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| United States Patent | 6048690 |
| Link to this page | http://www.wikipatents.com/6048690.html |
| Inventor(s) | Heller; Michael J. (Encinitas, CA); Tu; Eugene (San Diego, CA); Sosnowski; Ronald G. (Coronado, CA); O'Connell; James P. (Del Mar, CA) |
| Abstract | Methods for electronic perturbation of fluorescence, chemilluminescence and
other emissive materials provide for molecular biological analysis. In a
preferred method for hybridization analysis of a sample, an electronic
stringency control device is used to perform the steps of: providing the
sample, a first probe with a fluorescent label and a second probe with a
label under hybridization conditions on the electronic stringency control
device, forming a hybridization product, subjecting the hybridization
product to an electric field force, monitoring the fluorescence from the
hybridization product, and analyzing the fluorescent signal. The label
preferably serves as a quencher for the fluorescent label. In yet another
aspect of this invention, a method for achieving electronic fluorescence
perturbation on an electronic stringency control device comprising the
steps of: locating a first polynucleotide and a second polynucleotide
adjacent the electronic stringency control device, the first
polynucleotide and second polynucleotide being complementary over at least
a portion of their lengths and forming a hybridization product, the
hybridization product having an associated environmental sensitive
emission label, subjecting the hybridization product and label to a
varying electrophoretic force, monitoring the emission from the label, and
analyzing the monitored emission to determine the electronic fluorescence
perturbation effect. In yet another aspect of this invention, a method is
provided for electronic perturbation catalysis of substrate molecules on
an electronic control device containing at least one microlocation
comprising the steps of: immobilizing on the microlocation an arrangement
of one or more reactive groups, exposing the reactive groups to a solution
containing the substrate molecules of interest, and applying an electronic
pulsing sequence which causes charge separation between the reactive
groups to produce a catalytic reaction on the substrate molecules. |
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Title Information  |
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| Publication Date |
April 11, 2000 |
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| Parent Case |
RELATED APPLICATION INFORMATION
This application is a continuation-in-part application of application Ser.
No. 08/534,454, filed Sep. 27, 1995, entitled "Methods for Hybridization
Analysis Utilizing Electrically Controlled Hybridization" U.S. Pat. No.
5,849,486, which is a continuation-in-part of application Ser. No.
08/304,657, filed Sep. 9, 1994, entitled, as amended, "Molecular
Biological Diagnostic Systems Including Electrodes", U.S. Pat. No.
5,632,957, which is a continuation-in-part of application Ser. No.
08/271,882, filed Jul. 7, 1994, entitled, as amended, "Methods for
Electronic Stringency Control for Molecular Biological Analysis and
Diagnostics", now allowed, which is a continuation-in-part of application
Ser. No. 08/146,504, filed Nov. 1, 1993, entitled, as amended, "Active
Programmable Electronic Devices for Molecular Biological Analysis and
Diagnostics", U.S. Pat. No. 5,605,662, and also application Ser. No.
08/703,601, filed Aug. 23, 1996, entitled "Hybridization of Polynucleotide
Conjugated with Chromophores and Fluorophores to Generate Donor-to-Donor
Energy Transfer System", U.S. Pat. No. 5,849,489, which is a continuation
of application Ser. No. 08/232,233, filed May 5, 1994, entitled
"Hybridization of Polynucleotide Conjugated with Chromophores and
Fluorophores to Generate Donor-to-Donor Energy Transfer System", now
issued as U.S. Pat. No. 5,565,322, which is a continuation-in-part of
application Ser. No. 07/790,262, filed Nov. 7, 1991, entitled
"Self-Organizing Molecular Photonic Structures Based on Chromophore- and
Fluorophore-Containing Polynucleotide and Methods of Their Use", now
issued as U.S. Pat. No. 5,532,129 (via continuation application Ser. No.
08/250,951, filed May 27, 1994) and also application Ser. No. 08/258,168,
filed Aug. 25, 1994, entitled "DNA Optical Storage", now issued as U.S.
Pat. No. 5,787,032, all incorporated herein by reference as if fully set
forth herein. |
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Title Information |
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Description |
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FIELD OF THE INVENTION
This invention relates to systems, devices, methods, and mechanisms for performing multi-step molecular biological analysis, nucleic acid hybridization reactions, nucleic acid sequencing, and the catalysis of biomolecular, organic and inorganic
reactions. More particularly, the molecular biological type analysis involves electronic fluorescent perturbation mechanisms for the detection of DNA hybrids, point mutations, deletions or repeating sequences in nucleic acid hybridization reactions,
electronic fluorescent perturbation mechanisms for sequencing of DNA and RNA molecules, and electric field based catalytic mechanisms for biomolecular, biopolymer and other chemical reactions.
BACKGROUND OF THE INVENTION
Molecular biology comprises a wide variety of techniques for the analysis of nucleic acid and protein. Many of these techniques and procedures form the basis of clinical diagnostic assays and tests. These techniques include nucleic acid
hybridization analysis, restriction enzyme analysis, genetic sequence analysis, and the separation and purification of nucleic acids and proteins (See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2 Ed., Cold
spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
Most of these techniques involve carrying out numerous operations (e.g., pipetting, centrifugations, electrophoresis) on a large number of samples. They are often complex and time consuming, and generally require a high degree of accuracy. Many
a technique is limited in its application by a lack of sensitivity, specificity, or reproducibility. For example, these problems have limited many diagnostic applications of nucleic acid hybridization analysis.
The complete process for carrying out a DNA hybridization analysis for a genetic or infectious disease is very involved. Broadly speaking, the complete process may be divided into a number of steps and substeps. In the case of genetic disease
diagnosis, the first step involves obtaining the sample (blood or tissue). Depending on the type of sample, various pre-treatments would be carried out. The second step involves disrupting or lysing the cells, which then release the crude DNA material
along with other cellular constituents. Generally, several sub-steps are necessary to remove cell debris and to purify further the crude DNA. At this point several options exist for further processing and analysis. One option involves denaturing the
purified sample DNA and carrying out a direct hybridization analysis in one of many formats (dot blot, microbead, microliter plate, etc.). A second option, called Southern blot hybridization, involves cleaving the DNA with restriction enzymes,
separating the DNA fragments on an electrophoretic gel, blotting to a membrane filter, and then hybridizing the blot with specific DNA probe sequences. This procedure effectively reduces the complexity of the genomic DNA sample, and thereby helps to
improve the hybridization specificity and sensitivity. Unfortunately, this procedure is long and arduous. A third option is to carry out the polymerase chain reaction (PCR) or other amplification procedure. The PCR procedure amplifies (increases) the
number of target DNA sequences. Amplification of target DNA helps to overcome problems related to complexity and sensitivity in genomic DNA analysis. All these procedures are time consuming, relatively complicated, and add significantly to the cost of
a diagnostic test. After these sample preparation and DNA processing steps, the actual hybridization reaction is performed. Finally, detection and data analysis convert the hybridization event into an analytical result.
The steps of sample preparation and processing have typically been performed separate and apart from the other main steps of hybridization and detection and analysis. Indeed, the various substeps comprising sample preparation and DNA processing
have often been performed as a discrete operation separate and apart from the other substeps. Considering these substeps in more detail, samples have been obtained through any number of means, such as obtaining of full blood, tissue, or other biological
fluid samples. In the case of blood, the sample is processed to remove red blood cells and retain the desired nucleated (white) cells. This process is usually carried out by density gradient centrifugation. Cell disruption or lysis is then carried
out, preferably by the technique of sonication, freeze/thawing, or by addition of lysing reagents. Crude DNA is then separated from the cellular debris by a centrifugation step. Prior to hybridization, double-stranded DNA is denatured into
single-stranded form. Denaturation of the double-stranded DNA has generally been performed by the techniques involving heating (>Tm), changing salt concentration, addition of base (NaOH), or denaturing reagents (urea, formamide, etc.). Workers have
suggested denaturing DNA into its single-stranded form in an electrochemical cell. The theory is stated to be that there is electron transfer to the DNA at the interface of an electrode, which effectively weakens the double-stranded structure and
results in separation of the strands. See, generally, Stanley, "DNA Denaturation by an Electric Potential", U.K. patent application 2,247,889 published Mar. 18, 1992.
Nucleic acid hybridization analysis generally involves the detection of a very small number of specific target nucleic acids (DNA or RNA) with an excess of probe DNA, among a relatively large amount of complex non-target nucleic acids. The
substeps of DNA complexity reduction in sample preparation have been utilized to help detect low copy numbers (i.e. 10,000 to 100,000) of nucleic acid targets. DNA complexity is overcome to some degree by amplification of target nucleic acid sequences
using polymerase chain reaction (PCR). (See, M. A. Innis et al, PCR Protocols: A Guide to Methods and Applications, Academic Press, 1990). While amplification results in an enormous number of target nucleic acid sequences that improves the subsequent
direct probe hybridization step, amplification involves lengthy and cumbersome procedures that typically must be performed on a stand alone basis relative to the other substeps. Substantially complicated and relatively large equipment is required to
perform the amplification step.
The actual hybridization reaction represents the most important and central step in the whole process. The hybridization step involves placing the prepared DNA sample in contact with a specific reporter probe, at a set of optimal conditions for
hybridization to occur to the target DNA sequence. Hybridization may be performed in any one of a number of formats. For example, multiple sample nucleic acid hybridization analysis has been conducted on a variety of filter and solid support formats
(See G. A. Beltz et al., in Methods in Enzvmology, Vol. 100, Part B, R. Wu, L. Grossman, K. Moldave, Eds., Academic Press, New York, Chapter 19, pp. 266-308, 1985). One format, the so-called "dot blot" hybridization, involves the non-covalent
attachment of target DNAs to filter, which are subsequently hybridized with a radioisotope labeled probe(s). "Dot blot" hybridization gained wide-spread use, and many versions were developed (see M. L. M. Anderson and B. D. Young, in Nucleic Acid
Hybridization--A Practical Approach, B. D. Hames and S. J. Higgins, Eds., IRL Press, Washington, D.C. Chapter 4, pp. 73-111, 1985). It has been developed for multiple analysis of genomic mutations (D. Nanibhushan and D. Rabin, in EPA 0228075, Jul. 8,
1987) and for the detection of overlapping clones and the construction of genomic maps (G. A. Evans, in U.S. Pat. No. 5,219,726, Jun. 15, 1993).
New techniques are being developed for carrying out multiple sample nucleic acid hybridization analysis on micro-formatted multiplex or matrix devices (e.g., DNA chips) (see M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology,
pp. 757-758, 1992). These methods usually attach specific DNA sequences to very small specific areas of a solid support, such as micro-wells of a DNA chip. These hybridization formats are micro-scale versions of the conventional "dot blot" and
"sandwich" hybridization systems.
The micro-formatted hybridization can be used to carry out "sequencing by hybridization" (SBH) (see M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). SBH makes use of all possible n-nucleotide oligomers
(n-mers) to identify n-mers in an unknown DNA sample, which are subsequently aligned by algorithm analysis to produce the DNA sequence (R. Drmanac and R. Crkvenjakov, Yugoslav Patent Application No. 570/87, 1987; R. Drmanac et al., 4 Genomics, 114, 1989;
Strezoska et al., 88 Proc. Natl. Acad. Sci. USA 10089, 1992; and R. Dramanac and R. B. Crkvenjakov, U.S. Pat. No. 5,202,231, Apr. 13, 1993).
There are two formats for carrying out SBH. The first format involves creating an array of all possible n-mers on a support, which is then hybridized with the target sequence. The second format involves attaching the target sequence to a
support, which is sequentially probed with all possible n-mers. Both formats have the fundamental problems of direct probe hybridizations and additional difficulties related to multiplex hybridizations.
Southern, United Kingdom Patent Application GB 8810400, 1988; E. M. Southern et al., 13 Genomics 1008, 1992, proposed using the first format to analyze or sequence DNA. Southern identified a known single point mutation using PCR amplified
genomic DNA. Southern also described a method for synthesizing an array of oligonucleotides on a solid support for SBH. However, Southern did not address how to achieve optimal stringency condition for each oligonucleotide on an array.
Concurrently, Drmanac et al., 260 Science 1649-1652, 1993, used the second format to sequence several short (116 bp) DNA sequences. Target DNAs were attached to membrane supports ("dot blot" format). Each filter was sequentially hybridized with
272 labeled 10-mer and 11-mer oligonucleotides. A wide range of stringency condition was used to achieve specific hybridization for each n-mer probe; washing times varied from 5 minutes to overnight, and temperatures from 0.degree. C. to 16.degree. C.
Most probes required 3 hours of washing at 16.degree. C. The filters had to be exposed for 2 to 18 hours in order to detect hybridization signals. The overall false positive hybridization rate was 5% in spite of the simple target sequences, the reduced
set of oligomer probes, and the use of the most stringent conditions available.
A variety of methods exist for detection and analysis of the hybridization events. Depending on the reporter group (fluorophore, enzyme, radioisotope, etc.) used to label the DNA probe, detection and analysis are carried out fluorometrically,
colorimetrically, or by autoradiography. By observing and measuring emitted radiation, such as fluorescent radiation or particle emission, information may be obtained about the hybridization events. Even when detection methods have very high intrinsic
sensitivity, detection of hybridization events is difficult because of the back-ground presence of non-specifically bound materials.
In the many applications of DNA hybridization for research and diagnostics, the most difficult analysis involve the differentiation of a single base mismatch from a match target sequence. This is because the analysis involves discriminating a
small difference in one hybridized pair, the mismatch, from the match. The teachings of this invention are of particular relevance to these problems.
SUMMARY OF THE INVENTION
As a main aspect of this invention, it has been surprisingly discovered that the fluorescence signal obtained during the electronic denaturation or dehybridization of DNA hybrids is perturbed at or around the electronic power (current and
voltage) levels which are associated with the denaturation or dehybridization process. In one embodiment, the fluorescence signal perturbation phenomena appears as a rise or spike in fluorescence intensity prior to dehybridization of a fluorescent
labeled probe from a capture sequence attached to the microlocation test site. The power level, amplitude and slope of this fluorescence spike provide analytical tools for diagnosis. The combination of the fluorescence perturbation with other
measurements also indicative of the hybridization match/mismatch state, such as consideration of the electronic melting (50% fluorescence decrease during electronic stringency control) can in combination provide a more efficient and reliable
hybridization match/mismatch analysis.
In general, this controlled dehybridization or electronic stringency process results in a significant differential between the final fluorescent intensity values for the match versus the mismatch sequence. This difference in fluorescent
intensity values is used to determine a discrimination ratio, which confirms and identifies that a particular mismatch was present in the sample.
It has been discovered that the fluorescent perturbation effect (FPE) provides a powerful analytical tool for DNA hybridization analysis, particularly for the near instantaneous, e.g., less than one minute, and especially less than 5 seconds,
discrimination of match/mismatched DNA hybrids. Novel DNA sequencing applications are possible. New fluorescent donor/acceptor/quencher energy transfer mechanisms are created. New electronic catalytic mechanisms are created.
In one aspect, this invention relates to using precisely controlled electric or electrophoretic fields to cause or influence fluorophore or chromophore groups in special arrangements with molecular structures (such as nucleic acids), to produce
rapid signal variations (perturbations) which correlate with and identify small differences in these molecular structures. In a preferred method for hybridization analysis of a sample, an electronic stringency control device is used to perform the steps
of: providing the sample, a first probe with a fluorescent label and a second probe with a label under hybridization conditions on the electronic stringency control device, forming a hybridization product, subjecting the hybridization product to an
electric field force, monitoring the fluorescence from the hybridization product, and analyzing the fluorescent signal. The label preferably serves as a quencher for the fluorescent label.
Most broadly, this invention relates to integrated microelectronic systems, devices, components, electronic based procedures, electronic based methods, electronic based mechanisms, and flurophore/chromophore arrangements for: (1) molecular
biological and clinical diagnostic analyses; (2) nucleic acid sequencing applications; and (3) for carrying out catalysis of biomolecular, organic, and inorganic reactions.
More specifically, the molecular biological and clinical diagnostic analyses relate to the utilization of the electronic fluorescent perturbation based mechanisms for the detection and identification of nucleic acid hybrids, single base
mismatches, point mutations, single nucleotide polymorphisms (SNPs), base deletions, base insertions, crossover/splicing points (translocations), intron/exon junctions, restriction fragment length polymorphisms (RFLPs), short tandem repeats (STRs) and
other repeating or polymorphic sequences in nucleic acids.
More specifically, the nucleic acid sequencing applications involve utilization of the electronic fluorescent perturbation based mechanisms to elucidate base sequence information in DNA, RNA and in nucleic acid derivatives. Most particularly, to
elucidate sequence information from the terminal ends of the nucleic acid molecules. This method achieves electronic fluorescence perturbation on an electronic stringency control device comprising the steps of: locating a first polynucleotide and a
second polynucleotide adjacent the electronic stringency control device, the first polynucleotide and second polynucleotide being complementary over at least a portion of their lengths and forming a hybridization product, the hybridization product having
an associated environmental sensitive emission label, subjecting the hybridization product and label to a varying electrophoretic force, monitoring the emission from the label, and analyzing the monitored emission to determine the electronic fluorescence
perturbation effect.
More specifically, the catalytic reactions relate to the utilization of electronic based catalytic mechanisms for carrying out biomolecular, biopolymer, organic polymer, inorganic polymer, organic, inorganic, and other types of chemical
reactions. Additionally, the electronic based catalytic mechanisms can be utilized for carrying out nanofabrication, and other self-assembly or self-organizational processes. This method provides for electronic perturbation catalysis of substrate
molecules on an electronic control device containing at least one microlocation comprising the steps of: immobilizing on the microlocation an arrangement of one or more reactive groups, exposing the reactive groups to a solution containing the substrate
molecules of interest, and applying an electronic pulsing sequence which causes charge separation between the reactive groups to produce a catalytic reaction on the substrate molecules.
More generally, the present invention relates to the design, fabrication, and uses of self-addressable self-assembling microelectronic integrated systems, devices, and components which utilize the electronic mechanisms for carrying out the
controlled multi-step processing and multiplex reactions in a microscopic, semi-microscopic and macroscopic formats. These reactions include, but are not limited to, most molecular biological procedures, such as: (1) multiplex nucleic acid hybridization
analysis in reverse dot blot formats, sandwich formats, homogeneous/heterogeneous formats, target/probe formats, in-situ formats, and flow cytometry formats; (2) nucleic acid, DNA, and RNA sequencing; (3) molecular biological restriction reactions,
ligation reactions, and amplification type reactions; (4) immunodiagnostic and antibody/antigen reactions; (5) cell typing and separation procedures; and (6) enzymatic and clinical chemistry type reactions and assays.
In addition, the integrated systems, devices, and components which utilize electronic based catalytic mechanisms are able to carry out biomolecular, biopolymer and other types of chemical reactions: (1) based on electric field catalysis; and/or
(2) based on multi-step combinatorial biopolymer synthesis, including, but not limited to, the synthesis of polynucleotides and oligonucleotides, peptides, organic molecules, bio-polymers, organic polymers, mixed biopolymers/organic polymers, two and
three dimensional nanostructures, and nanostructures and micron-scale structures on or within silicon or other substrate materials.
Additionally, with respect to electronic fluorescent perturbation mechanisms, the present invention relates to unique intermolecular and intramolecular constructs and arrangements of chromophores, fluorophores, luminescent molecules or moities,
metal chelates (complexes),enzymes, peptides, and amino acids, associated with nucleic acid sequences, polypeptide sequences, and/or other polymeric materials. Of particular importance being those constructs and arrangements of fluorophores and
chromophores which produce fluorescent energy transfer, charge transfer or mechanical mechanisms which can be modulated or affected by electric or electrophoretic fields to produce fluorescent or luminescent signals which provide information about
molecular structure.
With respect to the electronic catalytic mechanisms in homogeneous (solution) or heterogeneous (solution/solid support) formats, the present invention relates to unique intermolecular and intramolecular constructs and arrangements of
chromophores, fluorophores, luminescent molecules or moities, metal chelates (complexes),enzymes, peptides, and amino acids, nucleophilic molecules or moities, electrophilic molecules or moities, general acid or base catalytic molecules or moieties, and
substrate binding site molecules and moities, associated with nucleic acid sequences, polypeptide sequences, other biopolymers, organic polymers, inorganic polymers, and other polymeric materials.
Additionally, this invention relates to the utilization of electric or electrophoretic fields to induce fluorescent perturbation based mechanisms in arrangements of fluorophores and chromophores in solid state or sol-gel state optoelectronic
devices and optical memory materials.
It is therefore an object of this invention to provide for methods and systems for improved detection and analysis of biological materials.
It is yet a further object of this invention to provide for methods which provide for the rapid and accurate discrimination between matches and mismatches in nucleic acid hybrids.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a plot of the relative fluorescent intensity as a function of applied power (microwatts) for a 20-mer oligomer duplex (100% AP).
FIG. 1B is a plot of the relative fluorescent intensity versus applied power (microwatts) for a 19-mer oligomer duplex (53% GC).
FIG. 2A is a graph of the relative fluorescent intensity verus applied power (microwatts) for a 20-mer oligomer duplex (100% AT).
FIG. 2B is a plot of the relative fluorescent intensity verus applied power (microwatts) for a 19-mer oligomer duplex (53% GC).
FIG. 3A shows a cross-sectional view of a mismatched test site having a capture probe, target DNA and a reporter probe.
FIG. 3B is a cross-sectional view of target DNA and a reporter probe with an associated fluorophore.
FIG. 3C is a graph of the fluorescent response graphing the relative fluorescent intensity as a function of time for a pulses sequence.
FIG. 4A is a cross-sectional view of a matched test site having a capture probe, target DNA and a reporter probe with an intercalated fluorophore.
FIG. 4B is a cross-sectional view of target DNA and a reporter probe with an intercalating fluorophore.
FIG. 4C is a graph of the fluorescent response showing the relative fluorescence intensity as a function of time for a pulsed sequence.
FIG. 5 shows the fluorescent intensity (% remaining Fluorescein) profiles as a function of time (seconds) for a one base mismatch and a match sequence for Ras G 22 mers during the basic electronic dehybridization process.
FIG. 6 shows the fluorescent intensity (% remaining fluorescence) as a function of time (seconds) observed during the general electronic dehybridization of match/mismatch hybrids for the Ras and RCA5 (HLA)systems.
FIG. 7A shows a graph of the normalized fluorescent intensity versus time (seconds) for match/mismatch profiles exhibiting the oscillating fluorescent perturbation effect.
FIG. 7B shows an expanded view graph of the first 12 seconds of the graph of FIG. 7A.
FIG. 8A shows a schematic representation for the hybridized arrangement of the target probe and the Bodipy Texas Red labeled reporter probe, and the position of the one base mismatch.
FIG. 8B shows a schematic representation of FIG. 8A, but where a mismatch between the target and probe is present.
FIG. 9 shows a graph of the normalized fluorescent intensity as a function of time (seconds) match/mismatch profiles exhibiting the oscillating fluorescent perturbation effect, in the presence of a second probe containing a quencher group
(Malachite Green).
FIG. 10A shows a schematic representation for the hybridized arrangement of the target probe, the Bodipy Texas Red labeled reporter probe, and the Malachite Green quencher probe.
FIG. 10B shows the schematic representation of FIG. 10A with a mismatch between the target and the probe.
FIG. 11A shows a schematic representation for the hybridized arrangement of a target probe, a labeled reporter probe and a quencher probe.
FIG. 11B shows the schematic representation of FIG. 11A with a mismatch between the target and probe.
FIG. 12 shows a sequence of steps for electronic perturbation catalysis.
DETAILED DESCRIPTION OF THE INVENTION
The APEX device as described in the various parent applications has been utilized in novel ways resulting in methods which improve the analytical or diagnostic capabilities of the device. It has been surprisingly discovered that the fluorescent
signal is perturbed during the electronic dehybridization of DNA hybrids. This method has particular application to DNA hybridization and single-base mismatch analysis. Specifically, during electronic dehybridization, also known as stringency control
or electronic stringency control, a rise or spike in the fluorescence intensity has been observed just prior to the dehybridization of the fluorescent labeled probes from capture sequences attached to the APEX chip pad.
FIGS. 1A and 1B show the results of electronic denaturization experiments run on an APEX chip having 25 test microlocations with 80 micron diameter utilizing platinum electrodes. For this use, the chip was overlaid with a 1 micron thick
avidin/agarose permeation layer. Two 5'-labeled bodipy Texas Red (Ex 590 nm, EM 630 nm) target probes were used in the experiments. The probe of FIG. 1A was a 17 mer (5'-BYTRAAATTTTAATATATAAT-3') (SEQUENCE ID NO. 1) containing 100% AT, with a melting
temperature (Tm) of 33.degree. C. The probe of FIG. 1B was a 19 mer (5'BYTRCCACGTAGAACTGCTCATC-3') (SEQUENCE ID NO. 2) containing 53% GC, with a melting temperature (Tm) of 54.degree. C. (Melting temperature or Tm refers to the temperature at which the
dehybridization process is 50% complete). The appropriate complementary biotinylated capture sequences were attached to the avidin/agarose permeation layer over several of the test pads (on the same chip). The capture probe density was .about.10.sup.8
probes per pad. The fluorescent labeled target probes, at a concentration of .about.1.0 .mu.M in 50 mM sodium phosphate (pH 7.0), 500 mM NaCl were first hybridized to the attachment probes on the 5580 chips. The chips were then thoroughly washed with
20 mM NaPO4 (pH 7.0).
Electronic denaturation was then carried out by biasing the test pad negative, and increasing the power to the test pad from .about.10.sup.-1 microwatts (.mu.W) to .about.2.times.10.sup.2 microwatts (.mu.W) over a 90 second time period. Three
pads were tested for each of the target probes. The relative change in fluorescent intensity was plotted as a function of the increasing power. In general, the electrophoretic field, force or power necessary to dehybridize a probe from its
complementary sequence correlates with the binding energy or Tm (melting temperature) for the DNA duplex. In above experiments the overall power level (.mu.W) necessary to dehybridize the 19-mer probe with 53% GC probe (Tm of 54.degree. C.) was higher
than for the 20-mer probe with 100% AT (Tm of 33.degree. C.), that is, the equivalent electronic melting point (Em) at which dehybridization is 50% complete is higher for the 53% GC probe. Also, the fluorescent perturbation (FIGS. 1A and 1B, circled
region) for the 10-mer probe with 53% GC is observed to be significantly different from that associated with the 100% AT probe.
FIGS. 2A and 2B show the results of denaturation experiments run on the APEX chip having 25 test microlocations with 20 micron deep wells to the underlying platinum electrodes. The well structures on the chip were filled with avidin/agarose
composite, forming a 20 micron deep permeation layer. The same fluorescent target probes, capture probes and protocols were used in the deep well experiments as in the operation of the device resulting in the information of FIGS. 1A and 1B. As in the
first experiments, the overall power (.mu.W) necessary to dehybridize the 19-mer probe with 53% GC (Tm of 54.degree. C.), is higher than for the 20-mer probe with 100% AT (Tm of 33.degree. C.). Also, the slope for the 100% AT probe is much shallower,
then for the 53% GC probe. The fluorescent perturbation/spike phenomena is very pronounced for the 19-mer probe with 53% GC in the deep well experiments.
The fluorescent perturbation phenomena correlates well with the sequence specificity of the dehybridization process. The power level (.mu.W) value, amplitude and slope of the fluorescent spike are useful for many aspects of hybridization
analysis including single base mismatch analysis. The fluorescent perturbation (Fp) value, namely those values associated with the fluorescence perturbation, e.g., onset value, peak height and slope, combined with the electronic melting (Em) values,
namely, the half-height value of fluorescence, provide significantly higher reliability and additional certainty to hybridization match/mismatch analysis. By combining two or more analytical measurements, a more effective and precise determination may
be made.
In the above experiments, the target probes were labeled with a Bodipy Texas Red fluorophore in their 5' terminal positions. While Bodipy TR is not a particularly environmentally sensitive fluorophore it nevertheless showed pronounced effects
during electronic denaturation. More environmentally sensitive fluorophores may be used to obtain larger perturbations in their fluorescent properties during electronic dehybridization.
The placement of a sensitive fluorescent label in optimal proximity to the initial denaturation site is preferred. By associating certain fluorescent labels in proximity to the denaturation site, as opposed to labeling at the end of the target
or probe, increased specificity and enhanced effects may result. As shown in FIGS. 3A and 4A, an intercalating fluorophore 10 may be disposed between a reporter probe 2 and target DNA 4. FIG. 3A shows the condition in which the reporter probe 2 is
mismatched from the target DNA 4 by a mismatched base 6. In each of FIGS. 3A and 4A, the capture probe 8 serves to capture the target DNA 4, with the pad 12 providing the electrophoretic action. Preferably, the intercalating fluorophore 10 would be
placed next to the single base mismatch site 6 (FIG. 3A). The intercalating type fluorescent label could be, for example, ethidium bromide and acridine derivatives, or any other known fluorescent labels consistent with the objects of this device and its
use.
FIGS. 3B and 4B show the condition of the reporter probe 2, the target DNA 4 and the mismatch base site 6 after the application of a pulse at the fluorescent perturbation value via the pad 12. The change from intercalated to the non-intercalated
environment would produce a major change in fluorescent signal intensity for certain labels like ethidium.
Furthermore, the use of a mismatch site directed fluorophore label does not require that the hybrid be completely denatured during the process. As shown in FIG. 3C and FIG. 4C, an analysis procedure is preferred in which an appropriate pulsed
"Fp" power level is applied which causes a mismatched hybridization site to partially denature and renature relative to a matched hybridization site. The procedure results in an oscillating fluorescent signal being observed for mismatch hybrid site,
while the fluorescent signal for the matched hybrid site remains unchanged. FIGS. 3C and 4C shows the relative fluorescent intensity as a function of varied applied power. This procedure provides a highly specific and discriminating method for single
base mismatch analysis. Additional advantages include: (1) longer probes (>20-mer) than those used in conventional hybridization procedures can be used in this process, (2) Probe specificity is more determined by placement of the fluorescent label
(particularly for single base mismatches), and (3) as the procedure does not require complete denaturation of the hybrid structures, each sample can be analyzed repetitively for providing a higher statistical significant data, such as through standard
averaging techniques.
Referring to FIG. 5, in the process of carrying out electronic DNA hybridization and selective dehybridization (by electronic stringency) on active DNA chip devices (e.g., on an APEX chip), it was surprisingly discovered that the fluorescence
signal from labeled reporter probes or target DNAs was perturbed during the initiation of electronic dehybridization at or around the electronic power levels (current and voltage) associated with that dehybridization process. Specifically, this
fluorescence signal perturbation shows itself often as a rise or spike in the fluorescence intensity prior to dehybridization of the fluorescent labeled probe sequence from the DNA sequence attached to the microscopic test site (microlocation) on the DNA
chip surface. The main region of fluorescence perturbation is shown in the dashed circle.
The fluorescent perturbation effect (FPE) is usually most pronounced for a one base mismatched probe sequence relative to the match probe sequence. In the general electronic hybridization and dehybridization procedure, the precisely controlled
electronic stringency process results in a significant differential between the final fluorescent intensity values for the match versus the mismatch sequence. The mismatch sequence is more effectively dehybridized and more rapidly removed from the test
location than the match sequence. In the general electronic hybridization and dehybridization process this difference in fluorescent intensity values is used to determine a discrimination ratio, which confirms and identifies that a particular mismatch
was present in the sample. The particular parameters of electric field strength (current/voltage), solution conductivity, electrode geometry and pulsing time used to produce this selective dehybridization between the match and the mismatch occur at what
is called the electronic melting temperature (Etm). The electronic dehybridization and stringency process allows match/mismatch discriminations to be carried out very rapidly (within substantially 20 to 60 seconds), compared with the classical
hybridization stringency process, which involves temperature control and stringent washing procedures, which can take hours to complete. The single base pair mismatch (single BPM)sequence is observed to decrease faster than the match sequence allowing
one to obtain a match/mismatch discrimination ratio for the pair.
Initial observations of the fluorescent perturbation effect (FPE), which occurs almost immediately upon initiation of the electronic dehybridization process, indicated that it was possible to use the FPE as a way to distinguish match/mismatched
DNA hybrids even more rapidly, typically in less than a minute, and most preferably in several seconds or less. Another very powerful and novel feature of the FPE is that this technique does not require the removal of the probe or target sequence in
order to discriminate a match from the mismatch hybrid, whereas the general electronic dehybridization process and classical hybridization techniques typically require the removal of the mismatch sequence relative to the matched sequence. A further
advantage of the FPE technique is that probes of any size can potentially be used for match/mismatch hybrid discriminations or other applications. Longer probes sequences can provide overall better hybridization stability and selectivity.
Further investigations of the fluorescent perturbation effect has revealed other aspects and advantages of this unique phenomena which include: (1) that the amplitude, frequency, and slope of this fluorescent signal can provide a powerful
analytical tool for other types of DNA hybridization analysis, in addition to the near instantaneous discrimination of single base mismatched DNA; (2) that multiple probe systems, involving a quencher probe and fluorescent acceptor probe (and donor
probes), can be used to further enhance the FPE technique; (3) that a variety of electronic pulsing sequences (DC and AC variations) can be developed which further improve and broaden the scope of FPE based analysis of DNA and other molecular structures;
(4) that the electronic fluorescence perturbation mechanism could lead to DNA sequencing applications; (5) that new arrangements of fluorescent donor/acceptor/quencher groups could be created for improved energy transfer mechanisms and applications; and
(6) that novel electronic catalytic mechanisms could be created. These are the subjects of this invention.
The basic fluorescent perturbation effect occurs generally upon the initiation of electronic denaturation of match and mismatch hybrid pairs. In the case of the Ras (ras oncogene) hybrids in FIG. 5, the mismatch nucleotide is located
approximately in the middle of the probe sequence, and the fluorescent label (Bodipy Texas Red) is covalently attached to the terminal position of the oligonucleotide sequence, approximately 10 bases from the mismatched nucleotide (see Example 1, below). Upon initiation of dehybridization process the fluorophore responds to the changing environment of the dehybridizing DNA strands by brightening. Generally, most fluorophores are somewhat sensitive to their local physical, chemical, and thermal
environments; and a number of fluorophores are found to be extremely sensitive to changes in their environment. Environmental parameters such as hydrophilicity, hydrophobicity, pH, electrostat | | |
