Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-09-24
2004-03-09
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C536S024300, C536S024310, C536S024320, C536S024330, C702S019000, C702S020000
Reexamination Certificate
active
06703228
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and products associated with genotyping. In particular, the invention relates to methods of detecting single nucleotide polymorphisms and reduced complexity genomes for use in genotyping methods as well as to various methods of genotyping, fingerprinting, and genomic analysis. The invention also relates to products and kits, such as panels of single nucleotide polymorphism allele specific oligonucleotides, reduced complexity genomes, and databases for use in the methods of the invention.
BACKGROUND OF THE INVENTION
Genomic DNA varies significantly from individual to individual, except in identical siblings. Many human diseases arise from genomic variations. The genetic diversity amongst humans and other life forms explains the heritable variations observed in disease susceptibility. Diseases arising from such genetic variations include Huntington's disease, cystic fibrosis, Duchenne muscular dystrophy, and certain forms of breast cancer. Each of these diseases is associated with a single gene mutation. Diseases such as multiple sclerosis, diabetes, Parkinson's, Alzheimer's disease, and hypertension are much more complex. These diseases may be due to polygenic (multiple gene influences) or multifactorial (multiple gene and environmental influences) causes. Many of the variations in the genome do not result in a disease trait. However, as described above, a single mutation can result in a disease trait. The ability to scan the human genome to identify the location of genes which underlie or are associated with the pathology of such diseases is an enormously powerful tool in medicine and human biology.
Several types of sequence variations, including insertions and deletions, differences in the number of repeated sequences, and single base pair differences result in genomic diversity. Single base pair differences, referred to as single nucleotide polymorphisms (SNPs) are the most frequent type of variation in the human genome (occurring at approximately 1 in 10
3
bases). A SNP is a genomic position at which at least two or more alternative nucleotide alleles occur at a relatively high frequency (greater than 1%) in a population. SNPs are well-suited for studying sequence variation because they are relatively stable (i.e., exhibit low mutation rates) and because single nucleotide variations can be responsible for inherited traits.
Polymorphisms identified using microsatellite-based analysis, for example, have been used for a variety of purposes. Use of genetic linkage strategies to identify the locations of single Mendelian factors has been successful in many cases (Benomar et al. (1995),
Nat. Genet
., 10:84-8; Blanton et al. (1991),
Genomics
, 11:857-69). Identification of chromosomal locations of tumor suppressor genes has generally been accomplished by studying loss of heterozygosity in human tumors (Cavenee et al. (1983),
Nature
, 305:779-784; Collins et al. (1996),
Proc. Natl. Acad Sci. USA
, 93:14771-14775; Koufos et al. (1984),
Nature
, 309:170-172; and Legius et al. (1993),
Nat. Genet
., 3:122-126). Additionally, use of genetic markers to infer the chromosomal locations of genes contributing to complex traits, such as type I diabetes (Davis et al. (1994),
Nature
, 371:130-136; Todd et al. (1995),
Proc. Natl. Acad. Sci. USA
, 92:8560-8565), has become a focus of research in human genetics.
Although substantial progress has been made in identifying the genetic basis of many human diseases, current methodologies used to develop this information are limited by prohibitive costs and the extensive amount of work required to obtain genotype information from large sample populations. These limitations make identification of complex gene mutations contributing to disorders such as diabetes extremely difficult. Techniques for scanning the human genome to identify the locations of genes involved in disease processes began in the early 1980s with the use of restriction fragment length polymorphism (RFLP) analysis (Botstein et al. (1980),
Am. J. Hum. Genet
., 32:314-31; Nakamura et al. (1987),
Science
, 235:1616-22). RFLP analysis involves southern blotting and other techniques. Southern blotting is both expensive and time-consuming when performed on large numbers of samples, such as those required to identify a complex genotype associated with a particular phenotype. Some of these problems were avoided with the development of polymerase chain reaction (PCR) based microsatellite marker analysis. Microsatellite markers are simple sequence length polymorphisms (SSLPs) consisting of di-, tri-, and tetra-nucleotide repeats.
Other types of genomic analysis are based on use of markers which hybridize with hypervariable regions of DNA having multiallelic variation and high heterozygosity. The variable regions which are useful for fingerprinting genomic DNA are tandem repeats of a short sequence referred to as a mini satellite. Polymorphism is due to allelic differences in the number of repeats, which can arise as a result of mitotic or meiotic unequal exchanges or by DNA slippage during replication.
The most commonly used method for genotyping involves Weber markers, which are abundant interspersed repetitive DNA sequences, generally of the form (dC-dA)
n
(dG-dT)
n
. Weber markers exhibit length polymorphisms and are therefore useful for identifying individuals in paternity and forensic testing, as well as for mapping genes involved in genetic diseases. In the Weber method of genotyping, generally 400 Weber or microsatellite markers are used to scan each genome using PCR. Using these methods, if 5,000 individual genomes are scanned, 2 million PCR reactions are performed (5,000 genomes×400 markers). The number of PCR reactions may be reduced by multiplexing, in which, for instance, four different sets of primer are reacted simultaneously in a single PCR, thus reducing the total number of PCRs for the example provided to 500,000. The 500,000 PCR mixtures are separated by polyacrylamide gel electrophoresis (PAGE). If the samples are run on a 96-lane gel, 5,200 gels must be run to analyze all 500,000 PCR reaction mixtures. PCR products can be identified by their position on the gels, and the differences in length of the products can be determined by analyzing the gels. One problem with this type of analysis is that “stuttering” tends to occur, causing a smeared result and making the data difficult to interpret and score.
More recent advances in genotyping are based on automated technologies utilizing DNA chips, such as the Affymetrix HuSNP Chip™ analysis system. The HuSNP Chip™ is a disposable array of DNA molecules on a chip (400,000 per half inch square slide). The single stranded DNA molecules bound to the slide are present in an ordered array of molecules having known sequences, some of which are complementary to one allele of a SNP-containing portion of a genome. If the same 5,000 individual genome study described above is performed using the Affymetrix HuSNP Chip™ analysis system, approximately 5,000 gene chips having 1,000 or more SNPs per chip would be required. Prior to the chip scan, the genomic DNA samples would be amplified by PCR in a similar manner to conventional microsatellite genotyping. The gene chip method is also expensive and time-intensive.
SUMMARY OF THE INVENTION
The present invention relates to methods and products for identifying points of genetic diversity in genomes of a broad spectrum of species. In particular, the invention relates to a high throughput method of genotyping of SNPs in a genome (e.g. a human genome) using reduced complexity genomes (RCGs) and, in some exemplary embodiments, using SNP allele specific oligonucleotides (SNP-ASO) and specific hybridization reactions performed, for example, on a surface. The method of genotyping, in some aspects of the invention, is accomplished by scanning a RCG for the presence or absence of a SNP allele. Using this method, tens of thousands of genomes from one species may be simultaneously assayed for the presence or absence
Charest Alain
Housman David E.
Jordan Barbara
Landers John
Brusca John S.
Massachusetts Institute of Technology
Wolf Greenfield and Sacks, P.C.
Zhou Shubo (Joe)
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