Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-10-27
2003-04-08
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06544730
ABSTRACT:
OBJECTIVES AND SIGNIFICANCE
A need exists for characterized human nuclear loci that could be analyzed for diversity in a reasonably rapid way. Such diversity, or polymorphism, is useful for establishing human identity, often for forensic purposes. In addition, diversity is useful for establishing parentage. The use of polymorphism for these purposes and the methods to do so are described by the Committee on DNA Forensic Science,
The Evaluation of Forensic DNA Evidence
, National Academy Press, Washington, D.C., (1996) and by Walker,
Inclusion Probabilities in Parentage Testing
, American Association of Blood Banks, Arlington, Va. (1983). An in-depth analysis of genetic, ethnic and geographical variation is provided by Cavalli-Sforza et al. (
The History and Geography of Human Genes
. Princeton University Press, Princeton, N.J. (1994)). Nei (
Molecular Population Genetics
. Columbia University Press, New York (1987) provides a general discussion of the principles of population genetics.
Most human nuclear regions, however, show so little diversity that analysis requires sequencing of very long genomic regions to be informative. Regions of the genome that are hypervariable overcome this difficulty by allowing a significant amount of sequence variation in a shorter DNA sequence, providing a tremendous benefit for studies of human diversity and molecular anthropology.
Additional information can be derived from linkage disequlibrium of polymorphisms. Disequilibrium among polymorphisms can be correlated with ethnic origins and thus used to provide information about ethnic descent of an individual from his DNA.
Others have used length polymorphisms (e.g., VNTRS and microsatellites (Nakamura et al, 1987; Bowcock et al, 1994; Deka et al, 1995) or minisatellites (Amour et al, 1996), or combinations of markers for linkage disequilibirum studies (Tishkoff et al, 1996). These existing technologies are limited by low levels of polymorphism and complex analytical methods.
Analyses that utilize DNA sequence of point mutations directly avoid these problems, but only if the polymorphism density within the selected sequence is high. By analyzing a region with a high density of sequence variation, a large amount of useful information can be obtained from a short sequence.
BRIEF DESCRIPTION OF THE INVENTION
DEFINITIONS
All of the terms used in the specification and the claims are known to one skilled in the act. Nevertheless, in order to provide a clear and consistent understanding of the specification and the claims, including the scope given to such terms, the following definitions are provided.
Polymorphic locus or gene: A nucleic acid sequence localized in the diploid genome wherein the homologous copies are not identical.
Nucleotide diversity is the average number of nucleotide differences per site between any two randomly chosen sequences. This term and related concepts are further explained in Li and Grauer,
Fundamentals of Molecular Evolution
, Sinauer Associates, Inc., Pub., (1991).
The term “haplotype” means the set of alleles linked on a single chromosome.
The term “genotype” means the set of alleles present in an individual.
Alu sequences comprise a family of generally nonfunctional processed pseudogenes. Alu elements are DNA sequences that are approximately 300 bp long that belong to a family of repeated sequences. Alu family members appear more than 500,000 times in the human genome, constituting 5-6% of the genome (see Li and Grauer,
Fundamentals of Molecular Evolution
, Sinauer Associates, Inc., Pub., (1991).
The term “amplifying” which typically refers to an “exponential” increase in target nucleic acid is being used herein to describe both linear and exponential increases in the numbers of a select target sequence of nucleic acid.
The term “amplification” refers to any in vitro means for multiplying the copies of a target sequence of nucleic acid. Such methods include but are not limited to those discussed herein. Sequence-based amplification systems such as the polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA) (see Sooknanan and Malek, 1995, Biotechnology, 13: 563-564), and strand displacement amplification (SDA) (see Walker et al., 1994, Nucleic Acids Res.) amplify a target nucleic acid sequence. Signal-based amplification such as oligonucleotide ligation assay (OLA), Q.&bgr;. RNA replicase (Lizardi and Kramer. 1991. TIB 9: 53-58), cycling probe reaction (CPR) (Duck et al. 1991. Biotechniques 9: 142-147) and branched DNA (bDNA) (Urdea. 1993. Clin. Chem. 39: 725-726), amplify or alter a signal from a detection reaction that is target dependent.
The term “amplifying” which typically refers to an “exponential” increase in target nucleic acid is being used herein to describe both linear and exponential increases in the numbers of a select target sequence of nucleic acid.
The term “primer” as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is initiated, i.e., in the presence of four different nucleoside triphosphates and a DNA polymerase in an appropriate buffer (“buffer” includes pH, ionic strength, cofactors, etc.) and at a suitable temperature. Several methods of amplification that use primers have been devised, the best known being PCR. For example, in Stand Displacement Amplification (SDA), the 3′ end of the amplification primer (the target binding sequence) hybridizes at the 3′ end of the target sequence and comprises a recognition site for a restriction enzyme near its 5′ end.
The term “thermocycling profile” as used herein refers to the selected temperature parameters selected for “n” cycles of amplification. The thermocycling profile includes at least two temperatures, a high denaturation temperature, adequate for sample-template, and subsequent product, denaturation, and a low temperature appropriate for primer annealing and polymerase extension. Accordingly, particular thermocycling parameters are selected to control primer annealing and product denaturation and thus regulate accessibility and primer extension.
The choice of primers for use in PCR determines the specificity of the amplification reaction. Primers used in the present invention are generally oligonucleotides, usually deoxyribonucleotides several nucleotides in length, that can be extended in a template-specific manner by the polymerase chain reaction. The primer is sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization and typically contains 10-30 nucleotides, although that exact number is not critical to the successful amplification.
A primer is selected to be “substantially” complementary to a strand of the template having a specific sequence. For primer extension to occur, the primer must be sufficiently complementary to anneal to the nucleic acid template under the reaction conditions. Not every nucleotide of the primer must anneal to the template for primer extension to occur. The primer sequence need not reflect the exact sequence of the template. For example, in one embodiment of the invention, a non-complementary nucleotide fragment or tail is attached to the 5′ end of the primer with the remainder of the primer sequence being complementary to the template.
Alternatively, non-complementary bases can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the template for hybridization to occur and allow synthesis of complementary DNA strand.
As used herein in referring to primers, probes, or secondary oligonucleotides, the term “oligonucleotide” refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, and preferably more than three. Its exact size is not critical (except as noted herein), but the size depends upon many factors including t
Deininger Prescott
Kass David
Fredman Jeffrey
Murray Michael L.
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