WO1995021269A1 - Procede et appareil d'analyse de materiel genetique - Google Patents

Procede et appareil d'analyse de materiel genetique Download PDF

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Publication number
WO1995021269A1
WO1995021269A1 PCT/US1995/001395 US9501395W WO9521269A1 WO 1995021269 A1 WO1995021269 A1 WO 1995021269A1 US 9501395 W US9501395 W US 9501395W WO 9521269 A1 WO9521269 A1 WO 9521269A1
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dna
genetic material
pcr
chamber
nucleotide
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PCT/US1995/001395
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English (en)
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Mark K. Perlin
Michael B. Gorin
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Perlin Mark K
Gorin Michael B
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Publication of WO1995021269A1 publication Critical patent/WO1995021269A1/fr

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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
    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the present invention pertains to a process for determining inheritance patterns in eukaryotic DNA. More specifically, the" present invention is related to densely sampling the genome with polymorphic genetic markers using a hybridization-based genotyping method, and then using this genetic information to assess the trait inheritance, including disease susceptibility, mendelian genetic disorders, and complex traits relevant for plant or animal husbandry.
  • One such hybridization-based genotyping method entails forming mismatched heteroduplexes and quantitating single-stranded loop sizes.
  • the specific objective of the system is genome-wide high-resolution genotyping for the purpose of health risk assessment, including genetic susceptibility for disease, and identification of disease-associated genes.
  • the means for achieving this is genotyping polymorphic genetic loci by hybridization assays.
  • Genomic mismatch scanning (Nelson, S.F., McCusker, J.H. , Sander, M.A. , Kee, Y. , Modrich, P. , and Brown, P.O. 1993. Genomic mismatch scanning: a new approach to genetic linkage mapping. Nature Genetics , 4 (May) : 11-18.) , incorporated by reference, is one such approach, but has limited throughput since experiments are done on pairs (not sets) of individuals.
  • a sequence-tagged site is defined herein as a location on a genome characterized by at least one sequence. Much of this effort is done by Weissenbach's group at CEPH in France (Weissenbach, J., Gyapay, G., Dib, C. , Vignal, A., Morissette, J. , Millasseau, P., Vaysseix, G. , and Lathrop, M. 1992. A second generation linkage map of the human genome. Nature , 359: 794-801), incorporated by reference, and by Lander's group at the Whitehead Institute in Cambridge, Massachusetts. STSs are readily amplified by means of the polymerase chain reaction
  • VNTR variable nucleotide tandem repeat
  • the approach described herein centers on a detailed examination of such highly polymorphic intron genetic markers, rather than the highly conserved genes and their exon coding regions.
  • the method also applies to expanded repeats within genes, and specific nucleotide alterations of specific DNA sequences.
  • genotyping (1) an associated technology that will reduce the cost and error of the requisite genotyping, and thus enable widespread usage. Further, this technology must be coupled with (2) data acquisition and analysis methods that allow for fully automated error detection, risk analysis, and linkage analysis for both populations and families. Completion of this analysis generates a vast amount of data, hence the results must (3) be presented in a targeted fashion to disparate groups of end-users.
  • the novel parallel genotyping apparatus for polymorphic VNTRs The approach is to spatially localize each genetic locus in a two-dimensional array, and then locally aggregate PCR-amplified DNA products to the proper array regions. Then, perform DNA hybridization studies by means of a detection mechanism to quantitate properties of the PCR products, and thereby determine the alleles (i.e., the genotype) for every genetic locus.
  • a VNTR is a linear sequence of (deoxy)nucleotides of the pattern LW n R, where W is a short DNA sentence repeated n times, contained within two flanking regions of unique sequences: the left flanking region L, and the right flanking region R. These flanking sequences establish the singularity of a specific VNTR within a haploid genome. These unique sequences allow a VNTR to be associated with a specific location within the genome such that it can be physically or genetically mapped with respect to other DNA markers and/or genetic traits and disorders. Variations in the number of repetitive elements within the VNTR are common among individuals and allow specific alleles to be tracked as they are genetically transmitted from individuals to their offspring.
  • VNTRs are the short tandem repeat (STR), where n tends to be small (e.g., ⁇ 100), and repeating unit short (e.g., between two and five).
  • a CA-repeat is an STR where the dinucleotide CA is repeated n times, where n ranges in a human population from roughly ten to forty. There are an estimated 100,000 such CA-repeat loci in the human genome.
  • Other VNTRs include trinucleotide and tetranucleotide repeats. Following PCR, the allelic variation in tandem repeat number can be determined by DNA size measurements using polyacrylamide gel electrophoresis.
  • VNTRs are important for several reasons.
  • Many VNTRs have been associated with specific diseases (e.g., Huntington's disease, fragile X syndrome) (Kre er, I., Pritchard, M. , Lynch, M. , Yu, S., Holman, K. , Baker, E., Warren, S.T., Schlessinger, D. , Sutherland, G.R. , and Richards, R.I. 1991. Mapping of DNA instability at the Fragile X to a trinucleotide repeat sequence p(CCG) n . Science , 252: 1711-1714), incorporated by reference, where, in "anticipation", larger n often correlates with increased severity.
  • STRs serve as highly useful markers for specific diseases (Clemens, P., Fenwick, R. , Chamberlain, J. , Gibbs, R. , de Andrade, M. , Chakraborty, R. , and Caskey, C. 1991. Linkage analysis for Duchenne and Becker muscular dystrophies using dinucleotide repeat polymorphisms. Am J Hum Genet , 49: 951-960.), incorporated by reference.
  • (3) STRs are useful as sequence tagged sites (STSs) (Olson, M. , Hood, L. , Cantor, C. , and Botstein, D. 1989. A common language for physical mapping of the human genome.
  • genotyping is easily effected by measuring the total length of the PCR product. This is commonly done by spatially (or temporally) separating DNA molecules of different sizes (or conformations) using, for example, gel electrophoresis.
  • This invention therefore describes more cost effective approaches that enable higher throughput STR genotyping.
  • These methods employ nucleotide hybridization assays that directly measure the number of STR repeat units, rather than total fragment length.
  • Such detections by hybridization are miniaturizable, hence parallelizable (Monaco, A.P., Lam, V.M.S., Zehetner, G. , Lennon, G.G., Douglas, C. , Nizetic, D. , Goodfellow, P.N. , and Lehrach, H. 1991.
  • Nucleic Acids Res , 19(12): 3315-3318. incorporated by reference, and, ultimately, highly manufacturable. Further, they can be adapted to work in chemical solutions, or on substrates with small surface area.
  • the first method entails creating and detecting loop mismatches in heteroduplexes formed from the alleles* PCR products.
  • the second method uses hybridization panels to determine the alleles.
  • the present invention pertains to an apparatus for analyzing the genetic material of an organism.
  • the apparatus comprises means for amplifying the genetic material of the organism.
  • the apparatus also comprises means for characterizing the amplified genetic material.
  • the characterizing means is in communication with the amplifying means.
  • the characterizing means contains all of the genetic material within a region having a radius of less than two feet.
  • the amplifying means and characterizing means characterize the genetic material at a rate exceeding 100 sequence-tagged sites per hour per organism. The sequence-tagged sites are inherent to the genetic material.
  • the genetic material includes nucleotide sequences.
  • the amplifying means preferably includes a reaction plate with which the genetic material is in contact.
  • the reaction plate has a plurality of chambers, each of which is disposed in a unique location of the plate corresponding to a location within a genome having at least one nucleotide sequence.
  • the characterizing means preferably includes means for detecting whether a chamber contains a nucleotide sequence of the genetic material corresponding to the chamber's unique location.
  • the apparatus preferably also includes a thermocycler in thermal communication with the plate to heat and cool the plate.
  • the detecting means preferably includes a detector connected to the chambers which produces a chamber signal for each chamber corresponding to genetic material in each chamber.
  • the detecting means preferably also includes a processor in communication with the detector which receives the signal and identifies unique properties of the nucleotides in each chamber. The unique properties of the nucleotide of the genetic material in each chamber pertain to a number of nucleotides in any of the nucleotide sequences of the genetic material.
  • the amplifying means preferably includes at least one nucleotide sequence that corresponds to each chamber and which is in contact with the chamber. Each nucleotide sequence interacts with the nucleotide sequence of the genetic material of the nucleotide sequence if it is present.
  • the present invention also pertains to a method for analyzing genetic material of an organism.
  • the method comprises the steps of amplifying the genetic material. Then there is the step of characterizing the amplified genetic material in a region having a radius of less than 20 feet at a rate exceeding 100 sequence-tagged sites per hour per organism.
  • the genetic material includes RNA or DNA.
  • the characterizing step there preferably is the step of accessing risk of illness for which there is a genetic susceptibility in the organism. Such illnesses can include cancer, heart disease, etc.
  • the present invention also pertains to a method for manufacturing an apparatus for analyzing genetic material of an organism.
  • the method comprises the steps of placing corresponding sequence-tagged sites in contact with corresponding chambers of a plate. Then, there is the step of connecting detectors to the chambers which can detect where the nucleotide sequences of the genetic material of the organism, when placed in contact with the chambers, have reacted with the corresponding sequence-tagged sites in the corresponding chamber. Then, there is the step of placing a thermocycling device in contact with the plate to cause the sequence-tagged sites in the chambers to react with genetic material of the organism that is placed in contact with the chambers. Next, there is the step of connecting a computer to the detectors and to the thermocycling device to control operation of the thermocycling device, and to receive signals which correspond to the genetic material of the organism and the sequence-tagged sites of each chamber from the detectors.
  • the present invention also pertains to a method for determining the size of nucleotide sequences of an STR marker contained on genetic material comprising the steps of: amplifying the nucleotide sequences of the genetic material in a region relating to the STR marker. Then there is the step of performing nucleic acid hybridizations on the amplified nucleotide sequences. Then there is the step of producing signals corresponding to the hybridizations of the amplified nucleotide sequences. Then there is the step of determining the sizes of the nucleotide sequences contained in the genetic material.
  • Figure 1 is a schematic representation of a preferred embodiment of the apparatus.
  • Figure 2 is a schematic representation of parts of
  • FIGS 3a-3d list the steps for parallel genotyping of the present invention.
  • Figures 4a and 4b are schematic representations of mismatched loops formed from allele DNA.
  • Figure 5 includes figures 5a-5c and is a schematic representation of loop mismatch for determining a sum of STR alleles.
  • Figure 6 includes figure 6a and is a block diagram showing loop mismatch for determining a difference of STR alleles.
  • Figure 7 is a flow chart for determining the STR alleles from the sum and difference.
  • Figure 8 is a flow chart of loop mismatch protocol for a single STR locus.
  • Figure 9 is a flow chart for reducing the number of PCR experiments.
  • Figures lOa-lOc show representations for increasing measured signal from loops with respect to summation experiment.
  • Figures 11a and lib are representations for increasing measured signal from loops with respect to difference experiments.
  • Figure 12 is a flow chart of concordance mapping for genetic patterns.
  • Figure 13 includes parts a-c and is a flow chart for determining an STR allele sum from a nucleic acid synthesis step.
  • Figure 14 includes parts a-c and is a flow chart for determining an STR allele difference from a nucleic acid synthesis step.
  • Figure 15 is a flow chart for determining STR alleles from a nucleic acid synthesis step.
  • Figure 16 is a schematic representation of an assay for determining STR alleles from a nucleic acid ligation step.
  • Figure 17 includes parts a-b and is a schematic representation of an assay for determining STR alleles from a nucleic acid loop ligation step.
  • the apparatus comprises means for amplifying the genetic material of the organism.
  • the apparatus also comprises means for characterizing the amplified genetic material.
  • the characterizing means is in communication with the amplifying means.
  • the characterizing means contains all of the genetic material within a region having a radius of less than two feet. It should be noted that the region could have a radius of any reasonable size commensurate with the requirements of the task. For instance, the radius of the region could range from 1 cubic millimeter up to 10 feet by and anywhere in between.
  • the amplifying means and characterizing means characterize the genetic material at a rate preferably exceeding 100 sequence- tagged sites per hour per organism. It should be noted that the rate could be up to 100,000 sequence-tagged sites per hour per organism, or as slow as desired, or any rate in between. Also, per organism could also be defined to be the characterization of genetic material of multiple organisms. The sequence-tagged sites are inherent to the genetic material.
  • the genetic material includes nucleotide sequences.
  • the amplifying means preferably includes a reaction plate 102 with which the genetic material is in contact.
  • the reaction plate 102 has a plurality of chambers, each of which is disposed in a unique location of the plate 102 corresponding to a location within a genome having at least one nucleotide sequence.
  • the characterizing means preferably includes means for detecting whether a chamber contains a nucleotide sequence of the genetic material corresponding to the chamber's unique location.
  • the apparatus preferably also includes a thermocycler 104 in thermal communication with the plate 102 to heat and cool the plate 102.
  • the detecting means preferably includes a detector 108 connected to the chambers which produces a chamber signal for each chamber corresponding to genetic material in each chamber.
  • the detecting means preferably also includes a processor 110 in communication with the detector 108 which receives the signal and identifies unique properties of the nucleotides in each chamber.
  • the unique properties of the nucleotide of the genetic material in each chamber pertain to a number of nucleotides in any of the nucleotide sequences of the genetic material.
  • the amplifying means preferably includes at least one nucleotide sequence that corresponds to each chamber and which is in contact with the chamber. Each nucleotide sequence interacts with the nucleotide sequence of the genetic material of the nucleotide sequence if it is present.
  • the present invention also pertains to a method for analyzing genetic material of an organism.
  • the method comprises the steps of amplifying the genetic material. Then there is the step of characterizing the amplified genetic material in a region having a radius of less than 20 feet at a rate exceeding 100 sequence-tagged sites per hour per organism.
  • the genetic material includes RNA or DNA.
  • the characterizing step there preferably is the step of accessing risk of illness for which there is a genetic susceptibility in the organism. Such illnesses can include cancer, heart disease, etc.
  • the present invention also pertains to a method for manufacturing an apparatus for analyzing genetic material of an organism.
  • the method comprises the steps of placing corresponding sequence-tagged sites in contact with corresponding chambers of a plate 102. Then, there is the step of connecting detectors 108 to the chambers which can detect where the nucleotide sequences of the genetic material of the organism, when placed in contact with the chambers, have reacted with the corresponding sequence-tagged sites in the corresponding chamber. Then, there is the step of placing a thermocycling device 104 in contact with the plate 102 to cause the sequence-tagged sites in the chambers to react with genetic material of the organism that is placed in contact with the chambers.
  • thermocycling device 104 there is the step of connecting a computer 110 to the detectors 108 and to the thermocycling device 104 to control operation of the thermocycling device 104, and to receive signals which correspond to the genetic material of the organism and the sequence-tagged sites of each chamber from the detectors 108.
  • the present invention also pertains to a method for determining the size of nucleotide sequences of an STR marker contained on genetic material comprising the steps of: amplifying the nucleotide sequences of the genetic material in a region relating to the STR marker. Then there is the step of performing nucleic acid hybridizations on the amplified nucleotide sequences. Then there is the step of producing signals corresponding to the hybridizations of the amplified nucleotide sequences. Then there is the step of determining the sizes of the nucleotide sequences contained in the genetic material.
  • a parallel genotyping apparatus is described.
  • the purpose of said apparatus is to provide a physical, chemical, mechanical, and computational embodiment for performing simultaneous experiments on multiple genetic markers used for genetic characterization.
  • the apparatus is comprised of the following components:
  • thermocycling device 104 (1) A multi-chambered reaction plate 102. (2) A thermocycling device 104.
  • a computer device 110 with a memory.
  • the biochemical reactions occur in the chambers of the reaction plate 102, wherein a "chamber" denotes any localized region suitable for performing said reactions.
  • the thermocycling device 104 provides a means for PCR and hybridization experiments.
  • the robotic device 106 provides a means for transferring chemicals and performing other physical/chemical operations.
  • the detection device 108 is used to quantitatively measure the signals from DNA hybridization experiments.
  • the computer device 110 coordinates the activity of the other components, and performs any needed computations.
  • the primary requirement of the multi-chambered reaction plate 102 is a set of spatially arrayed chambers, each containing its own PCR primers for genome characterization, and providing operations for PCR amplification, DNA hybridization, and signal detection.
  • Any physical device, of any number of dimensions, in whole or in part, that provides this functionality can serve as a physical embodiment for the apparatus.
  • parallel synthesis methods for producing the oligonucleotides by spatially addressable masking techniques on a surface have been described (Fodor, ⁇ .P.A., Read, J.L., Pirrung, M.C., Stryer, L. , Lu, A.T., and Solas, D. 1991. Light-directed spatially addressable parallel chemical synthesis.
  • the basic container for the parallel genotyping reactions is a commercially available polystyrene or polycarbonate 384-chamber microtiter plate (USA Scientific Products, Ocala, FI) .
  • Alternative embodiments include 96-chamber and 864-chamber plates. Each chamber corresponds to one chamber. These plates occupy the space of standard 96-chamber microtiter plates and are compatible with current robotic systems such as the Beckman Biomek system.
  • the apparatus has one or more two-dimensional surfaces 102 comprised of reaction chambers.
  • Each STS genetic marker used from a genome corresponds to some reaction chamber.
  • This experimentation surface provides a means for performing parallel laboratory operations on all the chambers simultaneously.
  • five steps are performed: (1) A deposition of at least two oligonucleotides into the chamber. These oligonucleotides serve as PCR primers for the STS marker specific to the chamber.
  • Means are provided by the apparatus for PCR amplification, DNA hybridization, and signal detection. The following description relates these functions to the parts of the apparatus.
  • PCR Amplification The apparatus provides the means for amplifying the STS DNA region subsequent to presentation with genomic DNA.
  • PCR Innis, M.A. , Gelfand, D.H. , Sninsky, J.J., and White, T.J. 1990.
  • PCR Protocols A Guide to Methods and Applications . San Diego, CA: Academic Press. Mullis, K.B., Faloona, F.A. , Scharf, S.J., Saiki, R.K. , Horn, G.T. , and Erlich, H.A. 1986.
  • Specific enzymatic amplification of DNA in vitro the polymerase chain reaction. Cold Spring Harbor Symp. Quant . Biol .
  • thermocycling components for heating and cooling the reaction mixture.
  • the genomic DNA and PCR reagents are simultaneously transferred to the chambers by means of the robotic device.
  • thermostable polyermases can be used (Garrity, P.A. , and Wold, B.J. (1992). Effects of different DNA polymerases in ligation-mediated PCR: enhanced genomic sequencing and in vivo footprinting. Proceedings of the National Academy of Sciences of the United States of America , 89(3): 1021-5. Ling, L.L., Keohavong, P., Dias, C. , and Thilly, W.G. (1991) . Optimization of the polymerase chain reaction with regard to fidelity: modified T7, Taq, and vent DNA polymerases. Per Methods & Applications , 1(1): 63-9.), incorporated by reference.
  • thermocycling is done using a conventional programmable block thermal cycler 104 based on the heating and cooling of a metal block (using Peltier or fluid refrigerants for cooling) (R. Hoelzel, Trends in Genetics, August 1990, volume 6 #8; p 237-8), incorporated by reference.
  • the reaction plate is transferred to and from this computer-controlled thermal cycler by means of the robot 106.
  • a device 104 is used that heats and cools a rapidly circulating air mass around the plate (e.g., Biotherm PCR oven) (Garner, H.R. , Armstrong, B. , and Lininger, D.M. (1993). High-throughput PCR.
  • a robotic attachment (Beckman Biomek) , incorporated by reference, comprised of a thermocycling surface which has the same 384-chamber shape as the reaction plate is used to physically mate with the 384- chamber reaction plate, and provide the necessary heating and cooling operations under computer control.
  • heating and cooling elements such as Peltier junctions can be physically incorporated into the apparatus. This surface is suitable for transferring sample genomic DNA to many chambers simultaneously. Miniaturization enable shorter cycle times and greater homogeneity because of the rapid temperature equilibration of the thin films and small volumes.
  • DNA hybridization Sufficient volume and chemical composition is provided within each reaction chamber so that the requisite DNA hybridization (Ausubel, F.M. , Brent, R. , guitarist, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. , and Struhl, K. , ed. 1993. Current Protocols in Molecular Biology . New York, NY: John Wiley and Sons. Sa brook, J. , Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning, second edition . Plainview, NY: Cold Spring Harbor Press.), incorporated by reference, can occur.
  • the robotic component of the apparatus transfers the hybridization reaction mixture to the chambers, and provides means for heating and cooling the reaction chamber, as described above.
  • heteroduplexes include chemical derivatization and endonuclease digestion of single-stranded components.
  • the detection of the heteroduplexes and nucleotides within the loops is done with a commercially available spectrophotometric/fluorometric instrument 108 similar to that used for ELISAs (Dynatech Laboratories, Chantilly, Va) , incorporated by reference, modified to accomodate the larger number and smaller size chambers.
  • a scanning laser fluorimeter can also be employed over the plate surface. Because -the plate is flat and comprised of an optical grade surface, fluorescent detection is straightforward. The robot transfers the reaction plate to this optical detection device prior to the detection operation.
  • computerized fluorescent scanning microscopes are used that are capable of detecting and quantitating fluorescent signals and are suitable for the miniaturized system. These have been developed for immunological and genetic cytochemistry (Biological Detection Systems) , incorporated by reference.
  • a physical signal is measured from the reagent attached to a PCR primer.
  • detection reagents include (but are not limited to) radioactivity, fluorescence, phosphorescence, chemiluminescence, electrical resistivity, pH, and ionic concentration.
  • the direct electrical detection mechanisms are particularly attractive for direct coupling of the experiment onto a minaturized solid state detection device (Briggs, J. , Kung, V.T., Gomez, B., Kasper, K.C., Nagainis, P.A., Masino, R.S., Rice, L.S., Zuk, R.F., and Ghazarossian, V.E. (1990) .
  • the analysis of the signals is done by a computer device 110. Means are provided for the signals are transferred from the detector into the memory of the computer. A computer program for determining genotypes from the quantitative signals and calibrations curves resides in the memory of said computer.
  • the apparatus is manufactured by selecting a set of genetic markers, synthesizing both standard and derivatized oligonucleotide primers, and then depositing said oligonucleotide primers into the reaction chambers of a 384-chamber plate. This plate is then positioned with the other components of the apparatus, including the thermocycling device, the robotic device, the detection device, and the computer device.
  • a sufficient number of polymorphic genetic markers are chosen for unambiguously characterizing or tracing chromosomes in an organism containing DNA or RNA. Depending on the application, this can range from 10 centiMorgan (cm) to 0.001 cm. One cm is approximately one million megabases (Mb) . In a preferred embodiment, a resolution of 0.1 cm, or 100,000 base pairs (bp) , is used. In the human species, for example, which contains about 3 billion bp, this works out to 30,000 markers.
  • the genetic markers to be used for each STS are obtained as PCR primer sequences pairs from available databases (Genbank, GDB, EMBL; Hilliard, Davison, Doolittle, and Roderick, Jackson laboratory mouse genome database.
  • oligonucleotide primers for each STS are synthesized (Haralambidis, J. , Duncan, L. , Angus, K. , and Tregear, G.W. 1990. The synthesis of polyamide- oligonucleotide conjugate molecules. Nucleic Acids Research , 18(3): 493-9. Nelson, P.S., Kent, M. , and Muthini, S. 1992. Oligonucleotide labeling methods. 3. Direct labeling of oligonucleotides employing a novel, non-nucleosidic, 2- aminobutyl-1,3-propanediol backbone. Nucleic Acids Research , 20(23): 6253-9.
  • primers may be derivatized with a fluorescent detection molecule or a ligand for immunochemical detection such as digoxigenin. Derivatization of the primer for binding to the surface entails the incorporation of a biotinylated nucleotide at the 5' end of the synthetically made oligonucleotide. Additional biotinylated residues can also be incorporated (depending on the protocol) into this primer either at the time of biosynthesis or by secondary photo or chemical biotinylation.
  • oligonucleotides and their derivatives can be ordered from a commercial vendor (Research Genetics, Huntsville, AL) .
  • the oligonucleotide primer sets are deposited into each reaction chamber by means of a robotic system from source chambers containing a large store of presynthesized oligonucleotides. Said transferring can be effected in one or more operations, wherein oligonucleotide primers are deposited into multiple chambers in each transferring step, thereby creating a two-dimensional spatial array.
  • this deposition is effected by means of a parallel deposition device to which the 384-chamber plate is presented by means of a conveyor belt.
  • the deposition device has source chambers, each containing a large store of a unique oligonucleotides specific to a reaction chamber. Said source chambers are spatially arrayed to conform to the reaction chambers of the plate. Both the device and plate are properly positioned and made stationary, and then the chambers are filled in one or more more steps with the oligonucleotide.
  • the plates are dried and each chamber is then coated with a wax material, such as Ampliwax (Perkin-Elmer, Norwalk, CT) , incorporated by reference.
  • a wax material such as Ampliwax (Perkin-Elmer, Norwalk, CT) , incorporated by reference.
  • This material hardens at 4°C, is liquid throughout the temperature range of the PCR, and serves as a vapor barrier to prevent evaporation of the PCR reactions during the denaturation steps at 95 * C.
  • the oligonucleotides are covalently attached to a substrate such as glass by spatially addressable light-directed parallel DNA synthesis (Drmanac, R. , Drmanac, S., Strezoska, Z., Paunesku, T. , Labat, I., Zeremski, M. , Snoddy, J. , Funkhouser, W.K. , Koop, B. , and Hood, L. 1993. DNA Sequence Determination by Hybridization: a Strategy for Efficient Large-scale Sequencing. Science , 260: 1649-1652. Fodor, S.P.A., Read, J.L., Pirrung, M.C., Stryer, L.
  • Other components of the apparatus include resins and filters that will nonspecifically and reversibly bind double-stranded DNA, but not free nucleotides or short oligonucleotides (Molecular Biology LabFax, T.A. Brown, ed. Academic Press p281-4) , incorporated by reference. These are commercially available and can be readily modified to be fit within a manifold that will ensure leak-proof contact with the reaction chambers or plates. Uncharged nylon, charged nylon, and nitrocellulose are some of the filter materials in current use (Harley, C.B. , and Vaziri, H. (1991). Deproteination of nucleic acids by filtration through a hydrophobic membrane. Genetic Analysis, Techniques &
  • the commercially available polystyrene or polycarbonate 384- chamber microtiter plate 102 is arranged in a 24 by 16 array.
  • the commercially available robotic device 106 has a surface with 384 chambers arranged in a spatial configuration identical to that of the reaction plate 102.
  • all robotic actions e.g., for the steps of amplification, hybridization, and detection
  • robotic device 106 in mechanical juxtaposition with plate 102.
  • the commercially available programmable block thermal cycler 104 has a surface with 384 chambers arranged in a spatial configuration identical to that of the reaction plate 102. During thermocycling, every chamber of the plate 102 is in direct contact with its corresponding chamber in the thermocylcer 104.
  • the commercially available programmable oven thermocyler 104 is sufficiently large to accommodate the dimensions of 384-chamber reaction plate 102, and has sufficient uniformity to perform the necessary amplification reactions within each chamber.
  • a robotic device is used to transfer the reaction plate 102 to and from the oven thermocycler 104.
  • the commercially available ELISA-like spectrophotometric/fluorometric detection device 108 contains 384 chambers arranged in an spatial configuration identical to that of reaction plate 102. During the detection phase, the plate 102 is placed into the detector, with each chamber of plate 102 residing within its corresponding detection chamber of detector 108. This enables detections to be conducted simultaneously and independently for each chamber.
  • the computer device 110 coordinates the activities of the other components plate 102, thermocyler 104, robotic device 106, and detector 108. Note that most commercial thermocylers, robotic devices, and detectors include computational facilities for independently performing control, detection, and processing tasks, thus freeing the computer device 110 from such low-level processes.
  • the computer device 110 is connected to the detector 108. Signals obtained from the detector 108 are transferred to the memory of computer 110.
  • the computer 110 employs processing means for interpreting the signals in its memory, and determines and outputs the characteristics of nucleotide sequences in each chamber of the reaction plate 102.
  • genomic DNA is first extracted from an individual (say, by processing a blood sample) .
  • PCR reagents are then mixed with the genomic DNA, and a robotic device applies this PCR/DNA mixture to the chambers of the reaction plate of the apparatus. Every chamber has its own predeposited PCR primers that define a unique genetic marker.
  • PCR amplificiation of the genomic DNA marker region is then performed on every chamber using the thermocycling component of the apparatus.
  • a quantitative hybridization experiment is then conducted in ⁇ every chamber, possibly modifying the DNA.
  • the signals from these hybridization experiments are then measured from every chamber using the detection component, such as fluorescence measurements with a scanning light microscope. More than one (e.g., two or three) such parallel experiments may be needed to acquire all necessary genotyping data for one STR.
  • the measurements are then collected and analyzed by the component computer device to characterize the alleles at every marker.
  • the resulting genotyping information from the multiple alleles can be used for a number of applications, as described below.
  • One important use is the determination of genetic risk for phenotypic traits, including diseases.
  • haplotypes can be compared, and the shared genomic regions determined.
  • Correlating a shared trait and genotype commonalities enables a determination of genomic patterns that imply a quantitative risk for said trait.
  • These patterns can be applied to the genotypes of an individual and their relatives to compute a probability of expressing the trait.
  • the traits correspond to common multigenic multifactorial diseases, the highest risk entities are determined, and preventative measures undertaken, thereby improving the health of said individual.
  • Software systems are built to tailor the genotyping information for this advising task.
  • the quantitative hybridization experiment that is used in the preferred embodiment is a pair of loop mismatch assays.
  • the first assay measures the sum of the two STR allele loops, relative to a third (and smaller) STR.
  • the second assay measures the difference of the two STR allele loops relative to each other. By combining the sum and difference values, the two alleles can be determined.
  • the quantitative loop detection is effected by directly measuring the signals derived from the loops relative to the number of strands with loops (this is described in detailed later on) .
  • the loops are quantitated either by a chemical modification of the single-stranded loop DNA into a detectable state, or by incorporation of labeled DNA and subsequent digestion and detection of the single-stranded loop.
  • the number of strands is measured by using an end-labeled PCR primer.
  • the ratio of the (calibrated) loop measurements to the number of strands determines the loop size.
  • multiple hybridizations are performed for every STR, producing a patter that determines the genotype.
  • This system for performing multiple genotypings in parallel, with each STR in its separate cell, has many useful advantages over current genotyping methods, including the best gel-based multiplex methods. Specifically,
  • the experiment's architecture allows independent interchangeability of STR loci. Any STR(s) of the same class can be placed at any cell of the device.
  • the synthesis of oligonucleotides can be spatially or temporally separated from the execution of the PCR amplification and the detection.
  • Manufacturing enables miniaturization of the device, and the incorporation of detection machinery into the device.
  • the manual labor required for genotyping is greatly reduced, because the manufactured device eliminates the separate steps of handling multiple (e.g., thousands) specific STR primers. This includes synthesizing the oligonucleotides, performing the PCR, loading gels or other detection devices, and checking the genotyping results.
  • the CA (or GT) repeat region is of varying length.
  • Complementary strand A second strand having a Watson- Crick complementary DNA sequence to a first strand. However, the number of CA or GT repeats need not equal that of the first strand.
  • Upper strand The DNA strand 202 of the STR locus that contains the CA-repeat units.
  • Lower strand The DNA strand 204 complementary to the upper strand that contains the GT-repeat units.
  • the PCR oligonucleotide primer 206 that initiates the upper strand of the STR locus.
  • the PCR oligonucleotide primer 208 that initiates the lower strand of the STR locus.
  • the system is comprised of the following steps:
  • Step 1 entails the manufacture of an apparatus in which STR loci have been selected, and appropriate oligonucleotides (withmodifications) synthesized and deposited within each chamber.
  • Step 2a the process begins by extracting DNA from blood or tissue.
  • DNA There are numerous standard methods to isolate DNA including whole blood, isolated lymphocytes, tissue, and tissue culture (Ausubel, P.M., Brent, R. , guitarist, R.E., Moore, D.D., Seid an, J.G., Smith, J.A. , and Struhl, K. , ed. 1993. Current Protocols in Molecular Biology . New York, NY: John Wiley and Sons. Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning, second edition . Plainview, NY: Cold Spring Harbor Press. Nordvag 1992. Direct PCR of Washed Blood Cells.
  • DNA is extracted from anticoagulated human blood removed by standard venipuncture and collected in tubes containing either EDTA or sodium citrate.
  • the red cells are lysed by a gentle detergent and the leukocyte nuclei are pelleted and washed with the lysis buffer.
  • the proteinase K digestion is performed for 2 hours to overnight at 50 * C.
  • the solution is then extracted with an equal volume of buffered phenol-chloroform.
  • the upper phase is reextracted with chloroform and the DNA is precipitated by the addition of NaAcetate pH 6.5 to a final concentration of 0.3M and one volume of isopropanol.
  • the precipitated DNA is spun in a desktop centrifuge at approximately 15,000 g, washed with 70% ethanol, partially dried and resuspended in TE (lOmM Tris pH 7.5, 1 mM EDTA) buffer.
  • TE lOmM Tris pH 7.5, 1 mM EDTA
  • the reaction plates of the apparatus are maintained at 4"C at the time the genomic DNA has been mixed with the other components of the PCR reaction.
  • these other components include, but are not limited to, the standard PCR buffer (containing Tris pH8.0, 50 mM KCl, 2.5 mM magnesium chloride, albumin) , triphosphate deoxynucleotide ⁇ (dTTP, dCTP, dATP, dGTP) , the thermostable polymerase (Taq polymerase in this preferred embodiment, but others are available though buffer conditions are somewhat different) (Garrity, P.A. , and Wold, B.J. 1992.
  • the PCR primers for each locus are chosen for consistency with these uniform reaction conditions.
  • the total amount of this mixture is determined by the final volume of each PCR reaction (say, 10 ul) and the number of reactions (say, 384) .
  • This mixture can also be varied by including some of the constituents with the primers that are previously deposited in the microchambers. All of the necessary components for the PCR reactions are kept separate until the Ampliwax is melted and the aqueous phases reconstitute, each reaction cell receives a consistent and reproducible amount of the necessary components, and the combination of constituents does not compromise stability and biological activity (e.g., the Taq polymerase may be unstable if stored in a lyophilized state on the reaction plates) .
  • Step 2b the DNA/PCR mixture is applied to the reaction chambers with the Biomek robotics unit and the PCR is initiated by heating the plate rapidly to 95"C in order to melt the ampliwax, allow the DNA/PCR mixture to mix with the oligonucleotide primers (convection mixture is sufficent) , and denature the genomic DNA.
  • the ampliwax forms a stable vapor barrier over the chambers during the PCR reactions.
  • This method of initiating the PCR reactions is referred to as a "hot start" (D'Aquila et al., Nuc. Acid Res. 19 (13) 3749 ( 1 9 9 1)), incorporated by reference, and has the additional benefit of reducing the amount of nonspecific PCR products that are produced, thus improving the purity and amount of the final desired PCR signal that will be detected.
  • Step 2c the PCR reactions are performed on all of the reactions simultaneously by appropriately heating and cooling the plate to specific temperatures.
  • the plates are cooled to the annealing temperature (50 * -65 * C, typically 55°C) for a set time (0-100 seconds, typically 15 seconds) , warmed to the extension temperature which is optimal for the thermostable polymerase (e.g., 73 * C for Taq polymerase) and maintained for a set period of time (0-100 seconds, typically 30 seconds) .
  • the cycle is completed by elevating the temperature of the reaction to denature the DNA products (93-95"C for 0 - 60 seconds, typically 15 seconds) .
  • Step 2c the PCR cycles are completed, with each chamber containing the amplified DNA from a specific location of the genome.
  • Each mixture includes the DNA that was synthesized from the two alleles of the diploid genome (a single allele from haploid chromosomes as is the case with the sex chromosomes in males or in instances of cells in which a portion of the chromosome has been lost such as occurs in tumors, or no alleles when both are lost) .
  • the free triphosphate deoxynucleotides and the unused oligonucleotide primers are also in this mixture.
  • Step 2d is the last PCR step, which inactivates the thermostable polymerase, say, by the addition of EDTA. Ampliwax protects the integrity of the chambers and the mixing occurs at 37'C for several minutes.
  • Step 3a for quantitive loop mismatch genotyping, the DNA strands are allowed to reanneal at a temperature above the annealing temperature of the oligonucleotide primers, but below the melting temperature of perfectly matched complementary strands. In most instances, this will be between 65 and 75 * C, depending on the salt conditions of the buffer.
  • the annealing time can vary from 1 hour to 24 hours, with 2 hours selected in the preferred embodiment.
  • Step 3a the heteroduplex annealing is done with the original contents of the chamber for the "subtraction” assay of the loop detection method.
  • the “addition” assay that is required for the measurements of loop mismatches entails combining of the contents of a chamber with its counterpart from a control plate in which the PCR reaction has been carried out with a corresponding set of primers (same oligonucleotides, but with different primer modifications) on a target DNA that has the smallest possible number of repeated elements for the given DNA marker. These two assays are done in different chambers of the reaction plate, or on separate plates entirely.
  • the Left primer is linked to a detection molecule
  • the Right primer is covalently linked to a molecule necessary for binding (i.e., biotin in the preferred embodiment)
  • the unknown genomic DNA (Source DNA) is amplified using a Left primer that is labeled with the detection molecule and the Right primer is unmodified.
  • the control DNA (or Target DNA) is amplified with an unmodified Left primer and the Right primer contains the binding protein (such as biotin) .
  • the amplified DNA from the unknown source and the Target DNA are combined to form heteroduplexes, one will only detect the binding of the upper strand of the Source DNA to the immobilized lower strand of the Target DNA and homoduplexes of the Target DNA strands will be undetected as well as perfectly matched, creating no exposed loops for detection.
  • the corresponding Source and Target DNAs are appropriately combined using the Biomek robot though direct physical transfer methods (i.e., aligning the Source DNA plate on top of the Target DNA plate directly and mixing by melting the ampliwax) .
  • Steps 3b, 3c, 3d, 3e, and 3f the unwanted single strands, primers and free nucleotides are removed by using a 3 'to5'-specific exonuclease that will not cleave or disrupt internal single-stranded loop structures, in both the subtraction and addition assays.
  • Exonuclease VII from E. coli is capable of 3'- 5' exonuclease activity limited to single-stranded DNA (Ausubel, F.M. , Brent, R. , guitarist, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. , and Struhl, K. , ed. 1993.
  • the enzyme is added to the chambers over the Ampliwax surface, allowed to mix at 37'C and incubated for a brief period (1-60 minutes, 10 minutes in the preferred embodiment) and terminated by the addition of EDTA. At the same time, the buffer is adjusted to promote non-specific binding of the DNA to a resin or filter.
  • Step 3f free deoxynucleotides and primers that interfere with binding of the PCR products and the detection system are removed.
  • the purification of unincorporated DNA materials is combined with the elimination of single-stranded DNA species that remain after heteroduplex formation. In the preferred embodiment, this purification step is done after the heteroduplex formation, thereby also eliminating single-stranded DNA's.
  • heteroduplex formation may be somewhat inhibited by residual primers, combining of the steps greatly simplifies the method and aids in increasing the ⁇ ignal-to-noise ratio.
  • the separation of free deoxynucleotides and primers from the PCR products is achieved by filtration (the unwanted materials are significantly smaller than the final PCR products) using commercially available filters (Centricon 30 filters, A icon) , incorporated by reference, or by adsorption (Molecular Biology LabFax, T.A. Brown, ed. Academic Press p281-4) , incorporated by reference, which entails the nonspecific binding of the PCR products, double- stranded and single-stranded DNA to a matrix followed by removal of the supernatents containing the primers and nucleotides.
  • Step 3g the filter is set upon a plastic manifold that fits over the chambers of the 384 chamber plate, the apparatus is inverted so that the Ampliwax rises to the bottom surface of the chambers, and the DNA solution comes into contact with the filter.
  • Step 3h the filter is separated from the chamber and washed with a high salt buffer to remove the free nucleotides.
  • Step 3i this filter is then placed against a polystyrene surface (optical grade)
  • the heteroduplexes are bound to the polystyrene surface in an exact replica of their initial spatial orientation.
  • the heteroduplexes containing biotinylated primer will bind to the streptavidin surface under a wide range of buffers that are pH neutral.
  • the DNA is bound in the TE buffer and in Step 3j the plate is washed twice with 0.15 M phosphate buffer.
  • Step 3k the chemical derivatization of the C and A residues within the heteroduplex loops employs a modification of the method originally described by (Ki ura, K. , Nakanishi, M. , Yamamoto, T. , and Tsuboi, M. (1977).
  • the pH can be varied from 4.5 to 6.5 and alternative buffers can be used
  • the plate is then covered with the buffer containing a final concentration of CAA is 2.0% and incubated at 37"C for 4 hours (longer or shorter times may be used).
  • the reaction is terminated in Step 31 by washing with 0.01M Tris-HCl pH 7.0 and 1.0 M NaCl.
  • the NaCl prevents dissociation of the heteroduplexes during the etheno- dehydration step.
  • the plate is heated in the final wash volume at 85-90"C for 1 hour, which dehydrates the ethenoderivative.
  • loop-specific derivatization of the nucleotides with chloracetaldehyde or other chemical modification reagents provides an alternative means for eliminating background reagents prior to detecting nuclease-liberated free derivatized nucleotides.
  • the fluorescence of the primer detector molecule that is bound to the hybridized strand is measured at this time, or measured at a later stage in conjunction with the fluorescent adducts created within the loop structures.
  • the detection of the hybridized strands and the derivatized nucleotides within the loops are performed at the same time.
  • the method of detection is preferrably by fluorescence (Kimura, K. , Nakanishi, M. , Yamamoto, T. , and Tsuboi, M. (1977). A correlation between the secondary structure of DNA and the reactivity of adenine residues with chloroacetaldehyde. Journal of Biochemistry, 81(6): 1699-703.), incorporated by reference.
  • Alternative embodiments include chemiluminesence (Martin R. , Hoover, C. , Grimme, S., Grogan, Cl, Holtke, J. and Kessler, CF. (1990) Bio Techniques 9(6): 762-8), incorporated by reference, electrochemical coupling using silicon surfaces (Briggs, J. , Kung, V.T., Gomez, B., Kasper, K.C., Nagainis, P.A., Masino, R.S., Rice, L.S., Zuk, R.F., and Ghazarossian, V.E. (1990) . Sub-femtomole quantitation of proteins with Threshold, for the biopharmaceutical industry.
  • the etheno derivatives (primarily the ethenoadenine residues) within the loops are measured with the fluorimeter of the apparatus: excitation at 310 nm, and emission at 410 nm.
  • the degree of fluorescence and sen ⁇ ititivity of the fluorimeter is calibrated with a quinine sulfate standard (10' 5 - 10 "7 M in 0.1 N H 2 S0 4 ) .
  • the amount of direct etheno fluorescence is increased by a factor of 2 by completely digesting the samples with DNasel and phosphodiesterase, when a gel overlay is used to prevent diffusion of the signals and disruption of the two- dimensional array of markers.
  • the number of heteroduplexes is determined by the unique fluorescence of the adduct that was initially linked to the Left primers.
  • Rhodamine, fluorescein or isothiocyanine derivatives can all be used to obtain intense fluorescent signals that can be separately measured from the fluorescence of the etheno adducts.
  • Standard programs guantitate the two different signals by analyzing two or more regions of the emission and/or excitation spectra.
  • Alternative detection methods for the etheno- derivatives include the use of specific monoclonal antibodies (Eberle, G. , Barbin, A., Laib, R.J. , Ciroussel, F., Thomale, J. , Bartsch, H. , and Rajewsky, M.F.
  • Step 4a detection residues within a mismatch loop will display differing degrees of reactivity to the modifying reagents as well as interactions (including fluorescence quenching and energy transfer) between closely spaced ethenoderivatives. (Which is why the fluorescence of an etheno derivative in a polynucleotide is approximately half that of the free ethenonucleotide.)
  • systematic labeling is used to calibrate the fluorescent signal for each size of mismatch loop, thereby compensating for the nonlinearity of the fluorescent signal with respect to the loop size.
  • Step 4a* an alternative embodiment of the heteroduplex loop detection is accomplished by incorporating labeled nucleotides during the Step 2a PCR synthesis, and then in Step 31* digesting them out of the single-stranded loops of the heteroduplex.
  • Incorporating labeled nucleotides e.g., fluorescently or radioactively, using appropriate triphosphate deoxynucleotide precursors
  • the quantity of detectable freed label corresponds to the loop size.
  • SI nuclease from Aspergillus orcyze (Dodgson, J.B., and Wells, R.D. (1977). Action of single- strand specific nucleases on model DNA heteroduplexes of defined size and sequence. Biochemistry, 16(11): 2374-9. Gite, S., and Shankar, V. (1992). Characterization of SI nuclease. Involvement of carboxylate groups in metal binding. -European Journal of Biochemi ⁇ try , 210(2): 437-41. Shenk, T.E., Rhodes, C. , Rigby, P.W. , and Berg, P. (1975).
  • the gel prevents diffusion of the released nucleotides. (Diffusion is not an issue with direct detection of chemically modified nucleotides.)
  • the polystyrene plate is placed into a plastic manifold, recreating 384 separate chambers.
  • Step 4 detection chemical modification is combined with specific nuclease treatment.
  • SI or micrococcal nuclease can be used to enhance the fluorescence of the etheno-derivatized adenosines generated by the chloracetaldehyde reaction. This provides two sets of measures of the same residues, thus increasing accuracy and sensitivity.
  • the nuclease treatment can be used alone to liberate nucleotides from the loop. These free nucleotides are then separated from the retained double- stranded DNA of the heteroduplexes and quantitated. The spatial orientation of the reactions must be preserved as the nucleotides are released.
  • a gel such as polyacrylamide that is on a solid backing (available from FMC Corporation)
  • a manifold over the streptavidin plate to contain the solutions with the nuclease and free nucleotides.
  • a gel such as polyacrylamide that is on a solid backing (available from FMC Corporation)
  • FMC Corporation solid backing
  • a manifold over the streptavidin plate to contain the solutions with the nuclease and free nucleotides.
  • the polyacrylamide gel plate one takes a 0.1 - 0.5 mm polyacrylamide gel (ranging from 4-15%) bound to a plastic backing. The gel is slightly dehydrated with minimal surface moisture.
  • the nuclease solution is applied to the surface of the gel (the amount of SI or micrococcal nuclease must be titrated for the enzyme lot) and the gel is placed over the surface of the streptavidin plate to which the heteroduplexes are bound.
  • the gel layer is removed and the nucleotides embedded within the gel are quantitated by fluorescence, two-dimensional radioactivity counting, autoradiography, or immunochemical assays.
  • Steps 4b, 4c, and 4d An alternative detection mechanism is described in Steps 4b, 4c, and 4d.
  • the nucleotides within the heteroduplex loops are detected by distinguishing these nucleotides from those that are contained within the double-stranded portions of the DNA strands.
  • the chemical modification agent chloracetaldehyde that selectively reacts with the exposed nucleotides within the loops is employed to specifically modify the C and A nucleotides within the heteroduplex loops.
  • This reagent is preferrable to other chemical modification agents such as hydroxylamine, bisulfite, and osmium tetroxide because of its ease of use, and the fact that the derivatized nucleotides are fluorescent, while the chemical reagent and the unmodified nucleotides are not fluorescent.
  • chemical modification agents such as hydroxylamine, bisulfite, and osmium tetroxide because of its ease of use, and the fact that the derivatized nucleotides are fluorescent, while the chemical reagent and the unmodified nucleotides are not fluorescent.
  • a detection amplification method such as immunodetection of the adducts using a urease-conjugate and a silicon-based detection of a pH shift, contacts the polystyrene surface with an electronic silicon detector and a urea-containing gel interface using existing methods (Briggs, J. , Kung, V.T., Gomez, B. , Kasper, K.C., Nagainis, P.A., Masino, R.S., Rice, L.S., Zuk, R.F., and Ghazarossian, V.E. (1990). Sub-femtomole quantitation of proteins with Threshold, for the biopharmaceutical industry.
  • Step 5 the genotypes are determined for every STR.
  • the two signals for each locus represent the sum and difference between the alleles.
  • this representation becomes quantitative.
  • One allele is computed by adding the sum and difference values and then dividing by two, and the second allele is computed by subtracting the sum and difference values and then dividing by two. This genotype determination is done for every locus.
  • Phenotypic data is gathered on the individuals, animals, or plants which are genotyped. For humans, this includes the basic medical examination: history, physical, and laboratory data. Additional phenotypic markers for various genetic diseases (e.g., creatine kinea ⁇ e for Duchenne muscular dystrophy) can also be collected. Environmental risks and exposures are also recorded.
  • Genomic ⁇ , 18: 283-289. association, homozygosity mapping (Ben Hamida, C. , Doerlinger, N. , Belal, S., Linder, C. , and Reutenauer, L. 1993. Localization of Friedrich ataxia phenotype with selective vitamin E deficiency to chromosome 8q by homozygosity mapping. Nature Genetic ⁇ , 5: 195-200. Pollak, M.R. , Chou, Y.-H.W., Cerda, J.J., Steinmann, B. , LaDu, B.N. , Seidman, J.G. , and Seidman, C.E. 1993.
  • the result is one or more (with polygenic disease) peaks appearing at specific locations on the chromosome that both suggest specific gene regions, as well provide a signature pattern for phenotypic risk.
  • dense STS sampling along the genome i.e., x-axis
  • large numbers of individuals tested at these STSs with each STS's allele given a combined score (i.e., on a y-axis)
  • the conventional limitations of statistical linkage analysis are overcome, and the process becomes akin to a signal processing of genetic data in order to separate delta functions (i.e., the causative genes) from the background noise.
  • Risks of trait inheritance or disease can then be determined by probabilistic (e.g., Bayesian) techniques (Young, I.D. 1991. Introduction to Ri ⁇ k Calculation in Genetic Coun ⁇ elling. Oxford: Oxford University Press.), incorporated by reference, that correlate the available genotypic and phenotypic data and environmental factors with chance of disease occurrence.
  • the signatures of causative gene locations deduced from the population can be applied to each individual to ascertain risk.
  • one or more genetic loci can be associated with specific (desirable or undesirable) traits such as milk production or disease resistance. This information can be used for selective breeding.
  • the techniques of genotyping and phenotypic correlation can be similarly applied to the task of disease gene identification. Exploiting dense genotypic data is particularly advantageous over existing techniques in localizing the genes of complex multigenic diseases. Once genes have been localized on the genetic map, use of an integrated genetic/physical genome map allows the positional cloning (Kerem, B.-S., Rommens, J.M. , Buchanan, J.A. , Markiewicz, D. , Cox, T.K. , Chakravarti, A., Buchwald, M. , and Tsui, L.-C. 1989. Identification,of the cystic fibrosis gene: genetic analysis. Science , 245: 1073-1080.
  • the STR loop mismatch method employs heteroduplex hybridizations to directly measure the STR allele repeat number n.
  • n the two alleles at a given STR locus as the complementary strands in a heteroduplex DNA molecule.
  • one strand S contains s STR repeat units and its mismatched complementary strand T 1 contains t STR repeat units.
  • U' denotes the complementary strand of sequence U.
  • Each STR repeat unit is comprised of k nucleotides. Assume that the left and right flanking regions are identical (i.e., perfectly complementary).
  • SS-DNA single-stranded nucleic acid
  • subsequence L 402 is the left flanking region (with subsequence L' 404 complementary) and subsequence R 406 is the right flanking region (with subsequence R' 408 complementary) .
  • the s-t extra STR units form a single-stranded loop 410 of size (s-t)*k bases. Energetically, only one such loop is expected (Ninio, J. 1979. Biochimie , 61: 1133. Salser 1977. Cold Spring Harbor Symp. Quant . Biol . , 42: 985.), incorporated by reference; however, multiple loops would in no way change the results.
  • the complementary structure shown in subfigure 4B is formed.
  • the single-stranded loop 412 size (t- s)*k is on the complementary strand.
  • the key idea is this: by detecting the size of the single-stranded loop 410 or 412, the value s-t (or t- ⁇ ) can be determined. By comparing two unknown alleles with a known standard, and by also comparing the two alleles with respect to each other, these loop size measurements will precisely determine the two alleles, i.e., the genotype at the STR locus.
  • the signal strength from a loop of single-stranded DNA is proportional to the number of unmatched nucleotides in the heteroduplex ST'. This signal is measured by means of a first label (*) that corresponds to the number of unmatched nucleotides in the loop of ST'. This label is measured by means of a physical detection that preferentially detects specific nucleotides in single-stranded DNA.
  • the nucleotides in the S strand of the heteroduplex molecule are chemically modified after the PCR synthesis.
  • the modification to these nucleotides renders them detectable (e.g., by fluorescence).
  • the measured fluorescence of these modified S nucleotides is proportional to the size of the loop mismatch s-t.
  • the nucleotides in the S strand of the heteroduplex molecule are labeled (radiolabeled, or other detectable means) and then incorporated during the PCR synthesis. Subsequent digestion with an Sl-like endon ⁇ clease separates the mismatched (and labeled) S nucleotides from the heteroduplex. The measured signal of these released S nucleotides is proportional to the size s-t of the loop mismatch prior to enzymatic digestion.
  • Means of physical detecting a quantitative signal for determining the loop size include: radioactivity, fluorescence, optical density, ionic concentration, electromagnetic conductivity or susceptibility, electrochemical coupling, or other detection assays (all referred to previously in this description) .
  • the loop size is determined by the ratio of the (1) measured single-stranded loop signal strength to the (2) measured number of strands having a loop. Therefore, in addition to detecting loop size, accurate quantitation also requires determining the number of heteroduplex strands with measurable loops. This is done using an independent second label (#) on the S strands of the heteroduplex molecules. This label is comprised of a detectable molecule attached to the PCR primer of the S strand; subsequent measurement of this molecule quantifies the number of strands in heteroduplexes.
  • the loop with label (*) is indicated by A*s, which in the most preferred embodiment represent adenosine nucleotides on the single- stranded loop that are chemically modified by chloracetaldehyde into a detectable state.
  • A*s represent labeled (e.g., radiolabeled) nucleotides that are incorporated during PCR synthesis, and are then detected following endonuclease digestion.
  • the experiment consists of performing a PCR amplification of an unknown CA-repeat locus source S of the form L(CA) S R, and hybridizing it to a known complementary oligonucleotide target T' of the form [L(CA),R] ' in order to indu ce mismatch and quantitatively measure the loop.
  • a CA-repeat locus molecule is selected for analysis, and is defined by its unique left and right oligonucleotide primers.
  • the primers are synthesized with appropriate labeling and linking modifications (Haralambidis, J. , Duncan, L. , Angus, K. , and Tregear, G. . (1990) .
  • the target DNA TT 1 is constructed from a standard of known CA-repeat length t in a separate PCR experiment.
  • the allele size t is chosen sufficiently small, say between 0 and 10, so that s>t is always guaranteed.
  • Standard PCR amplification of genomically-derived or cloned DNA for 20-40 cycles is done using unlabeled primers and nucleotides, with a linker such as biotin on the right primer.
  • Step 2b the source DNA SS' is constructed from sample genomic DNA via a PCR experiment.
  • the CA-repeat locus molecule is defined by its unique left and right primers.
  • a standard PCR amplification of genomically derived DNA is done for 20-40 cycles using labeled (#) left primer in the presence of A* labeled nucleotides.
  • Step 3a the SS' and TT' duplex molecules are denatured to form single stranded DNAs. When renatured in solution, the hybridization pairs
  • the T strands of the TT 1 duplex are not detectable (since their loops match) , and can be factored out of the analysis.
  • the T strands can be removed by attaching the TT' duplex to solid support via the linker of T', and then denaturing T from T', and washing to remove T, thus purifying T*.
  • T can remain as a nondetectable competitive contaminant.
  • using an excess of SS' relative to TT' favors the production of ST' heteroduplexes. Therefore, the focus is on the hybridization pairs
  • the SS' contains no single-stranded loops, hence is not detectable. Further, since only the T' molecule has the linker for solid support, attaching the T' to a surface (e.g., the biotin of T' to a streptavidin-coated surface) and washing removes the SS' product. This leaves only ST 1
  • the heteroduplex molecule is comprised of an upper strand 502 and a complementary lower strand 504.
  • the hybridization product is as shown in subfigure 5A.
  • the upper source strand 502 is produced by a first PCR amplification of sample genomic DNA.
  • the single-stranded DNA loop 506 contains *- detectable A nucleotides. (Following chemical modification or by incorporation/digestion, the *-detectable A's are used to measure loop size via label *.)
  • a second label (#) 508 on the upper strand is for strand quantification, and is attached to the left PCR primer.
  • the complementary lower target strand 504 is produced by a different PCR amplification of a known STR locus, or by direct synthesis.
  • This lower strand has a linker 510 such as biotin attached to its 5' (right) end.
  • Step 3b chemical modification em b odiment, the exposed A*s on the single-stranded DNA loop are chemically modified by chloracetaldehyde, as shown in subfigure 5B; in Step 4a, detecting the fluorescence from the first label (*) on the A* 512 measures the magnitude of s-t.
  • Step 3c the exposed A*s on the single- ⁇ tranded DNA loop are digested from the heteroduplex into free A* 514 using an endonuclease, as shown in subfigure 5C; in Step 4b, the radioactive A* is then detected using a scintillation counter, thereby measuring the magnitude of s-t.
  • the allele is determined in Step 5. Calibrations done prior to the experiment ensure that these measurements provide precise quantitation. Since
  • Step 5 adds the known value t to s, forming the average (sl+s2)/2 of the alleles. Multiplying this average by 2 determines the allele sum sl+s2.
  • This experiment consists of performing a PCR amplification of an unknown CA-repeat locus with the (zero, one, or) two sources SI and S2 of the form L(CA),,R and L(CA) s2 R, and hybridizing them against each other's complementary strands. This induces a loop mismatch proportional to js2-slj, which is then quantitatively measured.
  • a CA-repeat locus molecule is selected for analysis, and is defined by its unique left and right oligonucleotide primers.
  • the primers are located far enough away from the CA-repeat region to assure a sufficiently long linear stretch of DNA in the homoduplex; this is done make the effect of different loop sizes on the free energy neglible.
  • the rationale is that the flanking regions and the complementary CA/GT repeat regions have a total free energy that is proportational to the number of matching nucleotides, whereas the single- stranded DNA loop of heteroduplex has a free energy that grows as the logarithm of the loop size (Ninio, J. 1979. Biochimie , 61: 1133. Salser 1977. Cold Spring Harbor Symp. Quant . Biol . , 42: 985.), incorporated by reference.
  • the free energy changes (and binding affinities) introduced by differing loop sizes is small.
  • target DNA TT' is constructed from a standard of known CA-repeat length t in a separate PCR experiment.
  • the allele size t is chosen sufficiently small, say between 0 and 10, so that s>t is always guaranteed.
  • Standard PCR amplification of genomically-derived or cloned DNA for 20-40 cycles is done using unlabeled primers and nucleotides. No labels or linkers are used.
  • Step 2b the two source alleles are constructed simultaneously in one PCR experiment: each allele serves as the hybridization target for the other.
  • a standard PCR amplification of genomically derived DNA is done for 20-40 cycles using labeled (#) left primer, and a right primer with a linker such as biotin, in the presence of A* labeled nucleotides.
  • Step 3a forms the heteroduplexes.
  • the SI,SI' and S2,S2' homoduplex molecules are denatured to form single stranded DNAs.
  • the hybridization pairs are renatured in solution.
  • SI,SI' S1,S2'; S2,S1'; and S2,S2'.
  • the heteroduplex molecule constructed after PCR amplifying the sample genomic DNA, and rehybridizing, is comprised of an upper strand 602 and a complementary lower strand 604.
  • the single-stranded DNA loop 606 contains *- detectable A nucleotide ⁇ . (Following chemical modification or by incorporation/digestion, the *-detectable A's are used to measure loop size via label *.)
  • a second label (#) 608 on the upper strand is for strand quantification, and is attached to the left PCR primer.
  • the lower strand also ha ⁇ a linker 610 such as biotin attached to its 5' (right) end.
  • the label is incorporated into both strands during the PCR by labeling the CA and/or the GT dN*'s.
  • both the S1,S2' and S2,S1' strands have detectable single-stranded loops. Since both have the same js2-slj loop ⁇ ize, there is a two- to four- fold increase in the desired measured signal.
  • Elimination can be done using a single- ⁇ trand ⁇ pecific 3' to 5* exonuclea ⁇ e that remove ⁇ SS-DNA but not internal loop ⁇ , such as E. coli exonuclease VII.
  • Step 3c's chemical modification embodiment the exposed A*s on the ⁇ ingle-stranded DNA loop are chemically modified by chloracetaldehyde; in Step 4a detecting the fluorescence from the first label (*) on A*s measures the magnitude of s-t.
  • Step 3d' ⁇ alternative ⁇ ynthe ⁇ i ⁇ /dige ⁇ tion embodiment the exposed A*s on the ⁇ ingle- ⁇ tranded DNA loop are dige ⁇ ted from the heteroduplex into free A* u ⁇ ing an endonuclea ⁇ e; in Step 4b the radioactive A* i ⁇ then detected using a scintillation counter, thereby measuring the magnitude of s- t.
  • Step 5 the allele difference i ⁇ determined. Calibration ⁇ done prior to the experiment assure that these measurements provide precise quantitation. Since
  • the genotype is computed from loop mismatch data, referring to figure 7, by combining the sum (from the figure 5 protocol) and difference (from the figure 6 protocol) of the allele size ⁇ ; thi ⁇ determination exploit ⁇ the elimination of PCR stutter artifact by pooling within each experiment, as described below.
  • the ⁇ ingle experiment of Step 1 accurately mea ⁇ ures the allele sum (sl+s2)
  • the single experiment of Step 2 accurately measures the allele difference js2- ⁇ l] .
  • Combining the ⁇ e in Step 3 determines the two alleles:
  • a detailed protocol i ⁇ given for the loop mismatch method The following steps referring to figure 8 are designed for measuring a single STR, rather than the multiple STRs assayed in figure 3.
  • Step 1 of figure 8 an STR locus is selected, and PCR primers are chosen to provide large flanking regions.
  • this protocol is not optimized for compatibility with the apparatu ⁇ of figure 1.
  • the primer ⁇ are ⁇ ynthe ⁇ ized derivatized to ⁇ upport the characterization experiment ⁇ .
  • a ⁇ econd right primer Rb containing one or more biotin residues at the 5 ' end or within the oligonucleotide.
  • Derivatizing the primer for binding to a surface entails incorporating a biotinylated nucleotide at the 5 ' end of the ⁇ ynthetically made oligonucleotide. Additional biotinylated re ⁇ idue ⁇ can be incorporated into thi ⁇ primer either at the time of bio ⁇ ynthe ⁇ is or by secondary photo or chemical biotinylation.
  • the preferred embodiment employs the direct addition of the 5* biotin by chemical synthe ⁇ is; alternatively, additional biotin molecules may improve the heteroduplex isolation effiency.
  • Step 2 three PCR amplifications are performed.
  • Source DNA from a genome to be characterized, and target DNA of known minimal repeat length t from an individual (or prepared in advance by cloning a segment of genomic DNA in a plasmid or phage vector) are prepared for PCR.
  • Three separate reactions are performed. These are identical, except for the following specific reaction mixtures:
  • PCR a TT' sum PCR mixture for Step 2.a target DNA, L, Rb, all dNTPs unlabeled
  • PCR b SS' ⁇ um PCR mixture for Step 2.b ⁇ ource DNA, L#, R, labeled a- 32 P-dATP, other dNTPs unlabeled
  • PCR c S2,S1' difference PCR mixture for Step 2.c source DNA, L#, Rb, labeled a- 32 P-dATP, other dNTP ⁇ unlabeled
  • each 0.2 or 0.5 ml tube contains the appropriate set of primers, followed by the ⁇ tandard PCR buffer containing Tri ⁇ buffer, KCl, MgCl 2 and dNTP (the four tripho ⁇ phate deoxynucleotide ⁇ ) .
  • the total size of each PCR reaction is 50 ul (though thi ⁇ can vary from 10-100 ul) .
  • Each ⁇ pecific PCR reaction contain ⁇ it ⁇ ⁇ pecific reaction mixture, the PCR buffer (ie lOmM Tri ⁇ pH8.0, 50 mM KCl, 2.5 mM magne ⁇ ium chloride, albumin), and thermo ⁇ table (e.g., Taq) polymera ⁇ e.
  • the PCR reaction i ⁇ overlayed with a thin layer of Ampliwax that ⁇ eparate ⁇ ⁇ ome of the components from each other so that the reaction begins when the temperature rises to a level that melt ⁇ the wax and allow ⁇ all of the component ⁇ to mix.
  • Thi ⁇ i ⁇ the "hot start” method of PCR which reduces nonspecific synthesi ⁇ products.
  • An initial heat denaturation of 93-95'C for 5 minutes is followed by the thermal cycles are performed 20-40 times.
  • Each cycle consists of a 30 sec denaturation step at 95'C, 15-30 second annealing ⁇ tep at 50-65 * C (typically 55 * C) and an exten ⁇ ion ⁇ tep at 73'C for 15-120 seconds (typically 45 second ⁇ ) .
  • 0.5M EDTA is added to a final concentration of 10 mM. This inactivates the Taq polymerase.
  • Step 3 the heteroduplex hybridizations and modifications are done.
  • Reaction ⁇ a and b are combined ( ⁇ ummation experiment) in Step 3a, and reaction c (difference experiment) i ⁇ kept ⁇ eparate in Step 3b. All the following operation ⁇ are done independently for the two reaction ⁇ (sum and difference).
  • the samples are then heated to 95"C for 5 minutes and allowed to anneal at a temperature of 75"C to discourage primer-strand annealing. After 2-24 hours, the temperature i ⁇ lowered to 4 * C to solidify the Ampliwax and the exonuclease VII (Gibco, BRL) , incorporated by reference, in the appropriate buffer i ⁇ added to the ⁇ urface.
  • the buffer condition ⁇ for the PCR are compatible directly with tho ⁇ e of exonuclea ⁇ e VII.
  • the reactions are initiated by heating to 37"C and incubated for a time ranging from 1-120 minute ⁇ .
  • the reaction ⁇ are terminated by the addition of chloroform to the tubes.
  • the supernatant ⁇ from the chloroform extraction ⁇ contained hetero- and homoduplexe ⁇ , digested single strand ⁇ , pri er ⁇ and free nucleotide ⁇ .
  • the double- ⁇ tranded DNA i ⁇ then purified u ⁇ ing a ⁇ pin column/filter (such as Centricon filters from Amicon) to remove the small molecular weight material and concentrate the samples.
  • the purified DNAs from experiment are then adsorbed to strepavidin paramagnetic bead ⁇ (DYNAL 1993. Dynabead ⁇ biomagnetic ⁇ eparation system, Technical Handbook: Molecular Biology, Dynal International, Norway.) to bind tho ⁇ e double-stranded DNAs that contain the biotinylated right primer.
  • the bead ⁇ are wa ⁇ hed several times with a neutral salt buffer to reduce non ⁇ pecific binding and not disrupt the double-stranded DNA.
  • the tubes are incubated at 37"C for 4 hours (longer or shorter times may be used) .
  • the reaction is terminated by wa ⁇ hing with 0.01M Tris-HCl pH 7.0 and 1.0 M NaCl.
  • the NaCl prevents dissociation of the heteroduplexes during the etheno-dehydration step.
  • the samples heated in the final wa ⁇ h volume at 85 * C for 1 hour (dehydrate ⁇ the ethenoderivative) .
  • Step 3 u ⁇ ing a ⁇ ingle- ⁇ trand ⁇ pecific endonuclea ⁇ e ⁇ uch a ⁇ SI nuclea ⁇ e or micrococcal nuclea ⁇ e, the original PCR products that have been treated with exonuclease and bound to the strepavidin beads are equilibrated in the endonuclease buffer and reacted for varying time ⁇ .
  • Step ⁇ 4a and 4b the ⁇ ignal ⁇ are detected.
  • the fluore ⁇ cence and radioactivity retained on the bead ⁇ are mea ⁇ ured.
  • the amount of floure ⁇ cein and 32 P can be independently determined.
  • the fluore ⁇ cence i ⁇ measured by heating the samples to 95"C and eluting the DNA from the bead ⁇ , taking the supernatents and measuring the fluore ⁇ cence with a fluorimeter (excitation at 310 nm emi ⁇ ion at 410 nm) .
  • the degree of fluore ⁇ cence and sensititivity of the fluorimeter is calibrated with a quinine sulfate standard (10 "5 - IO" 7 M in 0.1 N H 2 S0 4 ) .
  • the tubes can be counted again for the amount of retained floure ⁇ cein and 32 P label ⁇ .
  • the amount of radioactivity can be calibrated with known ⁇ tandards that account for tube geometry, sample volume and instrument counting efficiencies. Ba ⁇ ed upon the radioactivity and the fluorescence, the size of the loops can be establi ⁇ hed.
  • Step 5 the genotype is determined.
  • Step 5a the sum is computed from the Step 4a detection, and in Step 5b, the difference is computed from the Step 4b detection.
  • the result ⁇ are combined in Step 5c to determine the genotype of the STR, a ⁇ de ⁇ cribed. Thi ⁇ complete ⁇ the protocol.
  • DNA protection i ⁇ done to minimize ⁇ puriou ⁇ ⁇ ignal ⁇ from unhybridized ⁇ ingle- ⁇ tranded DNA, and exonucleases are not u ⁇ ed.
  • Step 3a a 10:1 excess of the SS' amplified product relative to the TT' amplified product i ⁇ preferably u ⁇ ed.
  • Step 3b when necessary, TT' (or fragments thereof) without labels or linker ⁇ i ⁇ added to block unhybridized SI' ⁇ trands.
  • the number of PCR reactions can be reduced by performing PCR reaction ⁇ b and c above a ⁇ a fir ⁇ t reaction u ⁇ ing a cleavable biotinylated right primer and modifying ⁇ everal steps.
  • the PCR product can then be combined with the second target PCR reaction a to allow ⁇ equential measurement of the sum and difference experiments. This i ⁇ accompli ⁇ hed by combining the two PCR reaction ⁇ for the Source and Target DNA' ⁇ in Step 2, preparing and i ⁇ olating the heteroduplexe ⁇ on the streptavidin beads in Step 3, and mea ⁇ uring the nucleotides within the loops by derivatization and fluorescence in Step 4.
  • Step 4 The initial measurements in Step 4 are then followed by the release of duplexes employing the immobilized Source strand by reduction of a disulfide linkage between the primer and the biotin.
  • a more sen ⁇ itive detection ⁇ y ⁇ tem for the chemical modification embodiment i ⁇ an antibody-enzyme conjugate that recognize ⁇ the derivatized DNA (i.e. , the etheno- derivatives created by chloracetaldehyde) and catalyzes a colorimetric reaction that can be measured in the supernatent.
  • the ⁇ imple ⁇ t form of thi ⁇ a ⁇ ay would be to u ⁇ e a betagalacto ⁇ ida ⁇ e-antibody conjugate that act ⁇ on a colorimetric substrate such a ⁇ X-gal or Blue-gal (BRL/Gibco) .
  • the fragments L(CA) n .,R, L(CA) n . 2 R, and ⁇ o on are al ⁇ o generated in addition to the main PCR product L(CA) ⁇ R.
  • the di ⁇ tribution of the smaller fragments generally follows a decay pattern, with the amount of L(CA) m R le ⁇ than L(CA) n R, when m ⁇ n.
  • Thi ⁇ decay pattern i ⁇ empirically ob ⁇ erved to differ from one genetic locu ⁇ to another, but remain ⁇ ⁇ table across unrelated individuals for any given locus.
  • the u ⁇ e of pooled target ⁇ in the preferred embodiment eliminates thi ⁇ artifact. Multiple sources hybridized against multiple targets, producing a quadratic number of heteroduplexes. The different CA-repeat ⁇ izes
  • the mismatch loop size of each hybrid (S-i)x(T-j) is (s- t-i+j).
  • Each mismatch loop larger by d than the mean s-t is mirrored by a roughly equal concentration in its symmetric matrix entry of a mi ⁇ match loop ⁇ maller by d than the mean ⁇ -t.
  • the total signal from the stuttered sources with the stuttered targets averages out to the mean value s-t.
  • STR Genotyping in a Combined Heteroduplex Experiment The sum and difference experiments * at a locus are done separately using separate PCRs: two for the sum, and one for the difference, as described above.
  • the first PCR to construct TT' is preferably done prior to the introduction of sample genomic DNA, and can be incorporated (or "compiled") into the apparatus.
  • the protocol of figure 8 employs two PCRs. The following describes how to reduce this to just one PCR experiment, thereby reducing operating time and space requirements.
  • Digoxigenin is u ⁇ ed a ⁇ a linker (The Geniu ⁇ Sy ⁇ tem User's Guide for Filter Hybridization, 1992. Boehringer Mannheim Corporation, Indianapolis, IN) , incorporated by reference.
  • Step 1 an STR locu ⁇ is selected and oligonucleotides prepared.
  • Step 2a unlabeled duplex TT' of a known small repeat size t is constructed by PCR or direct synthe ⁇ i ⁇ .
  • the right primer has a digoxigenin linker 1.
  • Step 2b the homoduplexes SI,SI 1 and S2,S2' of an uncharacterized genomic DNA sample are amplified via PCR.
  • the first label (*) is incorporated into the single- ⁇ tranded loop, the left primer ha ⁇ the ⁇ econd label (#) , and the right primer ha ⁇ a biotin linker 2.
  • Step 3 the duplexes are combined and denatured together at high temperature into their separate strands, yielding: S2 , SI , T , S2 ' , SI ' , and T ' .
  • the upper right triangle ⁇ ub atrix hybrid ⁇ provide all the detectable elements -
  • the hybrid ⁇ pecie ⁇ (S2,S2'; SI,SI'; T,T') along the matrix diagonal are not detectable, ⁇ ince the duplex ⁇ trand ⁇ are identical in size, and no loop mismatch is formed.
  • the lower left triangle sub atrix hybrid ⁇ are not detectable - (S1,S2') By as ⁇ umption, ⁇ l ⁇ s2, so no loop mismatch is formed.
  • the T' (pre-made, digoxigenin linker 1) lower DNA strand ⁇ from the SI' and S2' (locus-made, biotin linker 2) lower strands are spatially separated by using two different solid supports to specifically bind the digoxigenin and the biotin linkers in different measurable regions.
  • the ⁇ ignals required for measuring the sum and the difference are detected in spatially separated experiments.
  • Step 5 the usual analy ⁇ i ⁇ (which exploits the expected PCR stuttering and the pooled targets) is u ⁇ ed to compute the allele value ⁇ .
  • a cleavable biotinylated linker is used on the right primer of T' that allows separate PCRs of a target and of genomic DNA, combines the samples into a single heteroduplex reaction, and then detects all nine of the hybridization products listed above. The following are measured: (a) the number of SI and S2 strands bound, and (b) the number of nucleotides in the loops. Then, the S1,T' and S2,T' measurable heteroduplex species are liberated by reduction of the dissulfide linkage, followed by remea ⁇ uring the S2,S1' bound, and the number of nucleotides in the remaining loops.
  • a Scalable STR Genotyping Assay The methods described refering to figure 8 enable practical construction of the apparatus in figure 1 and sy ⁇ tem manufactured device de ⁇ cribed in figure 3 in which multiple STR loci are genotyped ⁇ imultaneou ⁇ ly.
  • Step (1) is specific for a given STR.
  • the other four step ⁇ are largely independent of the given STR. Therefore, the apparatu ⁇ in figure 1 i ⁇ constructed to spatially encode multiple genetic loci on a ⁇ urface, and place ⁇ Step (1) ' ⁇ specific STR oligonucleotides at each spatial location, prior to complete PCR processing.
  • Step (2a) in figure 8 depo ⁇ its the pooled targets TT', and then Step ⁇ (2b- 5) for the sample-dependent PCR proces ⁇ ing, DNA hybridization, ⁇ ignal detection, and genotype determination are performed simultaneously over the ⁇ urface.
  • Step ⁇ (2-5) for the sample-dependent PCR proce ⁇ sing, DNA hybridization, signal detection, and genotype determination are performed simultaneously over the surface.
  • the steps of figure 8 for single STR genotyping are related to the step ⁇ of figure 3 for multiple STR genotyping.
  • the three dimensions of ⁇ pace and one dimen ⁇ ion of time can be u ⁇ ed to multiplex the STR-specific oligonucleotides and the PCR processing.
  • multiple reaction chamber ⁇ in a three-dimen ⁇ ional arrangement would each contain STR-specific oligonucleotides over some time period.
  • the PCR processing would be done in parallel in multiple chambers, until all required signals were obtained. This physical arrangement can customize the PCR conditions, if neces ⁇ ary, to each STR.
  • 864-chamber plate ⁇ can be physically arranged to achieve over 100,000 simultaneou ⁇ characterization ⁇ .
  • Thi ⁇ i ⁇ done by con ⁇ tructing a surface of four plates in a 2x2 array, which provide ⁇ 3,456 chambers in a layer. Stacking thirty such layers provides 103,680 chambers.
  • This three dimen ⁇ ional arrangement is quite compact, with no chamber further than two feet from any other chamber.
  • thi ⁇ three dimen ⁇ ional organization fit ⁇ into a thermocycling PCR oven.
  • the hybridization, detection, and other steps are multiplexed in time, enabling efficient use of the robotic device, detection device, and computer to achieve a throughput commen ⁇ urate with the parallelization.
  • the signals from either the allele sum or allele difference experiments can be increased several-fold by detecting SS-DNA mismatch loops on Jot the upper and lower strands, rather than on just one strand.
  • the PCR stutter can again be eliminated by u ⁇ ing pooled targets.
  • the PCR primers 1002 (left) and 1004 (right) for the upper strand 1006 and the lower strand 1008 of the target TT' both contain linkers 1010 (e.g., biotin) for binding to solid support, but no (#) labels.
  • linkers 1010 e.g., biotin
  • the target TT' duplexes are constructed by standard PCR amplification of genomically derived DNA for 20-40 cycles using dNs without (*) label ⁇ .
  • Step 2b of figure 5 amplifie ⁇ the unknown homoduplexe ⁇ SI,SI' and S2,S2'.
  • the fir ⁇ t label (*) 1012 for loop quantitation is present on nucleotides (in equal proportions) in both strand ⁇ S 1014 and S' 1016.
  • the label (*) indicate ⁇ detectability, whether by chemical modification or by incorporation/dige ⁇ tion.
  • the second label (#) 1018 for strand quantitation is present on both the left 1020 and right 1022 PCR primers.
  • the source DNA SS' is developed by standard PCR amplification of genomically derived DNA for 20-40 cycles using (*) labeled dA*, dC*, dG*, and dT*.
  • Step 4 of figure 5's detection 4n loop size (*) ⁇ ignal ⁇ , and 2 ⁇ trand (#) ⁇ ignal ⁇ are measured per ST' molecule.
  • Step 5's allele determination this four-fold increase in label (*) and two-fold increase in label (#) is accounted for.
  • Steps in figure 5 applies to the case of two alleles SI and S2 for determining the allele sum.
  • Two ⁇ eparate PCR ⁇ are done, a ⁇ de ⁇ cribed: one for SI,SI' and S2,S2' labeled duplexes, and one for linker TT' targets.
  • the denaturation/reannealing experiment constructs nine hybridization products. However, only those containing an T or T' linker are detectable.
  • each SI (S2) or SI' (S2') acts as an S (S') strand, and the sum sl+s2 is measured.
  • the allele difference is determined using single-stranded loops from both the upper and lower ⁇ trand ⁇ . Thi ⁇ again ha ⁇ the advantage of ⁇ ignal amplification.
  • the genotyping i ⁇ done by cross- hybridizing SI,SI' with S2,S2'.
  • Step 1 the STR locus and its PCR primers are chosen.
  • Step 2 the two complementary strands are constructed in a single PCR amplification of sample genomic DNA.
  • the first loop quantitation label (*) is pre ⁇ ent on nucleotide ⁇ (in equal proportion ⁇ ) in both S and S'.
  • linker such as biotin, which i ⁇ attached to the 5' end of the right primer.
  • the hybridization product 1102 of the denaturation and reannealing is shown in subfigure 11A.
  • the various label and linker combinations are shown in the hybridization product table of subfigure 11B. Adding up the ⁇ ignals from the first label (*) 1104,
  • Step 5 the allele difference ⁇ 2-sl is computed as n, i.e., the normalized (and calibrated) ratio of loop size signal from the first label (*) to ⁇ trand number ⁇ ignal from the ⁇ econd label (#) .
  • step l is for identifying an STR, and synthe ⁇ izing suitable PCR reagent ⁇ .
  • the STR locus is identified by conventional techniques (Sambrook, J. , Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning, ⁇ econd edition . Plainview, NY: Cold Spring Harbor Pres ⁇ ; N. J. Dracopoli, J. L. Haine ⁇ , B. R. Korf, C. C. Morton, C. E. Seidman, J. G. Seidman, D. T. Moir, and D. Smith, ed. , Current Protocol ⁇ in Human Genetic ⁇ . New York: John Wiley and Son ⁇ , 1994), incorporated by reference.
  • preexi ⁇ ting STR loci for the genome of intere ⁇ t can be obtained from available databa ⁇ e ⁇ (Genbank, GDB, EMBL; Hilliard, Davi ⁇ on, Doolittle, and Roderick, Jack ⁇ on laboratory mou ⁇ e genome databa ⁇ e. Bar Harbor, ME; SSLP genetic map of the mouse, Map Pairs, Research Genetics, Huntsville, AL) , incorporated by reference.
  • the STR's repeat unit includes no more than three distinct nucleotides; for clarity in exposition, the following ⁇ pecification of the preferred embodiment assumes that the STR is a CA-repeat marker.
  • nucleic acid sequences flanking the CA-repeat region are determined by DNA sequencing methods (Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning, ⁇ econd edition . Plainview, NY: Cold Spring Harbor Press; United State ⁇ Biochemical 1994. USB Sequenase version 2.0 DNA sequencing kit, sequencing protocols, 9th edition, product number 70770, Amersham Life Science, Arlington Heights, IL) , incorporated by reference.
  • sequence of all or part of the STR locu ⁇ may reside in a preexisting available database, or in the original articles describing the locus.
  • oligonucleotide primers are designed for use with the DNA sequence using computer programs that facilitate PCR primer or DNA synthe ⁇ is oligonucleotide design, such a ⁇ MacVector 4.1 (Ea ⁇ tman Chemical Co., New Haven, CT) or Oligo 4.0 (National Biosciences, Inc., Madison, MN) , incorporated by reference.
  • the ⁇ e program ⁇ facilitate ⁇ electing lengths and positioning ⁇ of oligonucleotide ⁇ that are operative for enzymatic reactions.
  • the two PCR primers and the reaction conditions are designed to permit amplification of the DNA sequence, and include:
  • primer R 1 (L) a left PCR primer for the upper strand, and (R*) a right PCR primer for the complementary lower strand.
  • primer R 1 is biotinylated.
  • a third oligonucleotide for DNA sequencing primer and its reaction conditions are designed to permit sequencing of the DNA sequence:
  • (Q) a left (upstream) DNA sequencing primer that is directly adjacent to the CA-repeat region of the upper strand; this sequencing primer is designed to allow exten ⁇ ion acro ⁇ the entire tandem repeat sequence using nucleotides that are specifically limited to the repeat unit base composition.
  • the oligonucleotide primers for the CA-repeat genetic marker are synthe ⁇ ized (Haralambidi ⁇ , J. , Duncan, L. , Angu ⁇ , K. , and Tregear, G.W. 1990. The ⁇ ynthesis of polyamide- oligonucleotide conjugate molecules. Nucleic Acid ⁇ Re ⁇ earch , 18(3): 493-9. Nelson, P.S., Kent, M. , and Muthini, S. 1992. Oligonucleotide labeling methods. 3. Direct labeling of oligonucleotides employing a novel, non-nucleosidic, 2- aminobuty1-1,3-propanediol backbone.
  • the ⁇ e primer ⁇ may be derivatized with a fluore ⁇ cent detection molecule or a ligand for immunochemical detection ⁇ uch as digoxigenin.
  • these oligonucleotides and their derivative ⁇ can be ordered from a commercial vendor (Research Genetics, Huntsville, AL) .
  • a genetic material whose genotype is to be determined is selected for study. Thi ⁇ genetic material is then placed in contact with the PCR primers L and R', and PCR amplification i ⁇ performed.
  • the methods for this PCR amplification given here are standard, and can be readily applied to every CA- repeat or microsatellite marker that correspond ⁇ to a (relatively unique) location on a genome.
  • these other components include, but are not limited to, the standard PCR buffer (containing Tris pH8.0, 50 mM KCl, 2.5 mM magne ⁇ ium chloride, albumin) , tripho ⁇ phate deoxynucleotide ⁇
  • thermostable polymerase e.g., dTTP, dCTP, dATP, dGTP
  • the PCR reactions are performed on all of the reactions by heating and cooling to specific locu ⁇ -dependent temperature ⁇ that are given by the known PCR conditions.
  • the entire cycle of annealing, extension, and denaturation is repeated multiple times (ranging from 20-40 cycles depending on the efficiencies of the reactions and sen ⁇ itivity of the detection system) (Innis, M.A. , Gelfand, D.H. , Sninsky, J.J., and White, T.J. 1990. PCR Protocol ⁇ : A Guide to Method ⁇ and Application ⁇ . San Diego, CA: Academic Press.), incorporated by reference.
  • the thermocycling protocol on the Perkin- Elmer PCR System 9600 machine is:
  • each reaction tube containing the amplified DNA from a specific location of the genome.
  • Each mixture includes the DNA that was synthesized from the two alleles of the diploid genome (a single allele from haploid chromosome ⁇ as is the case with the sex chromosomes in males or in instance ⁇ of cell ⁇ in which a portion of the chromo ⁇ ome ha ⁇ been lo ⁇ t such as occurs in tumors, or no alleles when both are lost) .
  • the free deoxynucleotides and primers may be separated from the PCR products by filtration u ⁇ ing commercially available filter ⁇ (Amicon, "Purification of PCR Products in Microcon Microconcentrators,” Amicon, Beverly, MA, Protocol Publication 305; A. M. Krowczyn ⁇ ka and M. B. Henderson, “Efficient Purification of PCR Products Using Ultrafiltration,” BioTechnique ⁇ , vol. 13, no. 2, pp. 286-289, 1992) , incorporated by reference. Referring to figure 13, ⁇ tep 3 is for purification of the amplified complementary lower DNA strand.
  • the lower biotinylated strand is purified from the upper strand by using magnetic streptavidin coated beads (Dynal International, Oslo, Norway) .
  • magnetic streptavidin coated beads Disynal International, Oslo, Norway
  • the steps of Dynabead preparation, PCR product immobilization, DNA duplex melting using a 0.1M NaOH solution, and separation of the upper and lower DNA ⁇ trand ⁇ to purify the lower ⁇ trand are done, a ⁇ described (DYNAL 1993. Dynabeads biomagnetic ⁇ eparation system, Technical Handbook: Molecular Biology, Dynal International, Oslo, Norway) , incorporated by reference.
  • step 4 is for nucleic acid synthesis of the upper strand.
  • the purified amplified lower DNA strand serves as a template for a sequencing reaction.
  • the sequencing reaction provides a template-directed ⁇ ynthe ⁇ i ⁇ that extend ⁇ the upper strand across the CA-repeat region.
  • the nucleotides used are:
  • dNTP ⁇ Extension is largely restricted to the repetitive sequence by including only dNTPs that appear in the repeat unit.
  • dATP and dCTP are used.
  • One or both of these dNTPs are labeled with a detectable label *, preferably a radioisotope ⁇ uch as 35 S or 3 *-P (DuPont NEN Research Products, Boston, MA) , or a fluorescent probe (Biological Detection System ⁇ , Pitt ⁇ burgh, PA) .
  • a detectable label * preferably a radioisotope ⁇ uch as 35 S or 3 *-P (DuPont NEN Research Products, Boston, MA)
  • a fluorescent probe Biological Detection System ⁇ , Pitt ⁇ burgh, PA
  • Termination i ⁇ restricted to nucleotides not contained in the repetitive sequence.
  • ddGTP or ddTTP (ddUTP) are used, depending on the sequence of the marker.
  • the termination molecule is labelled with a second label **, that is distinct from the first label *, and can be independently detected.
  • fluorescein-labeled ddNTP (DuPont NEN Research Products, Boston, MA) is a convenient second label **.
  • a highly proce ⁇ sive polymerase enzyme having little or no exonuclease activity is preferably used, such as Sequenase 2 (U.S. Biochemical, Cleveland, OH) . Protocols optimized for the selected enzyme (United States Biochemical 1994.
  • USB Sequenase version 2.0 DNA sequencing kit sequencing protocols, 9th edition, product number 70770, Amersham Life Science, Arlington Heights, IL) , incorporated by reference, are applied, with the (labeled and unlabeled) dNTPs and ddNTPs de ⁇ cribed above ⁇ ub ⁇ tituted for the dNTP ⁇ and ddNTP ⁇ contained in the conventional ⁇ equencing protocol.
  • the u ⁇ e of Mn buffer can be helpful when synthesizing ⁇ hort sequences.
  • step 5 is for detecting signals from the synthesized nucleic acids.
  • the newly ⁇ ynthe ⁇ ized upper DNA sequence formed by means of the DNA sequencing reaction remains hybridized to the biotinylated lower strand, which in turn is tightly bound to the streptavidin beads.
  • the DNA ⁇ equencing primer ⁇ , nucleotide ⁇ , and other reagent ⁇ are removed by repeated gentle wa ⁇ hing with a buffer that promote ⁇ double stranded DNA, such as the Dynabead binding and washing buffer (DYNAL 1993. Dynabeads biomagnetic ⁇ eparation ⁇ yste , Technical Handbook: Molecular Biology, Dynal International, Oslo, Norway) , leaving only the bound duplex DNA containing the desired purified product.
  • Fluorescence signals are detected and quantitated, preferably by means of a fluorimeter.
  • Radioactive signal ⁇ are detected and counted, preferably by mean ⁇ of a scintillation counter.
  • ⁇ tep 6 i ⁇ for analyzing the detected ⁇ ignal ⁇ to determine the genotype ⁇ um (or average) .
  • Precalibration with a set of predetermined reference allele ⁇ can e ⁇ tabli ⁇ h the ⁇ cale factor, and any deviation ⁇ from linearity.
  • PCR ⁇ tutter artifact i ⁇ accounted for by deconvolution with the known stutter distribution (Perlin, M.W. , Burks, M.B., Hoop, R.C., and Hoffman, E.P. 1994.
  • this analysi ⁇ procedure compute ⁇ the genotype.
  • this procedure computes the average (or, equivalently, the sum) of the alleles.
  • step 4 ' is for nucleic acid synthesis of the upper strand, and is comprised of the steps:
  • the purified amplified lower DNA ⁇ trand serves as a template for a sequencing reaction.
  • the sequencing reaction provides a template-directed synthe ⁇ is that extends the upper strand across the CA-repeat region.
  • the nucleotides used are:
  • dNTP ⁇ Exten ⁇ ion i ⁇ largely re ⁇ tricted to the repetitive sequence by including only dNTPs that appear in the repeat unit. For a CA-repeat, only dATP and dCTP are used. These are both unlabeled.
  • a highly proce ⁇ ive polymera ⁇ e enzyme having little or no exonuclease activity is preferably used, such as Sequenase 2 (U.S. Biochemical, Cleveland, OH) . Protocols optimized for the selected enzyme (United States Biochemical 1994.
  • USB Sequenase version 2.0 DNA sequencing kit sequencing protocols, 9th edition, product number 70770, Amersham Life Science, Arlington Heights, IL
  • the unlabeled dNTPs described above are sub ⁇ tituted for the dNTPs and ddNTPs contained in the ⁇ tandard sequencing protocol. Washing with the stabilizing Dynabead binding and washing buffer is then done 2-4 times (DYNAL 1993. Dynabeads biomagnetic separation system. Technical Handbook: Molecular E ology, Dynal International, Oslo, Norway) to remove the unincorporated primers and dNTPs, and thereby purify the duplex DNA comprised of lower strand template and partially synthesized unlabeled upper strand DNA.
  • step 4 • b is for heteroduplex formation between different alleles of the upper and lower strands.
  • sodium hydroxide is used to melt the duplex, and an equimolar amount of hydrochloric acid is then subsequently used to reanneal (DYNAL 1993. Dynabead ⁇ biomagnetic separation system, Technical Handbook: Molecular Biology, Dynal International, Oslo, Norway) . Specifically (p. 23) , using the bead-immobilized double stranded product,
  • the denaturing and renaturing i ⁇ done by heating the duplex DNA solution to a temperature of 65°C to 95°C for a period of 2 to 30 minutes, and then gradually cooling the solution over a period of 15 to 90 minutes to a temperature between 25°C and 40°C.
  • step 4 'c is for labeled restricted synthe ⁇ i ⁇ of the upper ⁇ trand.
  • the purified amplified lower DNA ⁇ trand ⁇ erve ⁇ a ⁇ a template for continuing the ⁇ equencing reaction.
  • the template-directed ⁇ ynthe ⁇ i ⁇ continues the upper strand sequencing acro ⁇ the CA-repeat region.
  • the nucleotides used are:
  • dNTP ⁇ Extension i ⁇ largely re ⁇ tricted to the repetitive ⁇ equence by including only dNTPs that appear in the repeat unit.
  • dATP and dCTP are used for a CA-repeat.
  • dNTP ⁇ are labeled with a detectable label *, preferably a radioi ⁇ otope such as 35 S or 32 P (DuPont NEN Research Products, Boston, MA) , or a fluorescent probe (Biological Detection Systems, Pittsburgh, PA) .
  • a detectable label * preferably a radioi ⁇ otope such as 35 S or 32 P (DuPont NEN Research Products, Boston, MA) , or a fluorescent probe (Biological Detection Systems, Pittsburgh, PA) .
  • Termination is restricted to nucleotide ⁇ not contained in the repetitive sequence.
  • ddGTP or ddTTP (ddUTP) are used, depending on the sequence of the marker.
  • the termination molecule is labelled with a second label **, that is distinct from the first label *, and can be independently detected.
  • fluorescein-labeled ddNTP (DuPont NEN Research Products, Bo ⁇ ton, MA) i ⁇ a convenient ⁇ econd label **.
  • a highly proce ⁇ ive polymera ⁇ e enzyme having little or no exonuclease activity is preferably u ⁇ ed, ⁇ uch a ⁇ Sequena ⁇ e 2 (U.S. Biochemical, Cleveland, OH) . Protocol ⁇ optimized for the ⁇ elected enzyme (United State ⁇ Biochemical 1 994.
  • USB Sequena ⁇ e ver ⁇ ion 2.0 DNA sequencing kit, sequencing protocol ⁇ , 9th edition, product number 70770, Amersham Life Science, Arlington Heights, IL) are applied, and the (labeled and unlabeled) dNTPs and ddNTPs described above are sub ⁇ tituted for the dNTPs and ddNTPs contained in the standard sequencing protocol.
  • this heteroduplex product is comprised of unlabeled primer, an unlabeled repetitive sequence with about s repeated CA units, a *-labeled repetitive sequence with about (t-s) repeated CA units, and has a **-labeled terminator dye.
  • step 6* i ⁇ for analyzing the detected ⁇ ignal ⁇ to determine the genotype difference is analyzed.
  • Precalibration with a ⁇ et of predetermined reference allele ⁇ can e ⁇ tablish this scale factor, and any deviations from linearity.
  • PCR stutter artifact is accounted for by deconvolution with the known stutter distribution (Perlin, M.W. , Burks, M.B., Hoop, R.C., and Hoffman, E.P. 1994.
  • thi ⁇ analy ⁇ i ⁇ procedure computes the difference between the two alleles of the genotype.
  • a method is de ⁇ cribed for determining STR allele ⁇ by nucleic acid ⁇ ynthe ⁇ is that is comprised of the steps:
  • dNTPs Nucleotide ⁇ that are restricted to the composition of the repetitive unit, at least one of which is labeled with the repeat counter first label *.
  • thi ⁇ could be *-dATP and dCTP.
  • reporter R A biotinylated reporter R that i ⁇ added after the reducing agent ha ⁇ cleaved the biotinylated PCR primer from the streptavidin bead ⁇ .
  • the reporter R i ⁇ a biotinylated terminating ddNPT that is added by means of a sequencing enzyme.
  • reporter R is a biotinylated oligonucleotide that i ⁇ added a ⁇ the right flanking sequence of the repetitive sequence by mean ⁇ of a ligation enzyme.
  • the detection reagent ⁇ used for the required labeling may include (but are not limited to) radioactivity, fluorescence, phosphorescence, chemiluminescence, electrical resistivity, pH, and ionic concentration.
  • the lower strand can be sequenced, instead of the upper strand.
  • a repetitive unit other than CA, but containing no more than three distinct nucleotides can be used.
  • dNTPs are used for every nucleotide in the repetitive unit, with at least one of the repetitive unit nucleotides labeled with the first label *, and ddNTP( ⁇ ) are used for every nucleotide not in the repetitive unit, with the appropriate terminating nucleotide immediately following the repetitive sequence labeled with the second label **.
  • the hybridization panel method for genotyping STRs is distingui ⁇ hed from the loop mi ⁇ match method described previously in that the determination of an STR's allele ⁇ i ⁇ accompli ⁇ hed with an entire panel of hybridization probe ⁇ , rather than determining the allele ⁇ with only two loop mi ⁇ match hybridization experiment ⁇ .
  • This hybridization panel method generally entails more hybridization experiments per STR than the loop mismatch method.
  • this approach is applicable to the determination of specific nucleotide sequences realted to genomic DNA, specific genes, and known mutations.
  • the central idea of the hybridization panel method for genotyping STR alleles is to have a detection panel of DNA probes.
  • This panel measure ⁇ the extent of specific DNA binding of the patient's DNA against a set of probes.
  • a second coordinate of information can optionally be obtained by performing the reactions over a range of reaction ⁇ tringencie ⁇ (e.g., using temperature, ion concentration, or DNA denaturants) .
  • the re ⁇ ult is a mapping from one or two coordinates (probe and stringency) into the reaction energetics (binding affinity) .
  • L(CA) n R be one allele in the patient's PCR product for a given STR reaction chamber in the two dimensional array.
  • L is the left flanking region DNA ⁇ ubsequence
  • R is the right flanking region DNA subsequence
  • n is the number of allelically varying CA repeat ⁇ , ⁇ o that (CA) n i ⁇ the middle DNA ⁇ ubsequence of length 2n.
  • the right PCR primer is a suffix ⁇ ubsequence of the right flanking region R.
  • each detection panel is customized to the PCR product of it ⁇ STR allele.
  • Thi ⁇ i ⁇ done by providing a panel of allele ⁇ pecific oligonucleotide ⁇ (ASOs) (Lemna, W.K. , Feldman, G.L., Kerem, B.-S., Fernbach, S.D., Zevkovich, E.P., O'Brien, W.E., Riordan, J.R. , Collins, F.S., Tsui, L.- C, and Beaudet, A.L. 1990. Mutation analy ⁇ i ⁇ for heterozygote detection and the prenatal diagnosis of cystic fibrosis. N. E. J.
  • each ASO contains an allele-specific left flanking region, concatentated with a number n of repeat unit nucleotide ⁇ , concatentated with an allele- ⁇ pecific right flanking region.
  • the lengths of the left and right regions flanking the varying size repeat polymer are individually adjusted to ensure that the left and right oligomers have roughly the ⁇ ame DNA binding energies when hybridizing to their respective complementary DNA strand ⁇ .
  • thermodynamic ba ⁇ i ⁇ for thi ⁇ (and alternative) approaches is that while perfect DNA duplex matches will have minimum energy, mismatches will induce bulges or loops in the DNA duplex molecule that increase the free energy. A two base-pair bulge will have sufficiently increased free energy (Ninio, J. 1979. Biochimie , 61: 1133. Salser 1977. Cold Spring Harbor Symp. Quant . Biol . , 42: 985.), incorporated by reference, to reduce binding affinity by several kcal/mole relative to a perfect match; the larger the bulge, the more unfavorable the binding.
  • a panel of ASO ⁇ that provide for all value ⁇ of n is used to determine the m values expressed from the PCR product.
  • the panel of target probes is constructed as the set of DNA sequences formed by concatenating L 0 , (CA) n , and Ro, as
  • the complementary PCR ⁇ ource product ⁇ have the form
  • one detection panel i ⁇ provided for the PCR product ⁇ of each genetic marker.
  • Each detection panel corre ⁇ ponds to one marker locus, and is embedded at that locu ⁇ ' coordinate in the ⁇ patially localized PCR marker grid.
  • the two ⁇ urfaces (PCR and detection) may be separate or composite.
  • the oligomers flanking the STR region are (in general) different for every genetic marker. That is, the target probe panel sequences are cu ⁇ tomized to each genetic marker.
  • a ⁇ econd coordinate of hybridization ⁇ tringency would be added.
  • Thi ⁇ ⁇ tringency variation can be implemented by varying any of several factors in the hybridization, including temperature, ion concentration, formamide concentration, and nucleotide compo ⁇ ition (Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning, ⁇ econd edition . Plainview, NY: Cold Spring Harbor Pres ⁇ .), incorporated by reference.
  • the two coordinate ⁇ of differential target ⁇ and differential ⁇ tringency give an even clearer ⁇ ignature for STR allele ⁇ .
  • the signature of two alleles is formed by superimpo ⁇ ing those of single alleles.
  • unique signatures in one or two coordinates
  • the separation of the superimpo ⁇ ed pattern ⁇ to effect genotyping can be done without recourse to such a library of signature ⁇ by curve fitting or deconvolution proce ⁇ sing.
  • the ⁇ tringency variation can be effected by temperature ramp, or by changing the chemical environment of the hybridization over time.
  • An alternative embodiment uses an identical detection panel of target oligonucleotides for every genetic locus.
  • each grid is comprised of the target panel ⁇ (CA) n ! n varie ⁇ acro ⁇ all intere ⁇ ting polymorphisms ⁇ •
  • n could range from 10 to 40.
  • intentional DNA pairing mismatch is introduced to bias the hybridization again ⁇ t further STRs. This can be done by a three-fold expansion of these probes by adding a mismatching base pair at one end. For example, with CA-repeats as the STR, these four probe fa ilie ⁇ are po ⁇ ible for every n:
  • the same detection panel i ⁇ u ⁇ ed for every genetic locu ⁇ , but intentional mismatch is introduced by changing the target DNA composition.
  • CA- repeats as STRs, a family of (CA) D or (GT) n probes are used, but changes are introduced in specific bases. For example, some G's are changed to Cs, or to the energetically similar base inosine.
  • the doping i ⁇ introduced in the ⁇ ource molecule, rather than in the target ⁇ has the advantage of requiring ju ⁇ t one target DNA molecule (i.e., a very large repeated oligomer) for all the genetic loci. Thu ⁇ , the manufacturing co ⁇ t ⁇ are greatly reduced, ⁇ ince replicated complex panel ⁇ for each locus are not needed.
  • the extent of doping is introduced (say, with inosine) as a variable into the PCR reaction itself.
  • the doping i ⁇ random acro ⁇ the PCR product ⁇ , but ha ⁇ con ⁇ tant ⁇ tati ⁇ tic ⁇ , particularly in the repetitive unit region of the unknown STR PCR product molecule. If two coordinate signatures are de ⁇ ired, hybridization ⁇ tringency variation can be introduced a ⁇ well.
  • a single STR detection probe is used for all experiments.
  • Using a single probe, say (CA) n (n large and fixed) dramatically reduces manufacturing costs.
  • a temperature ramp experiment is then conducted in parallel for every genetic locu ⁇ by varying ⁇ tringency.
  • For each PCR product with GT-repeat length when its subpopulation of (GT) k ⁇ equence ⁇ rapidly melt ⁇ , there will be a ⁇ harp change in the melting profile.
  • Thi ⁇ will be detectable as a peak in the first derivative of the curve. The peaks provide a DNA size v ⁇ . concentration mapping that can then be used to determine the alleles.
  • STR repeat units of any size.
  • the newer trinucleotide repeat ⁇ , tetranucleotide repeat ⁇ , etc. are more favorable energetically, and provide greater allele differentiation.
  • the size of the bound detection oligonucleotide is adju ⁇ ted to maximally di ⁇ cri inate between a perfect match and a ⁇ ingle ba ⁇ e pair mi ⁇ match.
  • An alternative to detecting perfect v ⁇ . mismatched heteroduplexes is using chemical modification reagents (such as CII, CAA, Os0 4 , or hydroxylamine) that can react with single nucleotide mismatche ⁇ and then be detected.
  • Nested PCR (Yourno 1992. A Method for Nested PCR with Single Closed Reaction Tubes. PCR Meth . Appl . , 2(1): 60-65. Inni ⁇ , M.A. , Gelfand, D.H. , Snin ⁇ ky, J.J., and White, T.J. 1990. PCR Protocol ⁇ : A Guide to Method ⁇ and Application ⁇ . San Diego, CA: Academic Pre ⁇ s.) , incorporated by reference, can be done for a purer PCR amplification to reduce noise.
  • LCR Ligase chain reaction
  • both strands must be nucleic acid ⁇ . Whether these are comprised of DNA, RNA, or any other nucleic acid polymer is nonessential. The key requirement is the binding specifity of complete and partial sequence matches. Further, these nucleic acids are modified (e.g, with linker olecule ⁇ , biotin, detection moietie ⁇ ) to perform the detection component ⁇ of the method.
  • FIG 16 a schematic representation is shown of an assay for determining STR alleles from a nucleic acid ligation step.
  • Standard oligonucleotide ligation as ⁇ ay (OLA) a ⁇ ays for the exact match of a pair of oligonucleotides X and Y against a DNA template molecule previously amplified by PCR (Landegren, U. , Kaiser, R. , Sanders, J., and Hood, L. 1988. A liga ⁇ e-mediated gene detection technique. Science , 241: 1077-1080; Inni ⁇ , M.A. , Gelfand, D.H. , Snin ⁇ ky, J.J., and White, T.J. 1990. PCR Protocol ⁇ : A Guide to Methods and Application ⁇ . San Diego, CA: Academic Pre ⁇ ) , incorporated by reference. Following amplification with the PCR primer ⁇ L and R' , two ligation oligonucleotides are conventionally used:
  • (X) initiates the matching sequence from the 5' end, and is biotinylated
  • (Y) completes the matching ⁇ equence to the 3 ⁇ end, and is labeled (e.g., with radiolabel or fluorescent label).
  • the 5 1 end of Y is phosphorylated to allow ligation to X.
  • variable length repeat preclude ⁇ the de ⁇ cribed u ⁇ e of thi ⁇ a ⁇ ay.
  • CA-repeat allele ⁇ can be detected.
  • Zk bridges the gap between X and Y.
  • the 5' end of Zk is phosphorylated to allow ligation to X.
  • the phosphorylated Y is ligated to Zk.
  • This CA-repeat detection differs from conventional ligation as ⁇ ays in that (a) a three-way ligation is performed, (b) a set of intermediate molecules is u ⁇ ed, (c) these intermediate molecules are universally reusable for a ⁇ aying more than one CA-repeat marker, and (d) a ⁇ equence of varying length can be detected.
  • the best Zk's which have the ⁇ tronge ⁇ t ⁇ ignal ⁇ determine the allele ⁇ . This detection can be improved on by deconvolving the panel of signals with the known PCR stutter pattern of the alleles (Perlin, M.W. , Burks, M.B. , Hoop, R.C., and Hoffman, E.P. 1994.
  • ligation chain reaction is performed, rather than a PCR amplification followed by an OLA detection ⁇ tep.
  • Thi ⁇ embodiment u ⁇ es the three oligonucleotide ⁇ X, Y, and Z described above. Specific protocols can be found in (Ausubel, F.M. , Brent, R. , guitarist, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. , and Struhl, K. , ed. 1993. Current Protocol ⁇ in Molecular Biology . New York, NY: John Wiley and Sons; Dracopoli, N.J., Haines, J.L., Korf, B.R. , Morton, C.
  • FIG 17 a schematic representation i ⁇ ⁇ hown of an a ⁇ ay for determining STR alleles from a nucleic acid loop ligation step.
  • Two unique primers for a specific microsatellite are constructed.
  • the primer ⁇ are selected to flank the tandem repeat but to leave at least 15 to 20 bp of internal unique ⁇ equence flanking the repeat region.
  • the oligonucleotide is designed to have significant base mismatching if there is " ⁇ lippage" and a portion of the oligonucleotide extend ⁇ into the 5' and 3' portion ⁇ of the tandem repeat.
  • the degree of exten ⁇ ion into the repeat can be varied but i ⁇ done so that the bridging oligonucleotides are smaller, preferably 15-20 nucleotides than the loop oligonucleotide.
  • a melting temperature for the loop oligonucleotide that is about 10° higher than the largest bridging oligonucleotide is desirable.
  • the loop oligonucleotide is biotinylated or covalently bound to a support matrix or surface.
  • the loop oligonucleotide i ⁇ bound to paramagnetic beads that are covalently linked to strepavidin.
  • the loop oligonucleotide is phosphorylated at the 5' end.
  • the microsatellite marker is amplified using standard PCR primers and conditions.
  • the double-stranded DNA is denatured and annealed to the loop oligonucleotide.
  • the conditions of the annealing are such that the concentrations of the DNA and oligonucleotide are relatively low to discourage concatamer formation, the loop oligonucleotide should be pre ⁇ ent in exce ⁇ s with respect to the PCR product.
  • the hybridization is performed at a sufficient temperature (preferably 37°C) in O.lxSSC or a comparable buffer such that the annealed loop oligonucleotide and PCR strand are ⁇ table, but ⁇ imple annealing within the tandem repeat of the two PCR DNA ⁇ trand ⁇ i ⁇ di ⁇ favored.
  • the annealing i ⁇ performed at a low concentration in a minimum volume of 200 microliter ⁇ in order to di ⁇ favor concatamer formation.
  • part A the original PCR primers do not need to be removed prior to the annealing. After the annealing is completed, the unhybridized DNA and primers are eliminated by wa ⁇ hing with the hybridization buffer.
  • both specificity and sen ⁇ itivity i ⁇ achieved by hybridizing the PCR product with the loop oligonucleotide.
  • the ⁇ tructure i ⁇ annealed (in a ⁇ et of ⁇ eparate chamber ⁇ or po ⁇ ition ⁇ ) with a ⁇ et of bridging oligonucleotides that represent different multiples of the tandem repeat.
  • the bridging oligonucleotide i ⁇ complementary to the PCR'd DNA strand that is hybridized to the loop oligonucleotide.
  • the bridging oligonucleotide is labeled with radioactivity or another detection tag such as fluore ⁇ cein.
  • the bridging oligonucleotide is phosphorylated at the 5' end.
  • the exonuclease reaction is carried out to digest all noncircularized, single- or double-stranded DNAs and primers.
  • the remaining material on the support matrix represents the undigested circularized loop oligonucleotide and bridging oligonucleotide.
  • Bridging oligonucleotides that are too ⁇ hort or too long to perfectly clo ⁇ e the loop oligonucleotide are ligated to one end of the loop oligonucleotide but cannot allow the ⁇ tructure to circularize.
  • the ⁇ e partially ligated product ⁇ are then eliminated during the exonuclea ⁇ e ⁇ tep.
  • the nondegraded products (the circularized strands) are bound to the streptavidin-para agnetic beads in a 500 ⁇ l tube, washed three times with 200 ⁇ l of washing buffer and then counted directly or denatured off of the beads using the loading buffer/Dye for sequencing gels and run on a standard denaturing sequencing gel.
  • the annealing and ligation of the bridge and loop oligonucleotides to create a circular ⁇ tructure i ⁇ performed as a two-stage process to discourage concatemer formation.
  • this protocol only the bridge oligonucleotide is pho ⁇ phorylated.
  • the reaction is identical to that described until the end of the ligation ⁇ tep.
  • the ⁇ ample i ⁇ denatured at 95°C for 5 minute ⁇ and 0.1 unit of T4 Polynucleotide kinase is added at 37°C for 30 minute ⁇ . Thi ⁇ phosphorylates the 5' ends of the loop oligonucleotides.
  • the reaction is then again heated at 95°C for 2-5 minute ⁇ and the ⁇ ample ⁇ are diluted 100 fold in lx liga ⁇ e buffer to promote circularization.
  • IPM Inner Product Mapping
  • the probe' RH signature i ⁇ compared with the RH ⁇ ignature of every STS.
  • the ⁇ ignature ⁇ match at some RH (i.e. , ++ or —)
  • this indicate ⁇ concordance between the two ⁇ ignatures
  • a mismatch i.e., +- or -+
  • this indicates discordance between the signature ⁇ .
  • the sum of the matche ⁇ minus the sum of the mi ⁇ matches i ⁇ computed, which generates a profile curve acro ⁇ the chromo ⁇ ome.
  • the peak of this profile sugge ⁇ t ⁇ the location of the probe.
  • a feature of IPM is its ability to map accurately using few experiments: a logarithmic number of RHs provides linear resolving power.
  • Recombination events in meiosi ⁇ cause the founders' chromosomal region ⁇ to be retained or lo ⁇ t in progeny.
  • the location of thi ⁇ probe is suggested by the concordance of chromosomal regions that affected (or carrier) individuals ⁇ hare with founder( ⁇ ) (++) , or tho ⁇ e region ⁇ which unaffected individuals do not share with founder( ⁇ ) (—).
  • Step 1 phenotypic information i ⁇ obtained on a ⁇ et of related individual ⁇ .
  • Step 2 a den ⁇ e genotyping across a chromo ⁇ ome u ⁇ ing highly-polymorphic STS ⁇ i ⁇ obtained for all informative pedigree member ⁇ ; in the preferred embodiment, thi ⁇ i ⁇ done with the apparatu ⁇ of figure 1.
  • ⁇ ing pha ⁇ e known genotype ⁇ , haplotyping i ⁇ done wherever po ⁇ ible.
  • the founder genotype is obtained directly from the founder (if available) , or constructed indirectly as the union of alleles at each locus for every carrier or affected child of the founder.
  • Step 3 let v(i) be the sign of the phenotype of an individual i, where
  • w(i,m,a) the weight accorded the triple, as follows.
  • IPM identity-by-state
  • the w(i,m,a) term weights for the probability that an allele a was transmitted to individual i at marker m by the founder. That is, an accounting for identity-by-descent (IBD) is done.
  • IBD identity-by-descent
  • the probability of descent at a marker from the founder for an allele on the chromosome is computed.
  • the product of these link probabilitie ⁇ over every link in the inheritance path therefore provide ⁇ an estimate of the probability of descent. Linearly re ⁇ caling thi ⁇ descent probability from the range [0,1] by the function
  • Step 5 a concordance is computed for every allele of every STS marker by ⁇ umming over the individuals ⁇ i ⁇ chromosomes a ⁇
  • c(m,a) SUM (over i) [ v(i) * w(i,m,a) ].
  • Step 7 the genetic region ⁇ correlating with the trait are localized.
  • the concordance function C(m) computes a profile over the chromosome. Where this profile ⁇ how ⁇ a pattern on the chromosome that rises up to a peak, and then again descends from it, ⁇ ugge ⁇ ts the location of the gene (near the peak) .
  • the unaffected individuals are weighted to have les ⁇ influence.
  • Den ⁇ e genotype ⁇ are obtained for related ⁇ et ⁇ of individuals; in the preferred embodiment, this is done with the apparatus of figure 1.
  • Step 8 of figure 12 the genetic patterns obtained in Step 7 are used to as ⁇ e ⁇ the risk of individuals for various traits and diseases.
  • Step 9 the localization of disease genes on a genetic map is used to initiate the cloning of the gene via positional cloning techniques (Kerem, B.-S., Rommens, J.M. , Buchanan, J.A. , Markiewicz, D. , Cox, T.K. , Chakravarti, A., Buchwald, M. , and Tsui, L.-C. 1989. Identification of the cystic fibro ⁇ is gene: genetic analysi ⁇ .
  • Genotyping can be used for actuarial analysi ⁇ of health ri ⁇ ks in order to predict and reduce health care costs. Genotyping also finds application in transplantation (Scharf, S., Saiki, R. , and Ehrlich, H. 1988. New methodology for HLA class II oligonucleotide typing u ⁇ ing polymera ⁇ e chain reaction (PCR) amplification. Hum .
  • the loop mi ⁇ match method ⁇ de ⁇ cribed can detect exon repeat ⁇ that correlate with di ⁇ ease and prognosis, as well as exon alleles
  • Den ⁇ e genotyping can be u ⁇ ed to detect the occurrence of chromo ⁇ omal pattern ⁇ in a population.
  • Thi ⁇ applies in law enforcement applications (Jeffreys, A.J., Brookfield, J.F.Y., and Semeonoff, R. 1985. Po ⁇ itive identification of an immigration te ⁇ t-case using human DNA fingerprints. Nature, 317: 818-819.), incorporated by reference, for genetically fingerprinting individuals, a ⁇ well in paternity testing to asses ⁇ parenthood.
  • Genotyping can monitor the change ⁇ in the chromo ⁇ omal pattern ⁇ of population ⁇ , including:

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Abstract

La médecine moderne exploite relativement peu le matériel génétique d'un individu pour effectuer des interventions préventives, diagnostiques ou thérapeutiques. Cependant, retrouver l'origine de segments chromosomiques dans des familles et des populations, et les mettre ensuite en corrélation avec des traits phénotypiques, permettrait de déterminer avec précision les risques, pour les membres desdites familles et populations, de souffrir de maladies communes multifactorielles. On pourrait ensuite utiliser cette information pour adapter des interventions médicales aux états médicaux majeurs pour lesquels un individu présente un risque important. Le principal obstacle rencontré, dans ce contexte, a été le nombre très important d'expériences et de calculs relatifs à l'établissement de génotypes, requis pour échantillonner, de façon dense, des génomes. L'invention concerne un système qui permet l'établissement, à un rythme rapide, de génotypes, et ainsi la détermination efficace des risques susmentionnés et d'autres informations génétiques utiles. L'invention concerne également des procédés permettant de déterminer la taille d'allèles simples de répétition en tandem par hybridation d'acide nucléique, y compris la formation d'hétéroduplex non appariés et la quantification de la taille de leurs boucles monocaténaires.
PCT/US1995/001395 1994-02-04 1995-02-02 Procede et appareil d'analyse de materiel genetique WO1995021269A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO1996036731A2 (fr) * 1995-05-19 1996-11-21 Trustees Of Boston University Procedes de detection d'acides nucleiques
WO1998023776A1 (fr) * 1996-11-29 1998-06-04 Amersham Pharmacia Biotech Uk Ltd. Procede pour determiner la longueur de sequences repetes en tandem
US7972778B2 (en) 1997-04-17 2011-07-05 Applied Biosystems, Llc Method for detecting the presence of a single target nucleic acid in a sample

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996036731A2 (fr) * 1995-05-19 1996-11-21 Trustees Of Boston University Procedes de detection d'acides nucleiques
WO1996036731A3 (fr) * 1995-05-19 1997-02-06 Univ Boston Procedes de detection d'acides nucleiques
US5753439A (en) * 1995-05-19 1998-05-19 Trustees Of Boston University Nucleic acid detection methods
WO1998023776A1 (fr) * 1996-11-29 1998-06-04 Amersham Pharmacia Biotech Uk Ltd. Procede pour determiner la longueur de sequences repetes en tandem
US6083701A (en) * 1996-11-29 2000-07-04 Amersham Pharmacia Biotech Uk Limited Method for determining tandem repeat sequence length
US7972778B2 (en) 1997-04-17 2011-07-05 Applied Biosystems, Llc Method for detecting the presence of a single target nucleic acid in a sample
US8067159B2 (en) 1997-04-17 2011-11-29 Applied Biosystems, Llc Methods of detecting amplified product
US8257925B2 (en) 1997-04-17 2012-09-04 Applied Biosystems, Llc Method for detecting the presence of a single target nucleic acid in a sample
US8278071B2 (en) 1997-04-17 2012-10-02 Applied Biosystems, Llc Method for detecting the presence of a single target nucleic acid in a sample
US8551698B2 (en) 1997-04-17 2013-10-08 Applied Biosystems, Llc Method of loading sample into a microfluidic device
US8563275B2 (en) 1997-04-17 2013-10-22 Applied Biosystems, Llc Method and device for detecting the presence of a single target nucleic acid in a sample
US8822183B2 (en) 1997-04-17 2014-09-02 Applied Biosystems, Llc Device for amplifying target nucleic acid
US8859204B2 (en) 1997-04-17 2014-10-14 Applied Biosystems, Llc Method for detecting the presence of a target nucleic acid sequence in a sample
US9506105B2 (en) 1997-04-17 2016-11-29 Applied Biosystems, Llc Device and method for amplifying target nucleic acid

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