Classifications

• • 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 methods of this invention relate to systems for genetic identification for disease states and other gene related afflictions. More particularly, the methods relate to systems for the detection of single nucleic acid polymorphisms in nucleic acid sequences for the identification of polymorphisms in viruses, and eukaryotic and prokaryotic genomes.
• Molecular biology comprises a wide variety of techniques for the analysis of nucleic acid and protein sequences. 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, New York, 1989). Most of these techniques involve carrying out numerous operations (e.g., pipetting, centrifugation, and electrophoresis) on a large number of samples. They are often complex and time consuming, and generally require a high degree of accuracy.
• the complete process for carrying out a DNA hybridization analysis for a genetic or infectious disease is very involved.
• the complete process may be divided into a number of steps and sub-steps.
• the first step involves obtaining the sample (e.g., saliva, blood or tissue).
• the second step involves disrupting or lysing the cells which releases the crude DNA material along with other cellular constituents.
• several sub-steps are necessary to remove cell debris and to further purify the DNA from the crude sample. At this point several options exist for further processing and analysis.
• a third option involves denaturing the DNA and carrying out a direct hybridization analysis in one of many formats (dot blot, microbead, microplate, etc.).
• a second option called Southern blot hybridization, involves cleaving the DNA with restriction enzymes, separating the DNA fragments on an electrophoretic gel, blotting the DNA to a membrane filter, and then hybridizing the blot with specific DNA probe sequences. This procedure effectively reduces the complexity ofthe 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 an amplification procedure such as the polymerase chain reaction (PCR) or the strand displacement amplification (SDA) method.
• PCR polymerase chain reaction
• SDA strand displacement amplification
• 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.
• a reduction in the complexity ofthe nucleic acid in a sample is helpful to the detection of low copy numbers (i.e. 10,000 to 100,000) of nucleic acid targets.
• DNA complexity reduction is achieved to some degree by amplification of target nucleic acid sequences. (See, M.A. Innis et al, PCR Protocols: A Guide to Methods and Applications, Academic Press, 1990, Spargo et al, 1996, Molecular & Cellular Probes, in regard to SDA amplification). This is because amplification of target nucleic acids results in an enormous number of target nucleic acid sequences relative to non-target sequences thereby improving the subsequent target hybridization step.
• the actual hybridization reaction represents one ofthe most important and central steps in the whole process.
• the hybridization step involves placing the prepared DNA sample in contact with a specific reporter probe at set optimal conditions for hybridization to occur between the target DNA sequence and probe.
• Hybridization may be performed in any one of a number of formats. For example, multiple sample nucleic acid hybridization analysis has been conducted in a variety of filter and solid support formats (See G. A. Beltz et al, in Methods in Enzymology, Vol. 100, Part B, R. Wu, L. Grossman, K. Moldave, Eds., Academic Press, New York, Chapter 19, pp. 266-308, 1985).
• Dot blot hybridization involves the non-covalent attachment of target DNAs to a filter followed by the subsequent hybridization to a radioisotope labeled probe(s).
• "Dot blot” hybridization gained wide-spread use over the past two decades during which time 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).
• the dot blot method has been developed for multiple analyses of genomic mutations (D. Nanibhushan and D. Rabin, in EPA 0228075, July 8, 1987) and for the detection of overlapping clones and the construction of genomic maps (G. A. Evans, in US Patent Number 5,219,726, June 15, 1993).
• 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 (see R. Drmanac and R. Crkvenjakov, Yugoslav Patent Application #570/87, 1987; R. Drmanac et al, 4 Genomics, 114, 1989; Strezoska et al, 88 Proc. Natl. Acad. Sci.
• 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.
• Southern did not address how to achieve optimal stringency conditions for each oligonucleotide on an array.
• 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. Wide ranges of stringency conditions were used to achieve specific hybridization for each n-mer probe. Washing times varied from 5 minutes to overnight using temperatures from 0°C to 16°C. Most probes required 3 hours of washing at 16°C. The filters had to be exposed from 2 to 18 hours in order to detect hybridization signals. The overall false positive hybridization rate was 5% in spite ofthe simple target sequences, the reduced set of oligomer probes, and the use ofthe most stringent conditions available.
• detection and analysis ofthe hybridization events Depending on the reporter group (fluorophore, enzyme, radioisotope, etc.) used to label the DNA probe, detection and analysis are carried out fluorimetrically, 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 ofthe background presence of non-specifically bound materials. Thus, detection of hybridization events is dependent upon how specific and sensitive hybridization can be made. Concerning genetic analysis, several methods have been developed that have attempted to increase specificity and sensitivity.
• SNPs single nucleic acid polymorphisms
• STRs short tandem repeats
• a SNP is defined as any position in the genome that exists in two variants and the most common variant occurs less than 99% ofthe time.
• SNPs are crucial to be able to genotype them easily, quickly, accurately, and cost-effectively. It is of great interest to type both large sets of SNPs in order to investigate complex disorders where many loci factor into one disease (Risch and Merikangas, Science, Vol. 273, pp. 1516-1517, 1996), as well as small subsets of SNPs previously demonstrated to be associated with known afflictions.
• hybridization assays function by discriminating short oligonucleotide reporters against matched and mismatched targets. Due to difficulty in determining optimal denaturation conditions, many adaptations to the basic protocol have been developed. These include ligation chain reaction (Wu and Wallace, Gene, Vol. 76, pp. 245-254, 1989) and mini sequencing (Syvanen et al, Genomics, Vol. 8, pp. 684-692, 1990). Other enhancements include the use ofthe 5'-nuclease activity of Taq DNA polymerase (Holland et al, Proc. Natl. Acad. Sci. USA, Vol. 88, pp.
• K. Khrapko et al Federation of European Biochemical Societies Letters, Vol. 256, no. 1,2, pp. 118-122 (1989), for example, disclosed that continuous stacking hybridization resulted in duplex stabilization.
• J. Kieleczawa et al Science, Vol. 258, pp. 1787-1791 (1992), disclosed the use of contiguous strings of hexamers to prime DNA synthesis wherein the contiguous strings appeared to stabilize priming.
• L. Kotler et al Proc. Natl. Acad. Sci. USA, Vol. 90, pp.
• 10-mer DNA probes were anchored to the surface ofthe microchip and hybridized to target sequences in conjunction with additional short probes, the combination of which appeared to stabilize binding ofthe probes.
• short segments of nucleic acid sequence could be elucidated for DNA sequencing.
• Yershov further noted that in their system the destabilizing effect of mismatches was increased using shorter probes (e.g., 5-mers).
• Use of such short probes in DNA sequencing provided the ability to discern the presence of mismatches along the sequence being probed rather than just a single mismatch at one specified location ofthe probe/target hybridization complex.
• Use of longer probes e.g., 8-mer, 10-mer, and 13-mer oligos were less functional for such purposes.
• SNPs in a target nucleic acid are determined using a single capture site on an electronically addressable microchip (e.g, an APEX type microchip).
• an electronically addressable microchip e.g, an APEX type microchip.
• both wild type and mutant alleles are distinguished, if present in a sample, at a single capture site by detecting the presence of hybridized allele-specific probes labeled with fluorophores sensitive to excitation at various wave lengths.
• base-stacking energies of at least two oligonucleotides are used in conjunction with an APEX type bioelectronic microchip.
• the electronically facilitated method using an APEX type microchip offers several advantages over passive-based hybridization assays when base-stacking is employed.
• Electronically facilitated methods additionally allow multiple different amplicons to be addressed to discrete sites thereby greatly facilitating multiplexing of multiple patients or multiple amplicons on an open microchip.
• the amplicons ofthe target nucleic acid may be anchored to an electronic microchip capture site (i.e. "amplicon down" format) such that multiple amplicons may be placed at the same capture site.
• the amplicons may be anchored to the capture site on the microchip by attachment moieties located at the 5' end ofthe amplicon.
• attachment moieties can be binding agents such a biotin incorporated into one ofthe amplification primers.
• the anchored nucleic acids may in turn be probed simultaneously or sequentially.
• a target nucleic acid is first amplified, such as by PCR, SDA, NASBA, TMA, rolling circle, T7, T3, or SP6, each of which methods are well understood in the art, using at least one amplification primer oligomer that is labeled with a moiety useful for attaching the amplification product to a substrate surface.
• a biotin moiety can be attached at the 5' end ofthe primer.
• the labeled amplified dsDNA product may be denatured electronically or thermally and addressed to a specified capture site on the microchip surface, thereby making the amplicon behave as an anchored capture moiety.
• the complementary strand to the labeled amplification product (i.e., the non-labeled strand) is kept from reannealing to the labeled product by a "stabilizer” oligomer which is inputted into the process during electronic biasing ofthe labeled targeted amplicon to the capture site.
• a "stabilizer” oligomer as provided for in this invention, is unique in that unlike prior base-stacking inventions, it functionally serves two purposes (i.e., to hinder reannealing of complementary amplicons during electronic addressing ofthe biotinylated target amplicons, and to provide a base-stacking energy moiety for interaction with the second oligomer.
• electronic biasing equally facilitates distinguishing hybridization mismatches occurring at the terminal nucleic acid pairs of a hybridized duplex as well as destabilizing mismatches occurring internally (e.g., due to destabilizing caused by misalignment ofthe base pairs).
• This ability to detect mismatches allows the current invention to be less restricted in choices for positioning the location of SNP bases on probes although generally, for purposes of this invention, mismatches are desired to occur at the terminal base of a probe.
• the SNP relevant base may be inco ⁇ orated as the terminal base ofthe reporter probe such that when the stabilizer and reporter probes are annealed to the amplicon, the SNP relevant base will lie adjacent to one ofthe terminal bases ofthe stabilizer when both the stabilizer and reporter are annealed adjacently to one another on a target nucleic acid strand.
• Sensitivity and robustness may further be enhanced by the additional inclusion of yet another probe (i.e., the "interfering" probe) designed to be complementary to the non-labeled strand ofthe amplicon. Use of this probe further helps to compete away the undesired non-labeled amplicon strand from reannealing to the labeled strand.
• the stabilizer probe is anchored (i.e. "capture down” format)
• the system is also simple and multiple amplicons may be placed at the same capture site. These may then be probed simultaneously or sequentially.
• the stabilizer probe will be anchored to the substrate at its 5' end.
• the SNP base will be complementary to either the 3' base ofthe stabilizer/capture or the 5' base ofthe reporter probe.
• the SNP base will be complementary to either the 5' base ofthe stabilizer/capture or the 3' base ofthe reporter probe.
• a target nucleic acid is first amplified, such as by PCR or SDA.
• the amplified dsDNA product is then denatured and addressed to a specified capture site on the microchip surface that has an anchored stabilizer/capture moiety.
• the complementary strand to the desired amplification product strand is kept from reannealing to the desired strand by the stabilizer/capture oligomer that, as described above, serves as a first probe that also participates in base-stacking with a second reporter probe.
• the stabilizer/capture oligomer as provided for in this invention is unique in that unlike prior base-stacking inventions, it functionally serves two pu ⁇ oses (i.e., to hinder reannealing of complementary amplicons during electronic addressing ofthe target amplicons and to provide a base- stacking energy moiety for interaction with reporter oligomer thereby lessening the complexity of SNP determination in a microchip format).
• interfering probes may be used.
• multiple amplicons may be probed at any particular capture site.
• multiple SNPs in a target sequence may be detected.
• either ofthe above mentioned amplicon down or capture down formats may be employed.
• multiple base-stacking may be used to resolve the presence of closely spaced SNPs at a single locus. For example, where two SNPs are closely spaced, at least two short reporter oligonucleotides may be base-stacked against a longer stabilizer oligonucleotide. Each reporter may be labeled with a different fluorophore specific for the allele that occurs at each site.
• reporter probes inco ⁇ orating the wild-type and mutant bases of each SNP site, each containing a different fluorophore may be used to determine which allele is present.
• SNPs in a target nucleic acid are determined using combined base-stacking energies derived from both 5' and 3' ends of a single reporter probe.
• the target nucleic acid is amplified (such as by PCR and preferably via the strand displacement amplification (SDA) technique) such that two spaced amplicons ofthe target are generated.
• the two amplicons may be from the same genetic locus wherein the sequences are closely spaced, or may be from divergent or unrelated genetic loci.
• both the amplicon down and the capture down formats may be used.
• the stabilizer/capture is designed as a "bridging" stabilizer/capture probe to capture both amplicons in a spaced apart fashion so that at least one reporter probe, which may or may not contain SNP sequence at one or the other end, can be " «asteJ" between the amplicons.
• amplicon down format only one ofthe amplicons is anchored and a "bridging" stabilizer/capture probe having sequence complementary to the anchored amplicon and the non-anchored amplicon is employed to hybridize the amplicons in a spaced apart fashion allowing at least one reporter probe to be nested.
• a "bridging" stabilizer/capture probe having sequence complementary to the anchored amplicon and the non-anchored amplicon is employed to hybridize the amplicons in a spaced apart fashion allowing at least one reporter probe to be nested.
• more than one SNP containing reporter probe may be nested and take advantage of multiple base-stacking energies.
• the amplicons may be brought into close proximity with one another using either an anchored bridging stabilizer/capture probe, or an anchored amplicon and a bridging stabilizer/capture probe as described above.
• the presence of both amplicon sequences maybe detected using a reporter probe designed to nest between the captured amplicons using base- stacking energies to stabilize the reporter hybridization as described above.
• the reporter probe may inco ⁇ orate at either and/or both its 5' and 3' ends SNP or wild-type sequence associated with either or both loci.
• the SNP containing region may contain multiple SNPs and reporter probes can be designed so that more than one reporter probe is used to nest between the first and second amplicons such that each reporter has at least one nucleic acid base on either its 3' or 5' end corresponding to a SNP.
• the stabilizer oligomers are generally 20 to 44-mers and preferably about 30-mers, while the reporter probes are generally 10 to 12-mers and preferably about 11-mers. The lengths of such probes are highly effective in accordance with their use in an electronically addressable microchip format. Reporter probes shorter than 8-mers are generally not functional in the ionic environment ofthe current system.
• electronically aided hybridization is utilized in the process.
• electronic stringent conditions may be utilized, preferably along with other stringency affecting conditions, to aid in the hybridization. This technique is particularly advantageous to reduce or eliminate slippage hybridization among probes and target, and to promote more effective hybridization.
• electronic stringency conditions may be varied during the hybridization complex stability determination so as to more accurately or quickly determine whether a SNP is present in the target sequence.
• Hybridization stability may be influenced by numerous factors, including thermoregulation, chemical regulation, as well as electronic stringency control, either alone or in combination with the other listed factors.
• electronic stringency conditions include thermoregulation, chemical regulation, as well as electronic stringency control, either alone or in combination with the other listed factors.
• Electronic stringency hybridization ofthe target is one distinctive aspect of this method since it is amenable with double stranded DNA and results in rapid and precise hybridization ofthe target to the capture site. This is desirable to achieve properly indexed hybridization ofthe target DNA to attain the maximum number of molecules at a test site with an accurate hybridization complex.
• the initial hybridization step may be completed in ten minutes or less, more preferably five minutes or less, and most preferably two minutes or less.
• the analytical process may be completed in less than half an hour.
• the complex is labeled.
• a detection ofthe amount of labeled hybridization complex at the test site or a portion thereof is preferred.
• Any mode or modality of detection consistent with the pu ⁇ ose and functionality ofthe invention maybe utilized, such as optical imaging, electronic imaging, use of charge-coupled devices or other methods of quantification.
• Labeling may be ofthe target, capture, or reporter.
• Various labeling may be by fluorescent labeling, colormetric labeling or chemiluminescent labeling.
• detection may be via energy transfer between molecules in the hybridization complex.
• the detection may be via fluorescence perturbation analysis.
• the detection may be via conductivity differences between concordant and discordant sites.
• detection can be carried out using mass spectrometry.
• mass spectrometry In such method, no fluorescent label is necessary. Rather detection is obtained by extremely high levels of mass resolution achieved by direct measurement, for example, by time of flight or by electron spray ionization (ESI).
• ESI electron spray ionization
• reporter probes having a nucleic acid sequence of 50 bases or less are preferred. It is yet a further object of this invention to provide methods that may effectively provide for genetic identification.
• Yet a further object ofthe invention is to identify SNPs in infectious organisms such as those responsible for antibiotic resistance or that can be used for identification of specific organisms.
• FIG. 1A is a cross sectional view of one embodiment of an active matrix device useful in accordance with the methods of this invention.
• FIG. IB is a perspective view of an active array device useful with the methods of this invention.
• FIG. 2 is a schematic representation of one embodiment ofthe method of electronic SNP scoring by a dual fluorescent base-stacking format wherein the target amplicon population comprises wild-type and/or mutant alleles.
• the target amplicon population comprises wild-type and/or mutant alleles.
• one of the target strands is anchored to the capture site ("amplicon down" format).
• wild type and mutant alleles may be probed at a single capture site.
• the target includes both alleles, (i.e., heterozygote) reporter probes corresponding to each allele will be detected.
• only one allele is present, (i.e., homozygote) only one reporter probe will be detected.
• the figure represents detection of a homozygote population.
• FIG. 3 is a representation of one embodiment ofthe method of electronic SNP scoring by dual fluorescent base-stacking format wherein the stabilizing probe is anchored to the capture site ("capture down" format). Additionally this figure demonstrates the use of interfering probes to compete out undesired amplicon strands. As is similarly demonstrated in Fig. 2, wild type and mutant alleles may be detected.
• FIGs. 4a and 4b represent one embodiment ofthe method wherein base- stacking energies of multiple reporter probes are utilized.
• Fig. 4a shows the capture down format while Fig. 4b shows amplicon down format.
• This multiple base stacking approach is applicable where a target possesses closely spaced SNPs.
• FIG. 5 represents one embodiment ofthe invention wherein base-stacking energies are provided by nesting a reporter probe between two target amplicons.
• the stabilizer probe has nucleic acid base sequence complementary to both target amplicons.
• the figure depicts amplicon down format although capture down is equally applicable. Stabilization ofthe reporter probe in nested fashion signals the presence of both target species and/or any SNP integrated into the 5' or 3' terminus, or into both termini, ofthe reporter probe.
• FIG. 6 shows that the nested embodiment illustrated in Fig. 5 may also utilize multiple base-stacking energies of multiple reporter probes, each of which may include SNPs. As with the other formats, both amplicon and capture down formats are useful.
• FIG. 7 shows a nested format in which amplification is carried out using SDA.
• the termini ofthe amplicons necessarily possess sequence related to primers used in SDA that contain an endonuclease restriction site.
• SNPs may be either in the reporter termini or alternatively be in amplicon sequence immediately internal to the SDA primer sequence.
• amplicons generated using SDA may use either amplicon or capture down format (capture down shown) and may use multiple reporter stacking.
• FIGs. 8a and 8b are photographs showing hybridization results on the same microchip capture sites using reporter probes corresponding to wild type and mutant alleles labeled with fluorophores sensitive to two different wavelengths. Results show that homozygous mutant, homozygous wild type, and heterozygosity is clearly detectable. Specifically, the importance of stabilizer oligomer for scoring Factor V SNPs is represented. Five unknown Factor V samples (labeled A through E) were amplified using primers Seq. Id. No. 1 (Biotin-TGTTATCACACTGGTGCTAA) and Seq. Id. No. 2 (ACTACAGTGACGTGGACATC).
• the amplification product was then electronically targeted to 4 capture sites (columns 1,2, 4, and 5) using a direct current of 400 nAmps/site for 2 minutes.
• Column 3 was mock targeted and served as background control.
• the array was then treated with 0.5X SSC, pH 12 for 5 minutes to denature any rehybridized amplified products.
• 125nM Factor V stabilizer oligo, Seq. Id. No. 3 (TAATCTGTAAGAGCAGATCCCTGGACAGGC), was electronically biased using direct current of 400nAmps/site to all capture sites in column 1 for 15 seconds, column 2 for 30 seconds, and column 4 for 60 seconds.
• Column 5 was biased for 60 seconds with buffer only.
• the reporter oligomers were a CR6G labeled wild type reporter, Seq. Id. No. 4 (GAGGAATACAG-CR6G), and a Far- Red labeled mutant reporter, Seq. Id. No. 5 (AAGGAATACAG-Far-Red). Results indicate that Samples A and B are homozygous for mutant, Sample C is heterozygous for mutant and wild type, and Samples D and E are homozygous for wild type.
• FIG. 9 is a photograph showing that base-stacking energy stabilizes oligo reporters. Wild type Hemochromatosis sample was amplified using Seq. Id. No. 6 (Biotin-TGAAGGATAAGCAGCCAAT) and Seq. Id. No. 7
• CTCTCTCAACCCCCAATA The amplified sample was then mixed with either (i), no stabilizer oligo (column 1); (ii), l ⁇ M of the standard Hemochromatosis stabilizer oligomer in which case the stabilizer hybridizes adjacent to the reporter probe (column 2), Seq. Id. No. 8 (GGCTGATCCAGGCCTGGGTGCTCCACCTGG); (iii), a stabilizer oligomer that hybridizes to target with a one base gap between the stabilizer and reporter probe (column 4), Seq. Id. No. 9 (GGGCTGATCCAGGCCTGGGTGCTCCACCTG); or (iv), a stabilizer oligomer Seq. Id. No.
• the images represent the wild type reporter only, both before (initial signal) and after thermal denaturation (post-discrimination). Only in the situation where the stabilizer and reporter probes were adjacent was the hybridization stabilized. The same result is obtainable using mutant (data not shown).
• FIG. 10 shows the impact of stabilizer oligo on signal intensity.
• An amplified wild type Hemochromatosis sample was mixed with either the standard 30-mer stabilizer oligo (Seq. Id. No. 8), non-complementary random DNA (six different 20-mer to 24-mer oligos), or no DNA (water) at three concentrations (lOnM, lOOnM, and 1 ⁇ M). Each combination was biased to duplicate capture sites for 4 minutes using a biased alternating current protocol 800nAmps/site at 38 msec '+' and 10 msec '-'.
• Capture sites that received either no DNA or random DNA were subsequently biased for 1 minute with 125nM stabilizer oligo, while capture sites that already received stabilizer were biased for 1 minute with buffer only. Biasing conditions were direct current at 400nAmps/site.
• the histogram represents the signal intensities of both the wild type (Seq. Id. No. 11) and mutant (Seq. Id. No. 12, TACGTATATCT-Far Red) reporters post-discrimination, achieved at 28° C. Background from capture sites addressed with no DNA was subtracted.
• FIG. 11 is a chart illustrating that the allele content of unknown Hemochromatosis samples is readily determinable.
• Sixteen amplified, unknown Hemochromatosis samples were tested using 1 ⁇ M Hemochromatosis stabilizer oligo (Seq. Id. No. 8). The samples and stabilizer were electronically targeted to individual capture sites on a 25-site microarray. Biasing was carried out for 4 minutes using an alternating current of 700nAmps/site at 38 msec '+' and 10 msec '-'. Following passive reporting ofthe two allele-specific reporters (i.e., wild-type Seq. Id. No. 11 or mutant Seq. Id. No. 12), thermal discrimination was achieved at 29° C.
• the histogram represents the mean fluorescent intensities minus background (signal intensity from a mock targeted site). Results show that samples 1, 7, and 12 are heterozygous, samples 3, 4, 8, 9, 11, 13, and 16 are homozygous for wild type, and samples 2, 5, 6, 10, 14, and 15 are homozygous for mutant.
• FIG. 12 is a photograph showing multiplex analysis of Hemochromatosis and Factor V.
• the results for Factor V were derived from use ofthe opposite strand to the results shown in Fig. 8.
• Two known Hemochromatosis and Factor V samples were each amplified individually.
• Factor V samples were amplified using primers Seq. Id. No. 13 (Biotin- ACTACAGTGACGTGGACATC) and Seq. Id. No. 14 (TGTTATCACACTGGTGCTAA).
• the amplification products were then combined together along with 1 ⁇ M of each of their 30-mer stabilizer oligos (i.e., Seq. Id. No. 8 and Seq. Id. No.
• Figs. 1A and IB illustrate a simplified version ofthe active programmable electronic matrix hybridization system for use with this invention.
• a substrate 10 supports a matrix or anay of electronically addressable microlocations 12.
• the various microlocations in Fig. 1A have been labeled 12 A, 12B, 12C and 12D.
• a permeation layer 14 is disposed above the individual electrodes 12. The permeation layer permits transport of relatively small charged entities through it, but limits the mobility of large charged entities, such as DNA, to keep the large charged entities from easily contacting the electrodes 12 directly during the duration of the test.
• the permeation layer 14 reduces the electrochemical degradation that would occur to the DNA by direct contact with the electrodes 12, possibility due, in part, to extreme pH resulting from the electrolytic reaction. It further serves to minimize the strong, non-specific adso ⁇ tion of DNA to electrodes.
• Attachment regions 16 are disposed upon the permeation layer 14 and provide for specific binding sites for target materials. The attachment regions 16 have been labeled 16A, 16B, 16C and 16D to correspond with the identification ofthe electrodes 12A-D, respectively.
• reservoir 18 comprises that space above the attachment regions 16 that contains the desired, as well as undesired, materials for detection, analysis or use.
• Charged entities 20, such as charged DNA are located within the reservoir 18.
• the active, programmable, matrix system comprises a method for transporting the charged material 20 to any ofthe specific microlocations 12.
• a microlocation 12 When activated, a microlocation 12 generates the free field electrophoretic transport of any charged entity 20 that may be functionalized for specific binding towards the electrode 12. For example, if the electrode 12 A were made positive and the electrode 12D negative, electrophoretic lines of force 22 would run between the electrodes 12A and 12D. The lines of electrophoretic force 22 cause transport of charged entities 20 that have a net negative charge toward the positive electrode 12 A.
• single nucleic acid polymo ⁇ hism refers to a locus containing simple sequence motif which is a mutation of that locus.
• An “array” as used herein typically refers to multiple test sites, minimally two or more test sites wherein discrimination between wild type and mutant polymo ⁇ hisms can be carried out for any target sequence at each individual site.
• the typical number of test sites will be one for each locus to be tested such that heterozygocity or homozygocity for either allele are distinguishable at each site.
• the number of loci required for any particular test will vary depending on the application, with generally one for genetic disease analysis, one to five for tumor detection, and six, eight, nine, thirteen or more for paternity testing and forensics.
• the physical positioning ofthe test sites relative to one another may be in any convenient configuration, such as linear or in an arrangement of rows and columns.
• the hybridization complex is labeled and the step of determining amount of hybridization includes detecting the amounts of labeled hybridization complex at the test sites.
• the detection device and method may include, but is not limited to, optical imaging, electronic imaging, imaging with a CCD camera, integrated optical imaging, and mass spectrometry. Further, the detection, either labeled or unlabeled, is quantified, which may include statistical analysis.
• the labeled portion of the complex may be the target, the stabilizer, the reporter or the hybridization complex in toto.
• Labeling may be by fluorescent labeling selected from the group of, but not limited to, Cy3, Cy5, Bodipy Texas Red, Bodipy Far Red, Lucifer Yellow, Bodipy 630/650-X, Bodipy R6G-X and 5-CR 6G. Labeling may further be accomplished by colormetric labeling, bioluminescent labeling and/or chemiluminescent labeling.
• Labeling further may include energy transfer between molecules in the hybridization complex by perturbation analysis, quenching, electron transport between donor and acceptor molecules, the latter of which may be facilitated by double stranded match hybridization complexes (See, e.g., Tom Meade and Faiz Kayyem, Electron Transfer Through DNA.Site-Specific Modification of Duplex DNA with Ruthenium Donors and Acceptors, Angew. Chem. Int. Ed., England, Vol. 34, #3, pp. 352-354, 1995).
• detection may be accomplished by measurement of conductance differential between double stranded and non-double stranded DNA. Further, direct detection may be achieved by porous silicon-based optical interferometry or by mass spectrometry.
• the label may be amplified, and may include for example branched or dendritic DNA. If the target DNA is purified, it may be unamplified or amplified. Further, if the purified target is amplified and the amplification is an exponential method, it may be, for example, PCR amplified DNA or strand displacement amplification (SDA) amplified DNA. Linear methods of DNA amplification such as rolling circle or transcriptional runoff may also be used.
• SDA strand displacement amplification
• the target DNA may be from a source of tissue including but not limited to hair, blood, skin, sputum, fecal matter, semen, epithelial cells, endothelial cells, lymphocytes, red blood cells, crime scene evidence.
• the source of target DNA may also include normal tissue, diseased tissue, tumor tissue, plant material, animal material, mammals, humans, birds, fish, microbial material, xenobiotic material, viral material, bacterial material, and protozoan material.
• the source ofthe target material may include RNA.
• the source ofthe target material may include mitochondrial DNA.
• Base-stacking is dependent on the interactions ofthe ring structure of one base with the base ring of its nearest neighbor. The strength of this interaction depends on the type of rings involved, as determined empirically. While the applicants do not wish to be bound by any theory, among the possible theoretical explanations for this phenomenon are the number of electrons available between the two bases that participate in Pi bond interactions and the efficiency of different base combinations that exclude water from the interior ofthe helix, thereby increasing entropy. Although the above models are consistent with current data, the possible mechanisms of stacking interactions are not limited to these concepts.
• the first is based on Hereditary Hemochromatosis, an autosomal recessive disorder that may lead to cirrhosis ofthe liver, diabetes, hypermelanotic pigmentation ofthe skin, and heart failure.
• the disease is linked to a G to A nucleotide transition at position 8445 in the HLA-H gene (Feder et al, J. Biol. Chem., Vol. 272, pp. 14025- 14028, 1997). This locus was subsequently renamed HFE.
• the second assay centers on the Factor V gene.
• a mutation at position 1,691 (G to A substitution) leads to an increased risk of venous thrombosis (Bertina et al, Nature, Vol. 369, pp. 64-67, 1994).
• a SNP scoring methodology that offers both high throughput and cost effectiveness should allow implementation of routine tests for detecting individuals at risk for these, as well as other diseases that correlate to known SNPs, before disease onset.
• the utility of SNPs as genetic markers is therefore dependent, at least in part, upon the ability to provide accurate scoring of SNPs quickly. We have developed a novel scoring methodology, which fits these criteria.
• Hemochromatosis and Factor V yields discrimination values greater than 15-fold between match and mismatch.
• the poorest discrimination for a homozygote was ⁇ 6.3-fold.
• heterozygotes yielded ratios of approximately 1 :1, and never more than 2:1. Since the discrimination values are so disparate between homozygotes and heterozygotes, it allows us to call homozygotes even if the amplification is biased towards one strand (see Fig. 11).
• Hemochromatosis and Factor V We initially chose Hemochromatosis and Factor V to be analyzed as each SNP has been linked to a specific and important disease (Feder et al, 1996 Supra; Bertina et al, 1994 Supra). Moreover, both conditions are relatively prevalent in society. A recent AACC bulletin report suggests that Hemochromatosis may be more prevalent than previously believed (American Association for Clinical Chemistry, Inc., Clinical Laboratory News, Vol. 25, number 2, pp. 16, February 1999). The use therefore of a methodology for early genetic testing of people at risk for these two afflictions should become an important tool in determining people that are heterozygous or homozygous for the mutant allele. This will allow early treatment, thereby improving quality of life.
• the sample containing a nucleic acid population representing one of or both wild type and mutant alleles is amplified with two primers, one being biotinylated (i.e., amplicon down format).
• the amplification product 31, with its biotinylated moiety, and the complementary strand 32 are diluted 1 :2 in a final concentration of 50mM histidine.
• This solution also contains l ⁇ M of stabilizer oligomer 33.
• the stabilizer oligomer 33 is generally a 30-mer that is 100% complementary to both wild type and mutant alleles. This stabilizer directly abuts the polymo ⁇ hism site on the target amplicon such that when a perfectly matched mutant reporter 34 or wild-type 35 is added to the system, base-stacking will be present.
• the reaction solution is heated to 95°C for 5 minutes to allow the amplicon to denature.
• This sample after cooling, is then electronically biased to the capture site of choice on an APEX type microchip.
• the biotinylated amplicon strand 31 is attached to the microchip capture site via the biotin/streptavidin interaction with the permeation layer ofthe microchip.
• the 30- mer stabilizer oligomer 33 is hybridized to the amplicon strand 31 through hydrogen bonds.
• the 30-mer stabilizer 33 effectively blocks the binding ofthe fully complementary nonbiotinylated amplicon strand 32 due the relative higher concentration ofthe stabilizer 33 (The stabilizer is at luM concentration whereas the amplicon is generally between 500pM and 5nM).
• Imaging is then performed using two different lasers, one corresponding to the fluorophore on the wild-type reporter and one to the fluorophore on the mutant reporter. From these signal intensities, backgrounds are subtracted and specific activities are taken into account. A ratio of wild type to mutant signal is achieved from which the allelic composition ofthe amplicon products are determined.
• EXAMPLE I a. Assay for the discrimination of single-nucleotide polymorphisms.
• SNP scoring on an active matrix chip was accomplished as exemplified by the methodology illustrated in Figure 2.
• the target was amplified with one biotinylated primer.
• a high concentration of 30-mer stabilizer oligo was added to the denatured amplicon and the mixture was electronically addressed to capture sites of interest on the array. Because DNA could be rapidly concentrated and hybridized, this process took place in a period as short as two minutes.
• the stabilizer oligomer was complementary to the biotinylated amplicon strand (the strand being probed).
• the stabilizer prevented the rehybridization ofthe complementary target amplicon strand thereby allowing the two allele-specific fluorescently-labeled reporter oligos access to the biotinylated strand.
• the stabilizer oligo was designed such that its 5 '-terminus abutted the polymo ⁇ hism of interest.
• the reporter oligos one perfectly complementary to the wild type allele and one to the mutant allele, were designed such that their 3 '-termini encompassed the polymo ⁇ hism.
• the stabilizer and reporter oligomers perfectly matched the target in an adjacently hybridized format, strong base-stacking energy phenomena were realized.
• the reporters were 11 bp in length which provided excellent base-stacking differential signal between perfect matches and SNP mismatches, notwithstanding the results disclosed by prior researchers as mentioned above.
• the mismatched reporter has one less nucleotide hydrogen bonded to its complement than the matched reporter. Upon stringent discrimination conditions, the perfectly matched reporter remains bound to its complement while the mismatched reporter readily dissociates.
• the stabilizer can be designed to anneal to the amplicon at a position nearer the 3' end ofthe amplicon thereby necessitating that the 3 '-terminus of the stabilizer abut the polymo ⁇ hism and the 5 '-terminus ofthe reporter encompass the polymo ⁇ hism.
• the stabilizer oligo enhances SNP discrimination by imparting base-stacking energy.
• the increased stabilization for perfectly matched complexes can also be demonstrated in the augmented signal intensities of samples that received more stabilizer oligo (compare Factor V mutant samples A and B, column 1 (least stabilizer) and column 4 (most stabilizer) Fig. 8).
• the discrimination values (Table 1) in the presence of stabilizer are excellent.
• the allelic makeup of all five unknown Factor V samples are unambiguous with A and B being homozygous mutant, C being a heterozygote, and D and E being homozygous wild type. All results were independently confirmed by allele-specific amplification.
• stabilizer oligomers to Hemochromatosis were designed such that a 1 bp or a 10 bp gap would exist between the stabilizer and reporter. These stabilizers were compared with the standard Hemochromatosis stabilizer that directly abuts the reporter. In this experiment, the stabilizer oligomers and sample, specifically a Hemochromatosis wild type, were concomitantly biased to duplicate capture sites. The results are shown in Figure 9. In the case of no stabilizer (column 1), the initial wild type reporter signal is substantially reduced.
• a stabilizer oligo prevents rehybridization of the complementary nucleic acid strand.
• a difficulty in directing one strand of an amplification product following denaturation to a specific capture site of interest is that under most conditions the complementary strand will anneal back to its cognate partner.
• a high concentration of stabilizer oligomer was included with the amplification product during electronic addressing.
• SNPs as genetic markers requires that their presence in a sample be accurately and quickly determined via a high throughput system. By taking advantage of an electric field to rapidly concentrate and hybridize nucleic acid, we are able to achieve discrimination results very efficiently. The accuracy of this SNP scoring method is demonstrated in the following experiment.
• throughput is increased for multiplex analysis of target sequences by electronically targeting more than one amplicon product to a single capture site. This both enhances the speed ofthe assay and increases the information yield ofthe microarray.
• Hemochromatosis and Factor V sa iples and their respective stabilizer oligos Two such combinations were tested in quadruplicate. One contained a Hemochromatosis wild type and a Factor V mutant ( Figure 12, columns 1 and 2). The other contained Hemochromatosis and Factor V heterozygotes ( Figure 12, columns 4 and 5). Reporting and stringent washing was carried out first with Hemochromatosis reporters, followed by repeating the process with Factor V reporters.
• a Reporter nucleotide represents the 5 '-terminus ofthe reporter oligo.
• ''Stabilizer nucleotide represents the 3'-terminus ofthe stabilizer oligo.
• Nucleotide represents mismatched nucleotide on target sequence. For example, if the reporter is an A, then the match on the target nucleic acid is a T, and the mismatches are A, C, and G.
• ⁇ Values are fold discrimination between the matched target nucleic acid and designated mismatch.
• the base-stacking was a 3'-T (stabilizer oligo) abutting a 5'-A (reporter oligo), the weakest of all base-stacking interactions (R. Sinden, DNA Structure and
• the mismatch on the target DNA was a G, a nucleotide known to form weak bonds with an opposing A.
• the non-optimal discrimination achieved here could easily have been overcome by analyzing the opposite amplicon strand.
• EXAMPLE II Besides the amplicon down format described in Fig. 2, a second format is useful wherein the stabilizer is anchored to specified capture sites (i.e., capture down format). As shown in Fig. 3, amplicon strands 90 and 91 may be denatured and combined with biotin labeled stabilizer oligo 92. Additionally, further enhancement of signal may be derived from the inclusion of an "interfering" oligomer 93 designed to be complementary to the undesired amplicon strand.
• This format has been successfully used for the detection of Hemochromatosis, Factor V, and EH1 mutations.
• addressing ofthe amplicon occurs after denaturation.
• a specific interference oligonucleotide may be added to the protocol at the time of addressing to the capture site. This oligonucleotide is designed to be complementary to the undesired amplicon strand and should be present in molar excess. It should be designed to hybridize to the region outside ofthe stabilizer/reporter complementary region.
• the interference oligonucleotide may be placed 5' or 3' to the base-stacked complex site.
• FIG. 4 sets forth a format wherein multiple SNP containing reporter probes are used with one another to provide multiple base-stacking energies.
• Fig. 4a shows the capture down format while Fig. 4b shows the amplicon down format.
• amplicon 42 is stabilized with stabilizer 41 that is anchored to a capture site via biotin moiety 40, and two reporter probes 43 and 44 are hybridized to detect the presence of at least two SNPs.
• Fig. 4b is similar except that the amplicon 45 is biotin labeled 40' and anchored to the capture site while stabilizer 46 is unlabeled. This format is useful where there are multiple closely spaced SNPs at a single genetic locus.
• Mannose Binding Protein gene locus that co ⁇ elates with susceptibility to sepsis in leukopenic patients. In this case there are 4 SNPs spaced within 15 bases of each other.
• Another example is the human HLA locus in which there are a large number of naturally occurring variants scattered within 3 exons.
• the reporter probes are base-stacked against a stabilizer oligo and each ofthe reporters may be labeled with a different fluorophore specific for an allele that occurs at these sites.
• Fig. 5 depicts a nested format wherein the target nucleic acid may be amplified using standard primers, one of which may be labeled (e.g., 52) for application ofthe amplicon down format.
• amplicons 50' and 51' may be denatured and mixed with stabilizer (and interfering oligo if desired) to yield stabilizer: amplicon hybridization complex (50"/56/51").
• This complex is then addressed to a specified capture site followed by introduction of reporter probe 58 that benefits from base- stacking energies due to stabilizing interactions at both its 5' and 3' termini.
• amplicon down format is illustrated, nested base-stacking can also be carried out using the capture down format.
• This nested method is useful where there are multiple SNPs at a single genetic locus as described in EXAMPLE in as well as in situations where it is desired to detect SNPs from remote genetic loci. Moreover, this method is functional where it is desired to detect the presence of different and genetically unrelated amplicons whose coincident identification may provide useful information.
• Such information can be defined as "target-specific nucleic acid information" which provides some degree of identification ofthe nature ofthe target sequence.
• a first region of a target nucleic acid may provide an amplicon used to identify the source ofthe nucleic acid (e.g., Staphylococcus vs. E. coli).
• the second amplicon may be used to identify a particular trait such as antibiotic resistance (e.g., methicillin resistance).
• antibiotic resistance e.g., methicillin resistance
• the nesting reporter may provide additional data where SNPs are additionally associated with one or the other or both ofthe genetic loci from which the amplicons were generated.
• SNPs are additionally associated with one or the other or both ofthe genetic loci from which the amplicons were generated.
• An example of this is the identification of bacteria by polymo ⁇ hisms within a conserved gene sequence, such as 16S rDNA, or gyrase A sequences. In each one of these amplicons there may not be sufficient genetic divergence to uniquely identify all species or subspecies.
• use of a second independent locus can provide essential data.
• gyrase A is useful alone however, discrimination between closely related bacterial strains may be greatly augmented by inclusion of polymo ⁇ hisms in the gyrase B or par C loci.
• the reporter probe may inco ⁇ orate SNP or other specific bases at both its 5' and 3' termini.
• internal bases ofthe reporter oligo can be designed to inco ⁇ orate unique sequence complementary to internal base positions ofthe stabilizer, while the terminal bases ofthe reporter may comprise bases specific to stabilizer, SNPs, or other bases ofthe different genetic loci.
• Fig. 6 further depicts an additional aspect ofthe nested method wherein multiple reporters 63 may be nested to detect multiple SNPs that may be associated with either ofthe amplicon 60 and 62, or 65 and 66 species. As with single reporter nesting, both the amplicon down and the capture down formats are applicable.
• Fig. 7 further depicts a variation ofthe nested method wherein amplification ofthe target is carried out using SDA.
• the amplification primers inco ⁇ orate nucleic acid sequence related to the amplification process (i.e., restriction endonuclease sequence)
• the termini ofthe amplicons hybridized to the stabilizer do not represent target-specific sequence.
• primers 70 and 71 specific for target locus 74, and primers 72 and 73 for target locus 75 each contain necessary restriction sites (e.g., Bso BI).
• amplicons 74' and 75' are flanked by primer sequences 76, 77, and 81, 82 respectively. Internal to theses flanking sequences may be located the specific SNP containing sequences of interest 78,79, 83, and 84, which in turn flank target specific sequence 80 and 85.
• This arrangement requires that the stabilizer oligo be designed to inco ⁇ orate each ofthe above sequences in order to hybridize both amplicons and stabilizer into a complex. This additionally means that the stabilizer inco ⁇ orates SNP sensitive sequence rather than the reporter oligo.
• capture down format is depicted, the amplicon format is equally applicable.
• reporter probe 87 is hybridized to the complex in a nested fashion.
• the reporter may be designed to be stabilized where there is not any mismatches between the stabilizer and amplicon.
• hybridization between the stabilizer and amplicon would necessarily result in a "bubble" formation allowing such mismatches to provide the destabilization necessary to keep the reporter from hybridizing.
• base-stacking schemes are provided that achieve discrimination by breaking long regions of hybridization into two or more sequences.
• This methodology allows for discrimination of specific nucleic acid sequences from relatively short probes.
• the fact that short probes are used provides the opportunity to use detection mechanisms sensitive to both passive and electronic hybridization techniques.
• the use of short probes provides the opportunity to use detection mechanisms based solely on the probe's mass (i.e., mass spectrometry) where extremely high levels of mass resolution are achieved by direct measurement (e.g. by flight or ESI). In such case, reporter probes having a length of 50 bases or less are preferred. Detection using mass spectrometry could be carried out by separating the probe from the hybridization complex and launching it directly to the mass spec detector.

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