried through the probe and to the circuit. A
connection between any of the oligonucleotides in one probe with
any of the oligonucleotides in the other probe of the set will
complete the circuit producing an "on" signal. If the probes
consist of multiple copies of the oligonucleotides and/or if
multiple probes are used, the device can be used to quantitate the
level of the target nucleic acid molecule in the sample, by the
signal strength or the number of activated switches.
[0065] The number of probes may be increased in order to determine
concentrations of the target nucleic acid molecule. For example,
several thousand repeated probes may be produced in the detection
unit. The circuit would be able to count the number of occupied
sites. Calculations could be done by the unit to determine the
concentration of the target molecule.
[0066] The present invention can be used for numerous applications,
such as detection of pathogens. For example, samples may be
isolated from drinking water or food and rapidly screened for
infectious organisms. This invention may also be used for DNA
sequencing using hybridization techniques. Such methods are
described in U.S. Pat. No. 5,837,832, which is hereby incorporated
by reference. The present invention may be used to screen for
mutations or polymorphisms in samples isolated from patients.
[0067] The present invention may also be used for food and water
testing. In recent times, there have been several large recalls of
tainted meat products. The electronic DNA detection system can be
used for the in-process detection of pathogens in foods and the
subsequent disposal of the contaminated materials. This could
significantly improve food safety, prevent food borne illnesses and
death, and avoid costly recalls. Chips with probes that can
identify common food borne pathogens, such as Salmonella and E.
Coli, could be designed for use within the food industry.
[0068] In yet another embodiment, the present invention can be used
for real time detection of biological warfare agents: With the
recent concerns of the use of biological weapons in a theater of
war and in terrorist attacks, the device could be configured into a
personal sensor for the combat soldier or into a remote sensor for
advanced warnings of a biological threat. The devices which can be
used to specifically identity of the agent, can be coupled with a
modem to send the information to another location. Mobile devices
may also include a global positioning system to provide both
location and pathogen information.
[0069] In yet another embodiment, the present invention may be used
to identify an individual. A series of probes, of sufficient number
to distinguish individuals with a high degree of reliability, are
placed within the device. Various polymorphism sites are used.
Preferentially, the device can determine the identity to a
specificity of greater than one in I million, more preferred is a
specificity of greater than one in one billion, even more preferred
is a specificity of greater than one in ten billion.
[0070] As an example, a flow chart is provided indicating how a
cell sample can be tested for the presence of a target nucleic acid
molecule:
[0071] 1. Inject sample
[0072] 2. Lyse cells
[0073] 3. Process lysate
[0074] 4. Denature nucleic acid molecules
[0075] 4. Contact sample with probe sets--under stringent
conditions
[0076] 5. Determine whether current can travel between a probe
set
[0077] 6. Correlate the current signal with a positive
identification of the target DNA
[0078] Note that not all steps are required depending upon the
application. For example, lysis is only needed if the DNA is still
within a cell.
[0079] Control probe sets can be utilized to verify that the system
is working appropriately. The probe sets can recognize sequences
known to occur within the sample or be nucleic acid molecules which
are added to the sample.
[0080] Controls are especially useful to determine the presence of
sequences having a polymorphism. Control nucleic acid molecules
lacking the polymorphism may be compared in a separate test. In a
preferred embodiment, the control sequence is tested at the same
time in a separate chamber in the device.
[0081] The correct control sequence will hybridize to the probes at
a slightly higher temperature. This difference can be used to
differentiate the single base mutant from the correct sequence. The
device will indicate binding by the correct sequence at a
temperature where the mutant sequence cannot bind. However, at a
lower temperature, both sequences will bind.
[0082] In yet another embodiment, the nucleotide probes on the
substrate may be randomly chosen. A linker nucleic acid molecule
comprising a complimentary sequence to the substrate bound probe
and a sequence complimentary to the target nucleic acid molecule
(See FIG. 2). Thus the linker can be used to make the probe
sequence able to detect any target nucleic acid sequence without
having to modify the device itself. Rather the linker molecule may
be bound to the substrate bound nucleic acid molecule either before
or together with the sample to be tested. If desired the linker may
be ligated to the substrate bound probe. This would allow for the
reuse of the linker with multiple samples.
[0083] The present invention can be used to monitor gene expression
in cells. The level of RNA is determined using multiple switches
with probes complimentary to the target RNA molecule. Samples can
be taken at various times after a stimulus or at different stages
of development.
[0084] In yet another embodiment, the present invention can be used
to sequence nucleic acid molecules. Sequencing by hybridization
(SBH) is most efficiently practiced by attaching many probes to a
surface to form an array in which the identity of the probe at each
site is known. A labeled target DNA or RNA is then hybridized to
the array, and the hybridization pattern is examined to determine
the identity of all complementary probes in the array. Contrary to
the teachings of the prior art, which teaches that mismatched
probe/target complexes are not of interest, the present invention
provides an analytical method in which the hybridization signal of
mismatched probe/target complexes identifies or confirms the
identity of the perfectly matched probe/target complexes on the
array.
[0085] Techniques for sequencing a nucleic acid using a probe array
have been disclosed in PCT Application No. 92/10588, which is
hereby incorporated by reference. Each probe is located at a
positionally distinguishable location on the substrate. When the
labeled target is exposed to the substrate, it binds at locations
that contain complementary nucleotide sequences. Through knowledge
of the sequence of the probes at the binding locations, one can
determine the nucleotide sequence of the target nucleic acid. The
technique is particularly efficient when very large arrays of
nucleic acid probes are utilized.
[0086] In a preferred embodiment, the device consists of a
detection chip having the microfluidic structures needed to release
the nucleic acid molecules from a sample. The nucleic acid
molecules are introduced into a chamber with the detection system
having the probes. The detection switches are connected to a
processor which can analyze the results from the hybridization
reactions. A user interface, such as a screen is provided for the
user to read the results. In addition, the device may have
additional information in memory or accessible by modem regarding
the organism or individual from which the target nucleic acid
molecule was derived.
EXAMPLES
Example 1
Preparation of a Sample to Detect Pathogens
[0087] A sample to be tested is isolated. A common sample would be
a blood sample from a patient. The sample is injected into the
device. The sample moves into a chamber where it is treated
chemically, with detergents, and enzymatically, with proteases to
free nucleic acid molecules from cells in the sample. Heat
treatment is also used to facilitate the release of the nucleic
acid molecules. For that reason, proteins used in the present
invention are preferably thermostable. The mixture may then pass
though a filter on the chip to partially purify the nucleic acid
molecules.
Example 2
Preparation of Oligonucleotide Probe Sets
[0088] Each oligonucleotide probe set is selected so that the two
probes are complimentary to a portion of the target nucleic acid
molecule and so that the two portions of the target nucleic acid
molecule are located sufficiently far apart that the nucleic acid
molecule can bridge the gap between the two probes on the device
when they are both bound. The complimentary sequences will be
chosen such that there is some additional length to allow the
target nucleic acid molecule to move freely when bound by one
probe, so that it may access the second probe. Preferably the
molecule will not be much longer than needed to easily bridge the
gap. As the length of the molecule increases the chance of it
locating the second probe decreases, because the effective
concentration of the binding site on the target molecule decreases
as the volume in which it can move increases.
[0089] Each probe set will be attached to a substrate so that they
are positioned as discussed above.
Example 3
Testing for the Presence of the Target Nucleic Acid Molecule
[0090] The probe sets will be contacted with the nucleic acid
molecules. The test chamber has a small volume to facilitate
binding of the target to the probe. To increase the chance of
binding, the sample is circulated multiple times through the test
chamber. The sample will flow through a test chamber containing the
probe sets, at a flow rate sufficiently low to allow the target
nucleic acid molecules to bind to a probe. Conditions are
determined by the length and sequence of the probe.
[0091] The conditions will be set at a level where the stringency
is sufficient to eliminate non-specific binding to the probes. The
target nucleic acid molecule is contacted with the probes under
stringent conditions. The stringent conditions for hybridization
are by the nucleic acid, salt, and temperature. These conditions
are well known in the art and may be-altered in order to identify
or detect identical or related polynucleotide sequences. Numerous
equivalent conditions comprising either low or high stringency
depend on factors such as the length and nature of the sequence
(DNA, RNA, base composition), nature of the target (DNA, RNA, base
composition), milieu (in solution or immobilized on a solid
substrate), concentration of salts and other components (e.g.,
formamide, dextran sulfate and/or polyethylene glycol), and
temperature of the reactions. One or more factors be may be varied
to generate conditions of either low or high stringency different
from, but equivalent to, the above listed conditions.
[0092] The test chamber is then rinsed with a solution to remove
unbound nucleic acid molecules. A solution which is non-conducting
lowers the level of false positives by cutting down on conductivity
mediated by the buffer.
[0093] A current is then applied at one lead while a detector looks
for a signal at the other lead. A current between the two leads is
indicative of the presence of the target nucleic acid molecule.
[0094] This written description uses examples to disclose the
invention, including the best mode, and also to enable any person
skilled in the art to practice the invention, including making and
using any devices or systems and performing any incorporated
methods. The patentable scope of the invention is defined by the
claims, and may include other examples that occur to those skilled
in the art. Such other examples are intended to be within the scope
of the claims if they have structural elements that do not differ
from the literal language of the claims, or if they include
equivalent structural elements with insubstantial differences from
the literal language of the claims.
* * * * *