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EP1620575A2 - Procede de detection electrochimique de mutations de cellules somatiques - Google Patents

Procede de detection electrochimique de mutations de cellules somatiques

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Publication number
EP1620575A2
EP1620575A2 EP04760623A EP04760623A EP1620575A2 EP 1620575 A2 EP1620575 A2 EP 1620575A2 EP 04760623 A EP04760623 A EP 04760623A EP 04760623 A EP04760623 A EP 04760623A EP 1620575 A2 EP1620575 A2 EP 1620575A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
polynucleotide
probe
target
target polynucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04760623A
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German (de)
English (en)
Other versions
EP1620575A4 (fr
Inventor
Donald M. Crothers
R. Erik Holmlin
Chunnian Shi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GeneOhm Sciences Inc
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GeneOhm Sciences Inc
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Filing date
Publication date
Priority claimed from US10/429,293 external-priority patent/US20040086895A1/en
Application filed by GeneOhm Sciences Inc filed Critical GeneOhm Sciences Inc
Publication of EP1620575A2 publication Critical patent/EP1620575A2/fr
Publication of EP1620575A4 publication Critical patent/EP1620575A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to the detection of genetic mutations in somatic cells and methods of screening patients for cancer or precancer. Description of the Related Art
  • Somatic cells are definitionally distinguished from germ cells as the former are the cells which make up an individual's body while the latter are those cells which can participate in sexual reproduction. Both types of cells can experience genetic mutations under a variety of circumstances. Mutations in somatic cells are typically not passed to an individual's offspring; they are, however, often passed within an individual to the daughter cells of the mutated somatic cell through mitosis. The frequency with which somatic cells reproduce is generally related to the type of cell and to various abnormalities caused by genetic mutations in the cell. The propagation of somatic cell mutations is a principal mechanism behind most cancers.
  • Tissue of this type may be referred to as a tumor. If the cells remain in their place of origin and do not directly invade surrounding tissues, the tumor is said to be "benign.” If the tumor invades neighboring tissue and causes distant secondary growths (called metastasis), it may be termed "malignant.”
  • Such a method could facilitate earlier and more effective treatments for patients having, or at risk of developing, mutation-related disorders, including various cancers.
  • One aspect of the invention is a method for detecting a target polynucleotide, including the steps of: synthetically producing an enlarged target polynucleotide; hybridizing the target polynucleotide to a probe polynucleotide in a detection zone; and detecting the amount of polynucleotide in the detection zone to ascertain whether target polynucleotide has hybridized in the detection zone.
  • target polynucleotide is enlarged by attaching one or more polynucleotide strands to the target. Further the target polynucleotide can be enlarged by attachment of a plurality of polynucleotide strands, producing a branched structure.
  • the target polynucleotide is hybridized to more than one probe polynucleotide in the detection zone.
  • a further aspect of the invention is a method for detecting a nucleic acid analyte, including: generating an elongated reporter nucleic acid if the nucleic acid analyte is present; capturing the reporter nucleic acid with an immobilized probe that is substantially shorter than the reporter nucleic acid; and generating a signal that is a function of the size of the captured reporter nucleic acid to indicate the presence or absence of the nucleic acid analyte.
  • FIG. 1A depicts short strand duplex melting curves.
  • FIG. IB depicts long strand duplex melting curves.
  • FIG. 1C depicts melting curves in which an elongated target strand is hybridized to multiple short strand probes.
  • FIG. 2A illustrates on-chip amplification using head-to-tail polymerization.
  • FIG. 2B illustrates on-chip amplification using rolling circle amplification.
  • FIG. 2C illustrates on-chip amplification using a branch technique in conjunction with rolling circle amplification.
  • FIG. 3 shows a voltammagram which illustrates the signal enhancing effect of on- chip amplification.
  • the present invention is generally related to the detection of somatic cell mutations.
  • Preferred embodiments include the isolation of oligonucleotides from biological samples and an analysis of various oligonucleotide sequences for complementarity using an electrochemical hybridization assay. Accordingly, the knowledge that an oligonucleotide of unknown sequence is complementary to an oligonucleotide of known sequence can be used to identify the unknown sequence. Similarly, comparing to an oligonucleotide of unknown sequence to an oligonucleotide known to be healthy or "wild" can be used to characterize the unknown sequence as either wild or mutated.
  • Preferred embodiments of the present invention include the detection of polynucleotide hybridization in a detection zone.
  • Particularly preferred embodiments feature the use of a ruthenium complex in conducting an electrochemical assay.
  • an electrochemical assay detects nucleic acid hybridization using the general technique of Steele et al. (1998, Anal. Chem. 70:4670-4677), hereby expressly incorporated by reference in its entirety.
  • a plurality of nucleic acid probes which are complementary to a sequence of interest are used.
  • probes range in length from about 10 to 25 base pairs, with a length of about 17 base pairs being most preferred.
  • the probe strands are positioned within a detection zone.
  • the detection zone includes a surface, such as an electrode, in contact with a liquid medium, wherein the probe strands are immobilized on the surface such they are also in contact with the liquid medium.
  • the surface is a gold or carbon electrode that is coated with a protein layer such as avidin or streptavidin to facilitate the attachment of the nucleic acid probe strands to the electrode.
  • This protein layer should be porous, such that it allows ions to pass from the liquid medium to the electrode and vice versa.
  • probe strands can be attached directly to the surface, for example by using a thiol linkage to covalently bind nucleic acid to a gold electrode. Carbon electrodes or electrodes of any other suitable conductor can also be used.
  • a target strand (a nucleic acid sample to be interrogated relative to the probe) can be contacted with the probe in any suitable manner known to those skilled in the art.
  • a plurality of target strands can be introduced to the liquid medium described above and allowed to intermingle with the immobilized probes.
  • the number of target strands exceeds the number of probe strands in order to maximize the opportunity of each probe strand to interact with target strands and participate in hybridization. If a target strand is complementary to a probe strand, hybridization can take place. Techniques for adjusting the stringency of hybridization and techniques for detecting hybridization are also discussed herein.
  • embodiments of the present invention can include any combination of the following steps: extracting a biological sample from a patient, purifying a nucleic acid from a biological sample, amplifying a nucleic acid, isolating a nucleic acid in single stranded form, cyclizing a nucleic acid, elongating a nucleic acid, controlling hybridization stringency, amplifying the nucleic acid on a chip, and detecting hybridization. Accordingly, preferred embodiments for each of these steps are discussed in the following sections.
  • references to extracting an oligonucleotide from a patient typically refer to obtaining a sequence that will form the basis of a target strand. However, in many embodiments, the same techniques, or those which are similar, will also be appropriate for obtaining a sequence that will form the basis of a probe strand. Those of skill in the art will recognize that various biological and/or artificial sources of oligonucleotides are available and will be able to decide which are most suitable for creating probes or targets depending on the particular goals of the assay to be conducted. Extracting a Biological Sample
  • Biological samples that are useful in the present invention can include any sample from a patient in which a nucleic acid is present. Such samples can be prepared from a any tissue, cell, or body fluid.
  • biological cell sources include blood cells, colon cells, buccal cells, cervicovaginal cells, epithelial cells from urine, fetal cells or cells present in tissue obtained by biopsy.
  • Exemplary tissues or body fluids include sputum, pancreatic fluid, bile, lymph, plasma, urine, cerebrospinal fluid, seminal fluid, saliva, breast nipple aspirate, pus, amniotic fluid and stool.
  • Useful biological samples can also include isolated nucleic acid from a patient. Nucleic acid can be isolated from any tissue, cell, or body fluid using any of numerous methods that are standard in the art.
  • a stool sample is taken from a patient as part of a method of screening for colorectal cancer.
  • methods of extracting biological samples from stool are described in U.S. Pat. No. 5,741,650 (Lapidus et al), herein incorporated by reference. Lapidus et al. teach sectioning a stool sample to extract cells and cellular debris that may be indicative of cancer or precancer. Such a method can be used to obtain biological material containing a nucleic acid for further use in accordance with the present invention.
  • nucleic acid can include DNA and RNA.
  • the particular nucleic acid purification method will typically depend on the source of the patient sample. Techniques for purifying nucleic acid are known in the art and can include the use of homogenization, centrifugation, extraction with various solvents, chromatography, electrophoresis, and other known techniques.
  • the biological sample is a stool sample and nucleic acid from colorectal tissue is isolated and purified from stool cross sections according to methods disclosed in U.S. Pat. No. 6,406,857 (Shuber et al), hereby expressly incorporated by reference in its entirety.
  • RCA is one technique that can be used, through
  • PCR is preferred. It is particularly advantageous to use a "digital PCR" technique. Digital
  • PCR refers to a PCR method in which a liquid sample containing nucleic acids of interest is so thoroughly diluted and partitioned that each partition contains at most one nucleic acid molecule. Accordingly, if subsequent PCR amplification on a partition is successful, all of the resulting strands will be derived from one strand. Hence all of the PCR products for a given partition will be identical. Because the partitions themselves are unlikely to be identical to all the other partitions, it will often be advantageous to study those partitions found to contain nucleic acids in separate assays to determine which warrant further attention. Digital PCR is discussed in greater detail in Vogelstein et al. "Digital PCR," Proc.
  • single stranded nucleic acid is isolated using a streptavidin-coated bead.
  • an amplification product is denatured to generate single-stranded products, wherein at least one strand contains an addressable ligand at one terminus.
  • a biotinylated single- stranded PCR product having a copy of the nucleotide sequence of interest is incubated with streptavidin-coated beads, under conditions such that the biotinylated PCR product is attached to a bead, forming a bead-target sequence complex.
  • one strand of a double stranded nucleic acid is removed, for example, by selective exonuclease digestion.
  • the remaining single stranded nucleic acid can further be used in accordance with the present invention.
  • Cvclizing the Nucleic Acid and Performing RCA hi performing an assay in accordance with the present invention it is possible to cyclize and elongate the target nucleic acid prior to hybridization.
  • “Cyclization” generally refers to the process of creating a polynucleotide circle (preferably containing a particular sequence)
  • “elongation” generally refers to the process of increasing the length of a polynucleotide.
  • elongation includes a rolling circle amplification (RCA) step with an appropriate polynucleotide circle and is used to create a long strand of target nucleic acid.
  • RCA rolling circle amplification
  • cyclization and elongation can be used to generate one or more long target strands in which a sequence being interrogated is repeated several times. Effectively, many copies of a small target strand are linked end to end to generate a large target strand.
  • cyclization/elongation can be used to add as little as one repetition, it is generally preferred that multiple repetitions be added, for example, approximately 10, 50, 100, 250, 500, 750, or 1000 repetitions or more may be attached.
  • Circle size is also adjustable according to the requirements of the assay. Preferred circle sizes are in the range of about 40 to about 1000 base pairs, with about 800 base pairs being most preferred.
  • the number of repetitions selected can depend on the length of the circle being used. Specifically, it will generally be preferable to use more repetitions with smaller circles and fewer repetitions with larger circles so that the strands produced will be appropriately manageable and functional according to the demands of the assay.
  • any one of the many repetitions of the sequence on a large strand would be able to hybridize to a probe just as if that sequence were alone on a standard short target strand. Further, just one large target strand can generally hybridize to multiple probes (by coiling back toward the electrode surface and allowing another identical region of the long strand to attach to another complementary probe).
  • Elongation and the use of long target strands has various advantages. Particularly favorable advantages are related to stringency.
  • Stringency refers to a measurement of the ease with which various hybridization events can occur. For example, two strands that are perfectly complementary generally form a more stable hybrid than two strands that are not.
  • Various stringency factors can be adjusted such that in a single environment, the perfectly complementary pair would stay together while the imperfect pair would fall apart.
  • Ideal conditions are generally those which strike a balance between minimizing the number of hybridizations between noncomplementary strands (false positives) and minimizing the number of probes which remain unhybridized despite the presence of eligible complementary target strands (false negatives). Other various techniques for controlling stringency are also discussed in the next section.
  • Elongation is one technique that is useful in improving the effectiveness of temperature as a stringency factor.
  • a perfect hybrid is typically more stable than an imperfect hybrid and will outlast the imperfect hybrid when the temperature is increased.
  • dehybridization in either case is not a single event when dealing with populations of molecules. Instead, more and more molecuies give up the hydrogen bonds that hold opposing base pairs together over a range of temperature. Perfect hybrids outlast imperfect hybrids, but it is often very difficult, if not impossible, to find a single temperature at which there are no imperfect hybrids while perfect hybrids abound.
  • nucleic acid molecules exhibit a less gradual transition between their hybridized and unhybridized states when the temperature is changed. This is to say that the melt curve for a given population of molecules is steeper and more decisive when the nucleic acid strand is longer.
  • the distance between the curves of perfect and imperfect hybrids of equivalent length tend to crowd in a smaller temperature range, frustrating the initial attempt to create ⁇ a stringency environment that will distinguish between them.
  • FIGS. 1A and IB illustrate this phenomenon.
  • FIG. 1A shows the melt curves of matched and mismatched short strand duplexes.
  • FIG. IB shows the melt curves for longer strands.
  • FIG. 1 A has a large ⁇ Tm, but the gradual melt of the duplexes makes it difficult to select and maintain a temperature range that has a maximum specificity ratio.
  • FIG. IB has steeper and more decisive melting curves, but the ⁇ Tm is very small, again making it difficult to select and maintain a temperature range that allows maximum specificity.
  • some embodiments of the present invention include cyclization and elongation steps to produce a target strand of increased length.
  • a sequence is repeated several times on the target strand such that one target strand can participate in hybridization with more than one immobilized probe.
  • this can be used to create a larger temperature window in which perfect hybrids remain and imperfect hybrids fall apart, it is advantageous to adjust the temperature of the assay environment to minimize false positives as well as false negatives.
  • a duplex polynucleotide with a single base mismatch has a melting temperature T ml and a duplex polynucleotide with no base mismatch has a higher melting temperature T ⁇
  • T ml melting temperature
  • T ⁇ melting temperature
  • the results of such an assay could indicate whether a single base mismatch exists in the duplex being interrogated.
  • a determination of the temperature at which a duplex falls apart can be used to evaluate the quantity, type, and or location of mismatches, if any.
  • Various techniques for detecting hybridization are discussed infra.
  • Some embodiments of the present invention include providing a polynucleotide sample and then performing an assay to determine whether it contains a sequence of interest.
  • a nucleic acid circle is prepared in connection with the polynucleotide sample that contains both a portion of a sequence complementary to the sequence being interrogated and an "address sequence."
  • the address sequence is typically an arbitrary sequence of nucleotides that will also appear on a probe strand.
  • the circle can be amplified by RCA to produce a long target that contains several repetitions of the complement to the address sequence. When the target strand is allowed to interact with a probe containing the address sequence, the two can hybridize. Detection of hybridization can be used as an indication of the presence of the sequence being interrogated in the original sample.
  • assays of this type can detect the presence of various sequences as well as the presence of single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • Each cyclizable strand should also have a unique address sequence. The one cyclizable strand that fits correctly with the sample can then cyclize and undergo amplification. Then, by determining which address sequence corresponds to a probe-target hybrid, the identity of the nucleotide at the SNP location can be determined.
  • the number of target strands used in an assay will exceed the number of probe strands in order to maximize the opportunity of each probe strand to interact with target strands and participate in hybridization. If a target strand is complementary to a probe strand, hybridization can take place when the two come into contact. However, in some cases, even strands which are not truly complementary may come together and stay together as an imperfect hybrid.
  • Whether or not various hybridization events occur can be influenced by various stringency factors such as temperature, pH, or the presence of a species able to denature various hybridized strands. Increasing the quantity of target strands is one technique that can be useful in minimizing the number of probes that should hybridize to targets, but do not (false negatives).
  • Preferred techniques for controlling stringency include setting and maintaining the temperature and pH of the liquid medium environment. More preferred techniques also incorporate introducing one or more chemical species as stringency agents that can minimize the number of false positives and/or false negatives. Agents that can be used for this purpose include quaternary ammonium compounds such as tetramethylammonium chloride (TMAC).
  • TMAC tetramethylammonium chloride
  • TMAC is particularly useful in minimizing false positives.
  • This species generally acts through a non-specific salt effect to reduce hydrogen-bonding energies between G-C base pairs. At the same time, it binds specifically to A-T pairs and increases the thermal stability of these bonds.
  • These opposing influences have the effect of reducing the difference in bonding energy between the triple-hydrogen bonded G-C based pair and the double-bonded A-T pair.
  • One consequence is that the melting temperature of nucleic acid hybrids formed in the presence of TMAC is solely a function of the length of the hybrid.
  • a second consequence is an increase in the slope of the melting curve for each probe. Together, these effects allow the stringency of hybridization to be increased to the point that single-base differences can be resolved, and non-specific hybridization minimized.
  • Various techniques for using a stringency agent such as TMAC are discussed in U.S. Pat. No. 5,849,483 (Shuber), herein incorporated by reference.
  • mutator cluster region of the APC gene wherein mutations are highly correlated with colon cancer, is approximately 800 base pairs in length. If a probe oligomer is approximately 17 base pairs in length, it will typically require approximately 44 oligomers to blanket the entire 800 base pair strand.
  • Mutations at the end of a fragment are often difficult to detect, so it can be beneficial to use a second series of oligonucleotides that also blanket that 800 base pair strand, but are offset such that the middle of the second series of oligonucleotides corresponds to the ends of the adjacent first series of oligonucleotides. Allowing for a gap of three base pairs between adjacent probe sequences, it will typically require 80 oligomers to test 800 base pairs for mutations.
  • Various high volume techniques for testing a mutator cluster region can be used, hi a preferred embodiment, standard multiwell plates having 96 wells and 20 electrodes per well can be used to test a particular region; assuming four wells are used to determine which one of the four bases appears at a particular point in the sequence, each 96 well plate can test the properties of 24 different molecules.
  • the first preferred method of on-chip amplification is depicted in FIG. 2A.
  • either the 3' or 5' end of the hybridized PCR product can be targeted for a head-to-tail polymerization that builds up the amount of DNA on the electrodes.
  • three different oligonucleotides (not counting the immobilized probe and the target strands) will be used as shown here: the first oligomer is complementary to the 3' end of the hybridized PCR product (targeting the complement of the primer sequence), and contains a sequence A at its 5' end; the second oligomer has a sequence 5'-A*B-3', where A* is complementary to A; the third oligonucleotide has sequence 5'-AB*-3'.
  • these oligomers can form a polymeric product as shown.
  • the head-to-tail polymerization can continue until the strand reaches a desired length.
  • the ultimate length of the polynucleotide is limited in part by a competing cyclization reaction of the head-to-tail oligomers.
  • a higher concentration of head-to-tail oligomers in the liquid medium will generally produce longer linear polymers attached to the electrode, however.
  • the second preferred method of on-chip amplification is depicted in FIG. 2B.
  • This method uses rolling circle amplification.
  • a preformed circle (approximately 40 to 300 nucleotides) that has a region complementary to the 3' end of the bound PCR product is hybridized to the PCR product as shown.
  • a processive DNA polymerase can then be added so that RCA results, elongating the bound PCR product.
  • the PCR product is elongated by approximately 10 to 100 copies of the circle.
  • FIG. 2C A further technique for on-chip amplification is depicted in FIG. 2C.
  • This technique may be used in conjunction with other on-chip amplification methods and is commonly referred to as "branch" amplification.
  • additional polynucleotides that are capable of hybridizing with the target strand in a region beyond the probe-target hybridization region can be added to the liquid medium and allowed to hybridize with the bound target to further increase the amount of bound polynucleotide material when probe- target hybridization occurs.
  • these branch polynucleotide strands are further amplified, for example by RCA as depicted in FIG. 2C.
  • branches on top of branches a technique known as hyperbranching. Additional discussion of branching and hyperbranching techniques can be found, for example, in: Urdea, Biotechnology 12:926 (1994); Horn et al, Nucleic Acids Res. 25(23):4835-4841 (1997); Lizardi et al., Nature Genetics 19, 225-232 (1998); Kingsmore et al. (U.S. Pat. No. 6,291,187); Lizardi et al.
  • the increased amount of DNA can generate a larger and more detectable signal. This can be advantageous for assay purposes since both the probe and the target typically produce some detectable signal. If the signal of the target is enhanced, the contrast between hybridized and unhybridized probes will be more profound, h some embodiments, however, nucleic acid analogs can be used as probes which do not contribute to the overall signal; such designs are discussed in the following section. Even when such nucleic acid analogs are used as probes, target elongation can still be desirable.
  • nucleic acid hybridization is tested electrochemically using a transition metal complex. More preferably, hybridization is detected by measuring the reduction of a ruthenium complex as described below. Detecting Hybridization
  • a transition metal complex can be used as a counterion to conduct an electrochemical assay using the general technique of Steele et al. (1998, Anal. Chem. 70:4670-4677), herein incorporated by reference.
  • Counterions such as Ru(NH 3 ) 6 3+ or Ru(NH 3 ) 5 py 3+
  • Ru(NH 3 ) 5 py 3+ can be introduced to the liquid medium surrounding the immobilized oligonucleotides.
  • Ru(NH ) 5 py 3+ is preferred because its reduction to a divalent ion does not occur at the same electrical potential as the reduction of molecular oxygen.
  • the counterions will tend to cloud around the negatively charged backbones of the various nucleic acid strands.
  • the counterions will accumulate electrostatically around the phosphate groups of the nucleic acids whether they are single or double stranded.
  • the hybridized nucleic acid will typically have more counterions surrounding it.
  • the target can be much longer than the probe, typically 2 to 100 times, in which case the counterion accumulation will be dominated by single stranded regions of the target.
  • the probe strands can be constructed such that they do not contain a negatively charged backbone, then they will not attract counterions. Accordingly, more of the detectable signal will be due to counterions associated with the target strands. In cases where hybridization has not occurred, the detectable signal will be measurably lower since the target strands are not present to participate in counterion attraction.
  • Probe strands without a negatively charged backbone can include peptide nucleic acids (PNAs), phosphotriesters, methylphosphonates. These nucleic acid analogs are known in the art.
  • PNAs are discussed in: Nielsen, “DNA analogues with nonphosphodiester backbones,” Annu Rev Biophys Biomol Struct, 1995;24:167-83; Nielsen et al, “An introduction to peptide nucleic acid,” Curr ssues MolBiol, 1999;l(l-2):89-104; Ray et al., "Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future," FASEB J, 2000 Jun;14(9):1041-60; all of which are hereby expressly incorporated by reference in their entirety.
  • Phophotriesters are discussed in: Sung et al, "Synthesis of the human insulin gene. Part II. Further improvements in the modified phosphotriester method and the synthesis of seventeen deoxyribooligonucleotide fragments constituting human insulin chains B and mini-CDNA," Nucleic Acids Res, 1979 Dec 20;7(8):2199-212; van Boom et al, "Synthesis of oligonucleotides with sequences identical with or analogous to the 3'-end of 16S ribosomal RNA of Escherichia coli: preparation of m-6-2-A-C-C-U-C-C and A-C-C-U-C- m-4-2C via phosphotriester intermediates," Nucleic Acids Res, 1977 Mar;4(3):747-59; Marcus-Sekura et al, "Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues
  • Methylphosphonates are discussed in: U.S. Pat. No. 4,469,863 (Ts'o et al); Lin et al, "Use of EDTA derivatization to characterize interactions between oligodeoxyribonucleoside methylphophonates and nucleic acids," Biochemistry, 1989, Feb 7;28(3): 1054-61; Vyazovkina et al, "Synthesis of specific diastereomers of a DNA methylphosphonate heptamer, d(CpCpApApApCpA), and stability of base pairing with the normal DNA octamer d(TPGPTPTPTPGPGPC),” Nucleic Acids Res, 1994 Jun
  • an appropriate nucleic acid analog probe will not contribute, or will contribute less substantially, to the attraction of counterions compared to a probe made of natural DNA.
  • the target strand will ordinarily feature a natural phosphate backbone having negatively charged groups which attract positive ions and make the strand detectable.
  • a probe may be constructed that contains both charged nucleic acids and uncharged nucleic acid analogs.
  • pure DNA probes can be used alongside probes containing uncharged analogs in an assay.
  • precision in distinguishing between single stranded and double stranded will generally increase according to the electrical charge contrast between the probe and the target strands.
  • the exclusive use of probes made entirely of an uncharged DNA analog will generally allow the greatest signal contrast between hybridized and non-hybridized molecules on the chip.
  • probe strands containing methylphosphonates are preferred when nucleic acid analogs are desired.
  • Ru NH ⁇ spy 3"1" is a preferred counterion, though any other suitable transition metal complexes that bind nucleic acid electrostatically and whose reduction or oxidation is electrochemically detectable in an appropriate voltage regime can be used.
  • amperometry is used to detect an electrochemical reaction at the electrode.
  • an electrical potential will be applied to the electrode.
  • the counterions undergo an electrochemical reaction, for example, the reduction of a trivalent ion to divalent at the electrode surface, a measurable current is generated.
  • the amount of current corresponds to the amount of counterions present which in turn corresponds to the amount of negatively-charged phosphate groups on nucleic acids. Accordingly, measuring the current allows a quantitation of phosphate groups and can allow the operator to distinguish hybridized nucleic acid from unhybridized nucleic acid and determine whether the target being interrogated is complementary to the probe (and contains the sequence of interest).
  • a mutation can be detected at a level of about 1 part in 10 (which means one mutant version of a gene in a sample per 100 total versions of the gene in the sample) or less, about 1 part in 10 3 or less, about 1 part in 10 4 or less, about 1 part in 10 5 or less, or about 1 part in 10 6 or less.
  • a detectable label can be attached to or otherwise associated with certain polynucleotides in the detection zone. Accordingly, such a label can then be detected as an indication of whether hybridization has occurred.
  • labels are well known in the art and can include, for example, chemical moieties, dyes, radioactive probes, quantum dots, and nanoparticles.
  • Techniques for detection of various labels can include, for example, chemical detection, radioactivity detection, UV and/or visible spectroscopy, fluorescence, and the like. Examples Example 1: Augmenting an Electrical Signal Using On-chip Amplification The following procedure was performed to determine the effectiveness of on-chip amplification for enhancing an electrical signal. The results are discussed with reference to FIG. 3.
  • a solution consisting of biotinylated capture probe and NeutrAvidin was deposited to the surface of a carbon electrode and allowed to air dry.
  • the carbon electrode was transferred to a solution containing 5 ⁇ M of Ru(NH ) 6 Cl 3 in 10 mM Tris + 10 mM NaCl.
  • the electrochemical response was detected and recorded using Osteryoung Square Wave Voltammetry (OSWV). This measurement corresponds to the quantity of nucleic acid immobilized on the electrode before RCA is performed. The resulting current is represented by the smaller curve (which peaks at approximately 0.100 ⁇ A) as depicted in FIG. 3.
  • the immobilized capture probe was then hybridized with a circularized DNA as follows.
  • the electrode was rinsed with Tris buffer solution.
  • 10 ⁇ l of solution containing circularized DNA in 10 mM Hepes + 1 M LiCl was applied to the surface of the carbon electrode.
  • the electrode was maintained at 60 °C for 5 minutes and then cooled to room temperature and maintained at the room temperature for 30 minutes.
  • RCA was then performed as follows. The electrode was rinsed with the Tris buffer.
  • the electrode was rinsed with tris buffer and transferred to a solution containing 5 ⁇ M of Ru(NH 3 ) 6 Cl 3 in 10 mM Tris + 10 mM NaCl.
  • the electrochemical response was again detected and recorded using OSWV.
  • the resulting current is represented by the larger curve (which peaks at approximately 1.800 ⁇ A) as depicted in FIG. 3.
  • a patient at risk for colorectal cancer can be screened for cancer or precancer as follows: 1. An oligonucleotide sequence indicative of colorectal cancer is identified; this sequence is approximately 20 base pairs in length and is known as the "sequence of interest.”
  • a probe strand having a length of approximately 20 base pairs is produced having a sequence that is complementary to the sequence of interest.
  • a patient suspected of having colorectal cancer or suspected of later developing colorectal cancer is identified.
  • a stool sample voided from the patient is collected and sectioned to extract cells and cellular debris containing nucleic acids from the epithelial cells of the patient's colorectal tract.
  • DNA is extracted and isolated from the cells and cellular debris using methods disclosed in U.S. Pat. Nos. 5,741,650 (Lapidus et al) and 6,406,857 (Shuber et al).
  • DNA molecules isolated from the stool sample are amplified using digital PCR.
  • Single stranded PCR products are isolated using a streptavidin-coated bead.
  • the isolated, single stranded nucleic acid is cyclized and subject to RCA to produce elongated target strands.
  • a plurality of probe strands containing a biotin complex are immobilized on a gold electrode coated with streptavidin.
  • a liquid medium is placed in contact with the electrode surface and with the immobilized probe strands.
  • a plurality of target strands are introduced to the liquid medium such that they are allowed to interact with the probe strands.
  • Hybridization stringency is controlled by adjusting temperature, pH, and the quantity of TMAC in the liquid medium. Hybridization stringency is set such that perfectly complementary sequences hybridize but that all others do not.
  • On-chip amplification is conducted using head-to-tail polymerization to increase the length of any hybridized target on the electrode. This amplification adds approximately 10,000 base pairs to each bound target strand.
  • Ru(NH ) 5 py 3+ ions in a liquid are added to the liquid medium as counterions able to form an electrostatic cloud around nucleic acids.
  • An electrical potential is applied to the electrode on which the nucleic acid probes are immobilized.
  • the hybridization status of the probes to the targets is used to evaluate the health of the patient with regard to colorectal cancer and to decide whether to administer further healthcare services to the patient, including, for example, counseling, additional testing, administration of pharmaceutical agents, surgery, etc.

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Abstract

La présente invention concerne la détection de mutations de cellules somatiques, notamment sous forme d'étape d'un procédé pour identifier un cancer ou un précancer. La présente invention concerne des techniques pour extraire et isoler des oligonucléotides d'un patient et pour effectuer des essais d'hybridation. Des modes de réalisation préférés concernent une combinaison des étapes suivantes : prélever un échantillon biologique d'un patient, purifier un acide nucléique d'un échantillon biologique, amplifier un acide nucléique, isoler un acide nucléique sous une forme à simple brin, cycliser un acide nucléique, allonger un acide nucléique, commander une rigueur d'hybridation, amplifier un acide nucléique sur une puce et détecter une hybridation.
EP04760623A 2003-05-02 2004-04-30 Procede de detection electrochimique de mutations de cellules somatiques Withdrawn EP1620575A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10/429,293 US20040086895A1 (en) 2002-11-06 2003-05-02 Method of electrochemical detection of somatic cell mutations
PCT/US2004/013222 WO2004099755A2 (fr) 2002-11-06 2004-04-30 Procede de detection electrochimique de mutations de cellules somatiques

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EP1620575A2 true EP1620575A2 (fr) 2006-02-01
EP1620575A4 EP1620575A4 (fr) 2007-02-07

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002083839A2 (fr) * 2001-03-30 2002-10-24 Amersham Biosciences Ab Analyse par biopuces de polymorphismes à simple nucléotide
US20020168645A1 (en) * 1998-04-16 2002-11-14 Seth Taylor Analysis of polynucleotide sequence
US20030073122A1 (en) * 1993-11-01 2003-04-17 Nanogen, Inc. Methods for determination of single nucleic acid polymorphisms using a bioelectronic microchip

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030073122A1 (en) * 1993-11-01 2003-04-17 Nanogen, Inc. Methods for determination of single nucleic acid polymorphisms using a bioelectronic microchip
US20020168645A1 (en) * 1998-04-16 2002-11-14 Seth Taylor Analysis of polynucleotide sequence
WO2002083839A2 (fr) * 2001-03-30 2002-10-24 Amersham Biosciences Ab Analyse par biopuces de polymorphismes à simple nucléotide

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of WO2004099755A2 *
STEEL A B ET AL: "Electrochemical Quantitation of DNA immobilized on Gold" ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. COLUMBUS, US, vol. 70, no. 22, 15 November 1998 (1998-11-15), pages 4670-4677, XP002285670 ISSN: 0003-2700 *

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