Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-20
2003-03-25
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300, C536S023400
Reexamination Certificate
active
06537751
ABSTRACT:
BACKGROUND OF THE INVENTION
Recent advances in genetic engineering and bioinformatics have enabled the manipulation and characterization of large portions of the human genome. While efforts to obtain the full sequence of the human genome are rapidly progressing, there are many practical uses for genetic information which can be implemented with partial knowledge of the sequence of the human genome.
As the full sequence of the human genome is assembled, the partial sequence information available can be used to identify genes responsible for detectable human traits, such as genes associated with human diseases, and to develop diagnostic tests capable of identifying individuals who express a detectable trait as the result of a specific genotype or individuals whose genotype places them at risk of developing a detectable trait at a subsequent time. Each of these applications for partial genomic sequence information is based upon the assembly of genetic and physical maps which order the known genomic sequences along the human chromosomes.
The present invention relates to an ordered set of human genomic sequences comprising single nucleotide polymorphisms, as well as the use of these polymorphisms as a high resolution map of the human genome, methods of identifying genes associated with detectable human traits, and diagnostics for identifying individuals who carry a gene which causes them to express a detectable trait or which places them at risk of expressing a detectable trait in the future.
Advantages of the Biallelic Markers of the Present Invention
The map-related biallelic markers of the present invention offer a number of important advantages over other genetic markers such as RFLP (Restriction fragment length polymorphism), VNTR (Variable Number of Tandem Repeats) markers and earlier STS-(sequence tagged sites) derived markers.
The first generation of markers, RFLPs, are variations that modify the length of a restriction fragment. However, methods used to identify and type RFLPs are relatively wasteful of materials, effort, and time. Since they are biallelic markers (they present only two alleles, the restriction site being either present or absent), their maximum heterozygosity is 0.5. The theoretical number of RFLPs distributed along the entire human genome is more than 10
5
, which leads to a potential average inter-marker distance of 30 kilobases. However, in reality, the number of evenly distributed RFLPs which occurs at a sufficient frequency in the population to make them useful for tracking of genetic polymorphisms is very limited.
The second generation of genetic markers were VNTRs, which can be categorized as either minisatellites or microsatellites. Minisatellites are tandemly repeated DNA sequences present in units of 5-50 repeats which are distributed along regions of the human chromosomes ranging from 0.1 to 20 kilobases in length. Since they present many possible alleles, their informative content is very high. Minisatellites are scored by performing Southern blots to identify the number of tandem repeats present in a nucleic acid sample from the individual being tested. However, there are only 10
4
potential VNTRs that can be typed by Southern blotting. Thus, the number of easily typed informative markers in these maps is far too small for the average distance between informative markers to fulfill the requirements for a useful genetic map. Moreover, both RFLP and VNTR markers are costly and time-consuming to develop and assay in large numbers.
Initial attempts to construct genetic maps based on non-RFLP biallelic markers have focused on identifying biallelic markers lying within sequence tagged sites (STS), pieces of genomic DNA having a known sequence and averaging about 250 bases in length. More than 30,000 STSs have been identified and ordered along the genome (Hudson et al.,
Science
270:1945-1954 (1995); Schuler et al.,
Science
274:540-546 (1996), the disclosures of which are incorporated herein by reference in their entireties). For example, the Whitehead Institute and Genethon's integrated map contains 15,086 STSs.
These sequence tagged sites can be screened to identify polymorphisms, preferably Single Nucleotide Polymorphisms (SNPs), more preferably non RFLP biallelic markers therein. Generally polymorphisms are identified by determining the sequence of the STSs in 5 to 10 individuals.
Wang et al. (Cold Spring Harbor Laboratory:
Abstracts of Papers Presented on Genome Mapping and Sequencing
p. 17 (May 14-18, 1997), the disclosure of which is incorporated herein by reference in its entirety) recently announced the identification and mapping of 750 Single Nucleotide Polymorphisms issued from the sequencing of 12,000 STSs from the Whitehead/MIT map, in eight unrelated individuals. The map was assembled using a high throughput system based on the utilization of DNA chip technology available from Affymetrix (Chee et al.,
Science
274:610-614 (1996), the disclosure of which is incorporated herein by reference in its entirety).
However, according to experimental data and statistical calculations, less than one out of 10 of all STSs mapped today will contain an informative Single Nucleotide Polymorphism. This is primarily due to the short length of existing STSs (usually less than 250 bp). If one assumes 10
6
informative SNPs spread along the human genome, there would on average be one marker of interest every 3×10
9
/10
6
, i.e. every 3,000 bp. The probability that one such marker is present on a 250 bp stretch is thus less than {fraction (1/10)}.
While it could produce a high density map, the STS approach based on currently existing markers does not put any systematic effort into making sure that the markers obtained are optimally distributed throughout the entire genome. Instead, polymorphisms are limited to those locations for which STSs are available.
The even distribution of markers along the chromosomes is critical to the future success of genetic analyses. In particular, a high density map having appropriately spaced markers is essential for conducting association studies on sporadic cases, aiming at identifying genes responsible for detectable traits such as those which are described below.
As will be further explained below, genetic studies have mostly relied in the past on a statistical approach called linkage analysis, which took advantage of microsatellite markers to study their inheritance pattern within families from which a sufficient number of individuals presented the studied trait. Because of intrinsic limitations of linkage analysis, which will be further detailed below, and because these studies necessitate the recruitment of adequate family pedigrees, they are not well suited to the genetic analysis of all traits, particularly those for which only sporadic cases are available (e.g. drug response traits), or those which have a low penetrance within the studied population.
Association studies enabled by the biallelic markers of the present invention offer an alternative to linkage analysis. Combined with the use of a high density map of appropriately spaced, sufficiently informative markers, association studies, including linkage disequilibrium-based genome wide association studies, will enable the identification of most genes involved in complex traits.
Single nucleotide polymorphism or biallelic markers can be used in the same manner as RFLPs and VNTRs but offer several advantages. Single nucleotide polymorphisms are densely spaced in the human genome and represent the most frequent type of variation. An estimated number of more than 10
7
sites are scattered along the 3×10
9
base pairs of the human genome. Therefore, single nucleotide polymorphisms occur at a greater frequency and with greater uniformity than RFLP or VNTR markers which means that there is a greater probability that such a marker will be found in close proximity to a genetic locus of interest. Single nucleotide polymorphisms are less variable than VNTR markers but are mutationally more stable.
Also, the different forms of a characterized singl
Blumenfeld Marta
Chumakov Ilya
Cohen Daniel
Genset S.A.
Goldberg Jeanine
Jones W. Gary
Saliwanchik Lloyd & Saliwanchik
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