Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Biological or biochemical
Reexamination Certificate
2000-02-15
2004-10-19
Brusca, John S. (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Biological or biochemical
C435S006120, C435S091200, C536S023100
Reexamination Certificate
active
06807490
ABSTRACT:
FIELD OF THE INVENTION
The present invention pertains to a process for analyzing a DNA molecule. More specifically, the present invention is related to performing experiments that produce quantitative data, and then analyzing these data to characterize a DNA fragment. The invention also pertains to systems related to this DNA fragment information.
BACKGROUND OF THE INVENTION
With the advent of high-throughput DNA fragment analysis by electrophoretic separation, many useful genetic assays have been developed. These assays have application to genotyping, linkage analysis, genetic association, cancer progression, gene expression, pharmaceutical development, agricultural improvement, human identity, and forensic science.
However, these assays inherently produce data that have signficant error with respect to the size and concentration of the characterized DNA fragments. Much calibration is currently done to help overcome these errors, including the use of in-lane molecular weight size standards. In spite of these improvements, the variability of these properties (between different instruments, runs, or lanes) can exceed the desired tolerance of the assays.
Recently, advances have been made in the automated scoring of genetic data. Many naturally occurring artifacts in the amplification and separation of nucleic acids can be eliminated through calibration and mathematical processing of the data on a computing device (M W Perlin, M B Burks, R C Hoop, and E P Hoffman, “Toward fully automated genotyping: allele assignment, pedigree construction, phase determination, and recombination detection in Duchenne muscular dystrophy,” Am. J. Hum. Genet., vol. 55, no. 4, pp. 777-787, 1994; M W Perlin, G Lancia, and S-K Ng, “Toward fully automated genotyping: genotyping microsatellite markers by deconvolution,” Am. J. Hum. Genet., vol. 57, no. 5, pp. 1199-1210, 1995; S-K Ng, “Automating computational molecular genetics: solving the microsatellite genotyping problem,” Carnegie Mellon University, Doctoral dissertation CMU-CS-98-105, Jan. 23, 1998), incorporated by reference.
This invention pertains to the novel use of calibrating data and mathematical analyses to computationally eliminate undesirable data artifacts in a nonobvious way. Specifically, the use of allelic ladders and coordinate transformations can help an automated data analysis system better reduce measurement variability to within a desired assay tolerance. This improved reproducibility is useful in that it results in greater accuracy and more complete automation of the genetic assays, often taking less time at a lower cost with fewer people.
Genotyping Technology
Genotyping is the process of determining the alleles at an individual's genetic locus. Such loci can be any inherited DNA sequence in the genome, including protein-encoding genes and polymorphic markers. These markers include short tandem repeat (STR) sequences, single-nucleotide polymorphism (SNP) sequences, restriction fragment length polymorphism (RFLP) sequences, and other DNA sequences that express genetic variation (G Gyapay, J Morissette, A Vignal, C Dib, C Fizames, P Millasseau, S Marc, G Bernardi, M Lathrop, and J Weissenbach, “The 1993-94 Genethon Human Genetic Linkage Map,” Nature Genetics, vol. 7, no. 2, pp. 246-339, 1994; P W Reed, J L Davies, J B Copeman, S T Bennett, S M Palmer, L E Pritchard, S C L Gough, Y Kawaguchi, H J Cordell, K M Balfour, S C Jenkins, E E Powell, A Vignal, and J A Todd, “Chromosome-specific microsatellite sets for fluorescence-based, semi-automated genome mapping,” Nature Genet., vol. 7, no. 3, pp. 390-395, 1994; L Kruglyak, “The use of a genetic map of biallelic markers in linkage studies,”
Nature Genet
., vol. 17, no. 1, pp. 21-24, 1997; D Wang, J Fan, C Siao, A Berno, P Young, R Sapolsky, G Ghandour, N Perkins, E Winchester, J Spencer, L Kruglyak, L Stein, L Hsie, T Topaloglou, E Hubbell, E Robinson, M Mittmann, M Morris, N Shen, D Kilburn, J Rioux, C Nusbaum, S Rozen, T Hudson, and E Lander, “Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome,” Science, vol. 280, no. 5366, pp. 1077-82, 1998; P Vos, R Hogers, M Bleeker, M Reijans, T van de Lee, M Hornes, A Frijters, J Pot, J Peleman, M Kuiper, and M Zabeau, “AFLP: a new technique for DNA fingerprinting,”
Nucleic Acids Res
, vol. 23, no. 21, pp. 4407-14, 1995; J Sambrook, E F Fritsch, and T Maniatis,
Molecular Cloning
, Second Edition. Plainview, N.Y.: Cold Spring Harbor Press, 1989), incorporated by reference.
The polymorphism assay is typically done by characterizing the length and quantity of DNA from an individual at a marker. For example, STRs are assayed by polymerase chain reaction (PCR) amplification of an individual's STR locus using a labeled PCR primer, followed by size separation of the amplified PCR fragments. Detection of the fragment labels, together with in-lane size standards, generates a signal that permits characterization of the size and quantity of the DNA fragments. From this characterization, the alleles of the STR locus in the individual's genome can be determined (J Weber and P May, “Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction,”
Am. J. Hum. Genet
., vol. 44, pp. 388-396, 1989; J S Ziegle, Y Su, K P Corcoran, L Nie, P E Mayrand, L B Hoff, L J McBride, M N Kronick, and S R Diehl, “Application of automated DNA sizing technology for genotyping microsatellite loci,”
Genomics
, vol. 14, pp. 1026-1031, 1992), incorporated by reference.
The labels can use radioactivity, fluorescence, infrared, or other nonradioactive labeling methods (F M Ausubel, R Brent, R E Kingston, D D Moore, J G Seidman, J A Smith, and K Struhl, ed., Current Protocols in Molecular Biology. New York, N.Y.: John Wiley and Sons, 1995; N J Dracopoli, J L Haines, B R Korf, C C Morton, C E Seidman, J G Seidman, D T Moir, and D Smith, ed., Current Protocols in Human Genetics. New York: John Wiley and Sons, 1995; L J Kricka, ed., Nonisotopic Probing, Blotting, and Sequencing, Second Edition. San Diego, Calif.: Academic Press, 1995), incorporated by reference.
Size separation of fragment molecules is typically done using gel or capillary electrophoresis (CE); newer methods include mass spectrometry and microchannel arrays (R A Mathies and X C Huang, “Capillary array electrophoresis: an approach to high-speed, high-throughput DNA sequencing,”
Nature
, vol. 359, pp. 167-169, 1992; K J Wu, A Stedding, and C H Becker, “Matrix-assisted laser desorption time-of-flight mass spectrometry of oligonucleotides using 3-hydroxypicolinic acid as an ultraviolet-sensitive matrix,”
Rapid Commun. Mass Spectrom
., vol. 7, pp. 142-146, 1993), incorporated by reference.
The label detection method is contingent on both the labels used and the size separation mechanism. For example, with automated DNA sequencers such as the PE Biosystems ABI/377 gel, ABI/310 single capillary or ABI/3700 capillary array instruments, the detection is done by laser scanning of the fluorescently labeled fragments, imaging on a CCD camera, and electronic acquisition of the signals from the CCD camera. Flatbed laser scanners, such as the Molecular Dynamics Fluorimager or the Hitachi FMBIO/II acquire flourescent signals similarly. Li-Cor's infrared automated sequencer uses a detection technology modified for the infrared range. Radioactivity can be detected using film or phosphor screens. In mass spectrometry, the atomic mass can be used as a sensitive label. See (A. J. Kostichka,
Bio/Technology
, vol. 10, pp. 78, 1992), incorporated by reference.
Size characterization is done by comparing the sample fragment's signal in the context of the size standards. By separate calibration of the size standards used, the relative molecular size can be inferred. This size is usually only an approximation to the true size in base pair units, since the size standards and the sample fragments generally have different chemistries and electrophoretic migration patterns (S-K Ng, “Automating computational molecular genetics: so
Brusca John S.
Schwartz Ansel M.
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