Awards for SNP RFAs HG-98-001

National Human Genome Research Institute

National Institutes of Health
U.S. Department of Health and Human Services


Awards For SNP RFAs (HG-98-001)

Click on the Principal Investigator (PI) names to view the application abstracts. Some awards were funded for less than the amount requested, so the scope of the research may be reduced.

PIs  PRINCIPAL INVESTIGATOR INSTITUTION TITLE IC FUNDING
(PRIMARY IC FIRST)
BARANY, FRANCIS Cornell Univ. Medical Ctr. Identifying genome changes in cancer development NCI
BOYCE-JACINO, MICHAEL Orchid (was Molec. Tool) SNP's on CHIPS NHGRI
CHAKRAVARTI, ARAVINDA Case Western Reserve Univ. Human genomic polymorphisms NIMH, NHGRI, NIA, NIAAA, NICHD, NIDA, NINDS
CHEE, MARK Illumina, Inc. Randomly ordered DNA arrays for SNP discovery and typing NHGRI
COX, DAVID Stanford Univ. A high resolution SNP map of the human genome NHGRI
DAVIS, RONALD Stanford Univ. Large-scale discovery of SNPs NHGRI, NIDCD, NCRR
GERAGHTY, DANIEL Fred Hutchinson Cancer Research Ctr. SNPs in the MHC NHLBI, NIDDK
HOGAN, MICHAEL Genometrix, Inc. The Risk/Tox chip program NIEHS
KWOK, PUI-YAN Washington Univ. Method for global and targeted discovery of SNP markers NIA
KWOK, PUI-YAN Washington Univ. New methods for high throughput genome analysis NEI
LANDER, ERIC Whitehead Institute SNPs in gene-coding regions NHGRI, NIA, NIDCR
MYERS, RICHARD Stanford Univ. A mismatch enrichment method for discovering human SNPs NIDCD
NICKERSON, DEBORAH Univ. of Washington Finding and genotyping SNPs by automated sequence analysis NIAID
NOLAN, JOHN Los Alamos Nat. Lab. High throughput SNP discovery and scoring using flow cytometry NCRR
OLSON, MAYNARD Univ. of Washington Methods for SNPs NIEHS
TON, CARL Univ. of Washington Discovery of cSNPs in nicotinic acetylcholine receptors NINDS, NIAAA
WEBER, JAMES Marshfield Medical Research Foundation Human diallelic insertion/deletion polymorphisms NHLBI

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BARANY, FRANCIS Grant Number: R01CA81467

Project Title: IDENTIFYING GENOME CHANGES IN CANCER DEVELOPMENT

Abstract: Human diseases arise from a complex interaction of DNA polymorphisms or mutations and environmental factors. Single nucleotide polymorphisms (SNPs) have recently been identified as potentially powerful means for genetic typing, and are predicted to supersede microsatellite repeat analysis as the standard for genetic association, linkage and mapping studies. Thus, there is an urgent need to develop robust tools for the rapid analysis of SNPs. Our laboratory has used thermostable ligase to successfully detect and quantify a wide range of polymorphisms and mutations in known oncogenes and tumor suppressor genes. The ligase detection reaction (LDR), combined with multiplex PCR reactions, has also been used to detect multiple polymorphisms for human identification. Thus, the methods developed in our laboratory are ideally suited for rapid scoring of single nucleotide polymorphisms throughout the genome.

The first goal of this proposal is to extend quantitative ligase-based detection to score SNPs on a new type of universal DNA array. As a model system, we plan to quantify gene amplification or loss of heterozygosity in the c-myc, K-ras, APC and p53 genes in microdissected tissue from colon tumors using LDR and DNA arrays. This approach will be used to help identify a new tumor suppressor gene at the 7q31 loci, which is deleted in 80 percent of colorectal cancers.

A second goal of this proposal is to use a thermostable Endonuclease V mismatch cleavage enzyme / thermostable DNA ligase combination to rapidly identify and precisely characterize unknown coding region SNPs. Such SNPs are present in known cancer genes at low frequency, and carriers are at a significantly higher risk of developing breast, ovarian, prostate or colon cancer. Using enzymatic methods to identify SNPs in single or pooled samples, we expect to greatly accelerate discovery of SNPs in cancer susceptibility genes.

Enzymatic discovery/identification combined with universal array-based scoring represents an integrated approach to harness the power of single nucleotide polymorphisms for genetic studies, and may ultimately lead to a reduction of cancer burden through early screening and prevention programs.

Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIVERSITY, NEW YORK, NY 10021
Department: MICROBIOLOGY
Fiscal Year: 1998
Project Start: 30-SEP-98
Project End: 29-SEP-01
ICD: NATIONAL CANCER INSTITUTE
IRG: ZHG1

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BOYCE-JACINO, MICHAEL

Grant Number: 1 R01 HG 02035-01
Project Title: SNPs on CHIPS

Abstract: A major effort in discovery and characterization of genetic variation will require innovative, low-cost, high throughput methods for highly accurate comparative sequencing of DNA. We propose to develop a novel combinatorial array to enable ultralow cost, sensitive and high throughput discovery of single nucleotide polymorphisms. This will be accelerated by the development of technology to resequence PCR products from multiple individuals. We will develop a low complexity array based on 4mer DNA sequences for use in a high throughput 96-array microplate format (one array per well) to screen candidate cSNPs. This SNP discovery system, including software analysis tools, will facilitate greatly the discovery and characterization of new SNPs and will be amenable to adaptation in large and small-scale laboratory settings.

Institution: Orchid Biosciences, Inc.
Department:
Fiscal Year: 2000
Project Start: 6/1/2000
Project End: 5/31/2001
ICD: National Human Genome Research Institute
IRG: ZHGI

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CHAKRAVARTI, ARAVINDA

Grant Number: R01MH60007
Project Title: HUMAN GENOMIC POLYMORPHISMS

Abstract: The major goal in human genetics is to ascertain the relationship between DNA sequence variation and phenotypic variation. For these studies, molecular polymorphisms are indispensable for conventional meiotic mapping, fine-structure mapping and haplotype analysis. However, with the contemplated sequencing of a reference human genome and identification of all human genes, studies of complex genetic disorders are expected to be more efficient if one were to systematically search all human genes for functional variants by association and linkage disequilibrium studies. This requires the development of technology and methods for the systematic discovery of genetic variation in human DNA, primarily the single nucleotide polymorphisms (SNPs) which are the most abundant. Since functional variants may not reside in coding sequences only, we need to understand the relationship between functional variants and neutral variation in contiguous genomic regions; that is, we require studies of the nature and extent of sequence polymorphisms and linkage disequilibrium in genes, intergenic regions and flanking sites.

This proposal is a collaborative effort, between human geneticists, a biotechnology company and population geneticists that aims to use large-scale DNA microarray technologies for efficient genome-scale SNP discovery. The first major goal is to ascertain and refine methods for SNP discovery in contiguous genomic segments in a mixed population resource, assess the density with which robust SNP assays can be developed, and determine the allele frequency distribution of these SNPs. The studies will be conducted on multiple genomic segments, 100 kilobases or greater, whose nucleotide sequences are available in sequence databases and are known to contain candidate genes for neuropsychiatric disorders. The second major goal is to evaluate, by developing and genotyping multiplexed sets of SNPs, in four population samples of differing ages, the scale of background linkage disequilibrium to assess the density of polymorphisms needed to allow association mapping for complex human diseases and traits. The second aim will allow us to accurately estimate the density of SNPs needed to efficiently develop haplotypes across the entire human genome for disease association studies.

Institution: CASE WESTERN RESERVE UNIVERSITY, 10900 EUCLID AVENUE, CLEVELAND, OH 44106
Department: GENETICS
Fiscal Year: 1998
Project Start: 30-SEP-98
Project End: 31-AUG-01
ICD: NATIONAL INSTITUTE OF MENTAL HEALTH
IRG: ZHG1

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CHEE, MARK S.

Grant Number: R21HG01911
Project Title: RANDOMLY ORDERED DNA ARRAYS FOR SNP DISCOVERY AND TYPING

Abstract: The broad aims of the project are to address the need for better technologies for analyzing genetic information. This project has three major specific aims: 1) The development of a novel randomly-ordered array-based technology for efficient and scaleable SNP analysis. This part of the project is intended to create libraries of oligonucleotide probes and arrange them on the ends of optical fibers to create high density probe arrays for genetic analysis. This will provide the basis for versatile, low-cost, high-throughput, small-format assays for the highly parallel analysis of genetic information. 2) The development of highly parallel, low-cost SNP genotyping assays based on randomly ordered oligonucleotide probe arrays. A system for typing single nucleotide polymorphisms will be developed and used to demonstrate cost- effective genotyping using the new technology. 3) The development of robust methods for discovering polymorphisms using randomly ordered oligonucleotide probe arrays. Arrays and associated methods will be developed to analyze nucleic acid sequences for variation. There is an urgent need for better access to genetic information. Better tools for genetic analysis will accelerate the understanding of complex genetic systems, help in discovering the relationships between genes and disease, and be useful in the diagnosis, management and treatment of disease at the level of the individual.

Institution: ILLUMINA, INC., 9390 TOWN CENTRE DRIVE, STE. 2, SAN DIEGO, CA 92121-3015
Fiscal Year: 1999
Project Start: 01-FEB-99
Project End: 31-JAN-00
ICD: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
IRG: ZHG1

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COX, DAVID

Grant Number: R01HG01919
Project Title: HIGH RESOLUTION SNP MAP OF THE HUMAN GENOME

Abstract: Our overall goal is to 1) identify a dense set of single nucleotide polymorphisms (SNPs) spanning the human genome; 2) identify two alleles at each variant site; 3) order these SNPs with high confidence on a single integrated high-resolution map of the human genome containing genes and simple sequence repeat (SSR) markers; and 4) place all of this information in a public database, accessible to a wide range of potential users. We propose a three-year collaborative effort between the Stanford Human Genome Center (SHGC) and Affymetrix, Inc. to screen 24,000 sequence tagged sites (STSs) per year for polymorphic variation using Affymetrix DNA arrays. Each STS will be derived from BAC clones and amplified from the genomic DNA of eight anonymous unrelated individuals. Our preliminary data suggest that we will identify 6,000 such polymorphisms per year, with one in every four STSs containing a common variant. We will use an independent technology (ABI gel-based fluorescent sequencing) on DNA from two informative individuals, identified by chip-based screening, to confirm each SNP. Each polymorphic allele will be placed in a publicly-accessible database within 90 days of demonstrated consistency between gel-based and chip- based analyses. Our final product will be a map of 72,000 STSs, including 18,000 SNPS, integrated with 30,000 previously-mapped genes and simple sequence repeat (SSR) markers, ordered with greater than 95 percent confidence at 100 kb resolution.

Institution: STANFORD UNIVERSITY, STANFORD, CA 94305
Department: GENETICS
Fiscal Year: 1998
Project Start: 30-SEP-98
Project End: 30-JUN-01
ICD: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
IRG: ZHG1

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DAVIS, RONALD W.

Grant Number: R01HG01932
Project Title: LARGE SCALE DISCOVERY OF SINGLE NUCLEOTIDE POLYMORPHISMS

Abstract: The aim of this proposal is to screen a minimum of 5,000 STSs in 450 individuals per year. To achieve this throughput, we will screen upfront a plate of 90 ethnically diverse individuals. Only in the case when a frequent polymorphism is seen, or several different DHPLC profiles are observed, indicating the presence of various polymorphisms, will we analyze the remaining 360 samples individually. However, if no or only a polymorphism of low frequency is observed, we will pool at least four chromosomes to achieve adequate throughput. Based on present experience, only every fourth or fifth STS will require individual analysis of all individuals. In order to maximize sample throughput on the DHPLC systems, we will tag amplicons with different fluorophores. Present technology allows simultaneous detection of at least two such tags without spectral overlap. Further increases in throughput are planned by means of mass spec tags. For this purpose, STSs that can be analyzed at the same temperature as determined by our DHPLC melting algorithm will be labeled with different mass spec tags. Following their amplification, denaturation and reannealing, amplicons will be combined and separated on a single DHPLC column. Following resolution of hetero- and homoduplex molecules, the separated species will pass through a photolysis unit, in which the mass spec tags are cleaved off. Only the mass spec tags will then be subjected to analysis in a quadrupole mass spectrometer to identify the eluted peaks. Since different mismatches have been shown to yield different profiles, only a few representative samples, an expected average of 5 samples per STS screened, need to be sequenced in order to establish the chemical nature of a mismatch.

Institution: STANFORD UNIVERSITY, STANFORD, CA 94305
Department: BIOCHEMISTRY
Fiscal Year: 1998
Project Start: 30-SEP-98
Project End: 30-JUN-01
ICD: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
IRG: ZHG1

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GERAGHTY, DANIEL E.

Grant Number: R01HL62682
Project Title: SINGLE NUCLEOTIDE POLYMORPHISMS IN THE MHC

Abstract: The central goal of this proposal is to provide a near complete description of the SNPs within a defined region and to build a database that will provide for the rapid and effective dissemination of the resultant data to the scientific community. This study will focus on the class I region within the major histocompatibility complex (MHC). Our target for SNP discovery consists of a 2.3 megabase segment that includes all of the HLA class I genes. We are near completion of a project to determine the nucleotide sequence of this contiguous 2.3 Mbp segment, thus making available a template for SNP discovery and a team of highly trained genomic scientists. We also have well established informatics capabilities from our genomic work and will capitalize on this and strong institutional support in order to deliver SNP data to the scientific community. To reach these goals we propose to accomplish the following specific aims: 1) To identify a large proportion of the SNPs in the 2.3 Mbp HLA class I region and 2) To develop a database designed to track, monitor and disseminate data and information associated with the HLA SNP project. This aim will include four major categories of development: i) data capture, ii) statistical process control, iii) analysis, and iv) presentation. We will collaborate with an independent group to develop needed software and capitalize on ongoing efforts at the FHCRC to build a strong informatics software and hardware support base. As the MHC contains the most polymorphic loci resident in the human genome, a catalogue of HLA SNPs has the potential for a broad impact on the scientific research community. The applicability extends from genetic matching of donor and recipient in unrelated transplants, to explaining the many MHC disease associations that remain unsolved today, and further to basic questions in anthropology and evolutionary biology and indeed to genomic sciences. An HLA SNP data set will help define new approaches to treating disease, interpreting population studies, and extending the abilities for gene discovery and understanding gene function.

Institution: FRED HUTCHINSON CANCER RESEARCH CENTER, 1100 FAIRVIEW AVENUE NORTH, BOX 19024,SEATTLE, WA 98109-1024
Fiscal Year: 1998
Project Start: 30-SEP-98
Project End: 31-AUG-01
ICD: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG: ZHG1

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HOGAN, MICHAEL E.

Grant Number: R01ES09910
Project Title: RISK/TOX CHIP PROGRAM

Abstract: We will develop the instrumentation, the techniques and the DNA microarray technology to enable very large scale analysis of inherited genetic variation, on as many as 50 genes at a time. We refer to such testing as population-wide "allele signature" analysis. Below we list the specific aims of the research plan.

SA 1. We will optimize DNA microarray fabrication so that it can supply up to 1,000,000 microarrays per year for allele signature analysis. We focus our development and testing on a panel of approximately100 alleles chosen with reference to their importance in chemical toxicology and environmental carcinogenesis of the lung. We refer to this as the "Risk-fox Chip."

SA 2. We will develop procedures and automated workstations which can extract DNA and perform the requisite microarray hybridization, detection and data analysis on up 950,000 blood samples per year. The workstations will be based upon the use of a Beckman robot, coupled to a Sagian robot arm and will exploit recent advances made in the theory of nucleic acid extraction and highly multiplex DNA amplification.

SA 3 & SA 4. First, traditional methods will be used to measure the genotype of a set of about 100 DNA samples from blood, for each of the 100 alleles on the Risk-Tox Chip. These standardized DNA samples will be then used to validate the performance of the Risk-tox Chip, and to test the approaches chosen for automated sample preparation, hybridization and detection. Secondly, the high throughput automation procedures and the quality of the DNA microarray product will be validated on a twenty-fold larger set of blood samples which will have been archived by the National Center for Toxicological Research and by M.D. Anderson Cancer Center. Special emphasis will be placed on development of the informatics required for organization of very large-scale blood and DNA sample archives and the mathematical methods required to evaluate the predictive power of an allele signature in the very large scale, population based applications which we envision.

Institution: GENOMETRIX, INC., 3608 RESEARCH FOREST DR, STE B7, THE WOODLANDS, TX 77381
Fiscal Year: 1998
Project Start: 30-SEP-98
Project End: 29-SEP-01
ICD: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
IRG: ZHG1

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KWOK, PUI-YAN

Grant Number: R01AG16869
Project Title: METHOD FOR GLOBAL AND TARGETED DISCOVERY OF SNP MARKERS

Abstract: To accomplish the stated goals of the Human Genome Project and to reap the fruits of this major undertaking, the complete sequence of a composite human genome will have to be deciphered by the year 2005 and a genetic map composed of a dense set of single nucleotide polymorphism markers must be developed for complex genetic trait analysis. There is general agreement that the technology is in place for large-scale sequencing but that sequence- ready large-insert clone retrieval will soon become the bottleneck in any significant large-scale sequencing project. Similarly, a new NHGRI initiative will undoubtedly lead to the development of a dense set of SNP markers for the human genome but it is unclear how one can best utilize these markers in population studies that require genotyping thousands of individuals with thousands of markers.

In this proposal, we plan to develop two simple, flexible, homogeneous methods for DNA analysis based on fluorescence polarization detection. Specifically, we aim to (1) develop a four-color DNA genotyping method based on fluorescence polarization detection and primer extension reactions; (2) develop a multi-color PCR detection method based on fluorescence polarization detection and the 5'-nuclease "TaqMan" assay for both library screening and for allelic discrimination.

Once developed, these methods will facilitate clone recovery for sequencing projects and for flexible SNP genotyping in population studies. As such, they will help define the genetic factors associated with common diseases with complex inheritance patterns and the relationship between genetic and environmental influences on human health and disease.

Institution: WASHINGTON UNIVERSITY, 660 SOUTH EUCLID AVE., ST. LOUIS, MO 63110
Department: INTERNAL MEDICINE
Fiscal Year 1999
Project Start: 01-APR-99
Project End: 31-MAR-02
ICD: NATIONAL INSTITUTE ON AGING
IRG: ZHG1

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KWOK, PUI-YAN

Grant Number: R01EY12557
Project Title: NEW METHODS FOR HIGH THROUGHPUT GENOME ANALYSIS

Abstract: To accomplish the stated goals of the Human Genome Project and to reap the fruits of this major undertaking, the complete sequence of a "composite" human genome will have to be deciphered by the year 2005 and a genetic map composed of a dense set of single nucleotide polymorphism markers must be developed for complex genetic trait analysis. There is general agreement that the technology is in place for large-scale sequencing but that sequence- ready large-insert clone retrieval will soon become the bottleneck in any significant large-scale sequencing project. Similarly, a new NHGRI initiative will undoubtedly lead to the development of a dense set of SNP markers for the human genome but it is unclear how one can best utilize these markers in population studies that require genotyping thousands of individuals with thousands of markers.

In this proposal, we plan to develop two simple, flexible, homogeneous methods for DNA analysis based on fluorescence polarization detection. Specifically, we aim to (1) develop a four-color DNA genotyping method based on fluorescence polarization detection and primer extension reaction; (2) develop a multi-color PCR detection method based on fluorescence polarization detection and the 5'-nuclease "TaqMan" assay for both library screening and for allelic discrimination.

Once developed, these methods will facilitate clone recovery for sequencing projects and for flexible SNP genotyping in population studies. As such, they will help define the genetic factors associated with common diseases, with complex inheritance patterns, and the relationship between genetic and environmental influences on human health and disease.

Institution: WASHINGTON UNIVERSITY, 660 SOUTH EUCLID AVE., ST. LOUIS, MO 63110
Department: INTERNAL MEDICINE
Fiscal Year: 1999
Project Start: 01-DEC-98
Project End: 30-NOV-01
ICD: NATIONAL EYE INSTITUTE
IRG: ZHG1

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LANDER, ERIC S.

Grant Number: R01HG01953
Project Title: SINGLE NUCLEOTIDE POLYMORPHISMS IN GENE CODING REGIONS

Abstract: The primary aim of the proposed project is to perform a large-scale survey to identify coding-region single-nucleotide polymorphisms (cSNPs) in 5,000 human genes. The survey will cover approximately 8.5 Mb of sequence on 80 chromosomes (representing a total of about 680 Mb) and is expected to result in the identification of at least 20,000 cSNPs.

The coding region of each gene will be amplified by RT-PCR from 40 individuals and the resulting products will be screened for polymorphisms by two independent methods: (a) high-density oligonucleotide arrays (DNA chips) and (b) denaturing high-pressure liquid chromatography (dHPLC). All genes will be screened by both methods. The use of two screening methods provides high sensitivity to ensure that the vast majority of cSNPs are identified. It also yields a continual cross-check on accuracy, allowing weaknesses of each method to be identified and improvements made.

Based on polymorphism frequencies observed in our preliminary studies, the survey is expected to identify at least 20,000 cSNPs with approximately 45 percent encoding an alteration in amino acid sequence. The cSNPs should provide a valuable resource for studying disease association. To facilitate this, all information (including sequence change, effect on protein sequence, observed allele frequencies, etc.) will be promptly deposited on our web site and in national databases and all cSNPs will be made freely available for research.

Institution: WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH, CAMBRIDGE, MA 02142
Fiscal Year: 1999
Project Start: 10-MAR-99
Project End: 28-FEB-02
ICD: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
IRG: ZHG1

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MYERS, RICHARD M.

Grant Number: R21DC04090
Project Title: MISMATCH ENRICHMENT METHOD FOR DISCOVERING HUMAN SNPS

Abstract: Single nucleotide variation occurs throughout the human genome and contributes to the remarkable phenotypic variation present in the human race. For studying this variation, particularly with an aim to identify genes involved in common, multigenic diseases, very large numbers of single nucleotide polymorphisms (SNPs) must be identified and mapped in the human genome. This proposal aims to develop a mismatch enrichment method that will increase the efficiency with which SNPs can be discovered in any complex genome. In this method, 300-base pair restriction enzyme-digested fragments representing about 5% of the human genome will be isolated from DNA of four unrelated individuals. Heteroduplexes will be formed between the different alleles in this mixture of DNA by denaturation and rehybridization. Those fragments containing a mismatch, estimated to be about one out of seven fragments, will be purified from the non-mismatched fragments by binding to MutS protein, which is involved in mismatch repair in E. coli. The DNA fragment population enriched for mismatches will be cloned into a plasmid in E. coli under conditions that maintain both alleles in each resulting bacterial colony. Four-color fluorescent sequencing will be performed from each colony, such that the DNA sequence of the 300 bp fragment, as well as the position and composition of the two alleles, will be determined. For those DNA segments containing a SNP, PCR primers will be designed so that the fragment can be specifically amplified from total human genomic DNA. During the two year project efficacy of this method will be tested by discovering and sequencing approximately 1,000 such SNPs, and analyzing about 100 of these further by determining their map positions in the human genome and their allele frequencies in ten unrelated people.

Institution: STANFORD UNIVERSITY, STANFORD, CA 94305
Department: GENETICS
Fiscal Year: 1998
Project Start: 30-SEP-98
Project End: 29-SEP-00
ICD: NATIONAL INSTITUTE ON DEAFNESS & OTHER COMMUNICATION DISORDERS
IRG: ZHG1

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NICKERSON, DEBORAH A.

Grant Number: R01AI45279
Project Title: FINDING AND GENOTYPING SNPS BY AUTOMATED SEQUENCE ANALYSIS

Abstract: Single nucleotide substitutions and small unique base insertions or deletions are the most frequent form of DNA polymorphism and disease- causing mutation in the human genome. Therefore, it is important to develop highly accurate and efficient methods to identify and type these changes in populations. In fact, our ability to evaluate the relevance of specific variations with regard to phenotype will likely hinge on these issues. The goal of this project is to increase the speed and accuracy of automated approaches for identifying and typing DNA variations by improving base-calling software for automated sequencers. We will focus the development of this software around a project designed to scan the major human T cell receptor loci for sequence variation. Since T cells play a central role in the generating and regulating an immune response, a greater understanding of the natural variation in these genes may provide new insights into differences in immune function among human populations particularly with regard to resistance to infection or cancer, or in terms of susceptibilities to disorders such as autoimmune diseases or immune mediated hypersensitivities. Lastly, this project focuses on the development of automated DNA sequencing as a highly sensitive approach that can be applied not only identifying DNA polymorphisms and mutations in T cell receptor genes but also broadly to any other gene for genetic and disequilibrium mapping, DNA diagnostics (genetic and infectious diseases), tissue typing, and forensic testing.

Institution: UNIVERSITY OF WASHINGTON, 3935 UNIVERSITY WAY, SEATTLE, WA 98195
Department: MOLECULAR BIOTECHNOLOGY
Fiscal Year: 1999
Project Start: 01-DEC-98
Project End: 30-NOV-01
ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
IRG: ZHG1

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NOLAN, JOHN P.

Grant Number: R01RR14101
Project Title: HIGH THROUGHPUT SNP DISCOVERY AND SCORING WITH FLOW CYTOMETRY

Abstract: Analysis of DNA sequence variation is important for identifying disease related genes and diagnosing disease susceptibility. The most common type of genetic variation is the single nucleotide polymorphism (SNP), which may occur as frequently as 1 in every 1000 DNA bases. The goal of this proposal is to develop, validate, and demonstrate new approaches to the discovery and scoring of single nucleotide polymorphisms (SNPs). The new methods will take advantage of a versatile and widely available measurement platform, flow cytometry, to provide rapid and sensitive sample analysis without wash steps. De novo SNP detection will be achieved using an immobilized mismatch binding protein to bind fluorescently labeled heteroduplex DNA to microspheres, which will then be analyzed by flow cytometry. We will evaluate different mismatch binding proteins, immobilization approaches, and labeling strategies to develop a homogeneous assay compatible with multiplexed analysis by flow cytometry. The optimized assay will be sensitive, rapid, and scaleable to scan amplified genomic sequence for SNPs at rates of greater than 1 megabases/day.

SNP scoring will be performed using microsphere-based minisequencing in which primers will be extended with fluorescent dideoxynucleotides using polymerase, and then captured on beads and analyzed by flow cytometry. By employing a unique scheme of capture sequences to address differently dyed microspheres, we will be able to simultaneously type dozens, and potentially hundreds, of SNPs from a single genomic sample in 1-2 minutes.

We will validate and demonstrate these new approaches in collaboration with laboratories currently using conventional technologies to discover and score SNPs for use in disease diagnostics, pharmacogenetics, and mapping and linkage studies. Both our SNP discovery and scoring methods will be compatible commercial flow cytometers present in most universities, research institutes, and clinical diagnostic laboratories.

Institution: UNIVERSITY OF CALIFORNIA, LOS ALAMOS NATIONAL LABORATORY, LOS ALAMOS, NM 87545
Fiscal Year: 1998
Project Start: 30-SEP-98
Project End: 29-SEP-01
ICD: NATIONAL CENTER FOR RESEARCH RESOURCES
IRG: ZHG1

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OLSON, MAYNARD

Grant Number: R01ES09909
Project Title: METHODS FOR SINGLE NUCLEOTIDE POLYMORPHISMS

Abstract: This RFA calls for 30,000 to 300,000 genetic markers, based on single-nucleotide-polymorphisms (SNPs), to be created over the next few years. In response, we propose a pilot program that we believe can be scaled up to a whole-genome level, and which will provide a particularly important category of SNPs: those occurring in cDNA sequences (cSNPs). Many cSNPs can be extracted from the EST databases and we will exploit these as much as possible. However, we will also fill in the gaps in the databases, which come in two forms. For many of the less abundantly expressed genes, not many individuals have been sampled; and many genes are incompletely covered by ESTs. So, we will scan for cSNPs along the entire length of selected cDNA sequences, across a sample of 25 ethnically diverse individuals, deriving the full-length consensus cDNA sequence when one is not already available. Our cSNP discovery process will be sequence-driven. Although this is probably the most expensive approach, it is also the most comprehensive, as it ensures that nearly all common cSNPs will be found. Such a thorough approach is justifiable as most common cSNP are likely to be useful for the population-based association studies that are being planned in the growing efforts to understand genetically-complex diseases. Over the course of this 3 year grant, we will re-sequence 500 genes and create markers for all the common cSNPs that are found in this combination of new sequence data and existing EST data. We will create cDNA resources to better sample the full length of the cDNA sequence. We will evaluate a scoring technology (TDI) that has the potential to be arrayed. And we will develop software to facilitate the process of cSNP discovery, marker creation, and TDI scoring.

Institution: UNIVERSITY OF WASHINGTON, 3935 UNIVERSITY WAY, SEATTLE, WA 98195
Department: MOLECULAR BIOTECHNOLOGY
Fiscal Year: 1998
Project Start: 30-SEP-98
Project End: 29-SEP-01
ICD: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
IRG: ZHG1

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TON, CARL C.

Grant Number: R01NS38754
Project Title: DISCOVERY OF CSNPS IN NICOTINIC ACETYLCHOLINE RECEPTORS

Abstract: The goal of this pilot-scale project is to exhaustively discover and score for coding single nucleotide polymorphisms (cSNPs) within the family of genes that encode subunits of the neuronal nicotinic acetylcholine receptor (nAChR). The family comprises eleven known members: eight alpha-type subunits (alpha2 - alpha9) and three beta subunits (beta2 - beta4). Members of this class of pentameric ligand-gated Ca channels are known or suspected of being involved in a wide range of human disorders ranging from epilepsy and schizophrenia to nicotine addiction and auditory dysfunction. Thus a comprehensive set of cSNPs for these genes would greatly assist in future genetic and/or biological studies on human disease, or of gene/environment interactions.

The specific aims of the project are: (1) gene structure: determination of the genomic organization (e.g. putative promoter region, intron/exon boundaries) of the 11 receptor genes, hence to develop robust PCR reactions for the amplification of all the exons and their flanking splice sites, as well as promoter regions. (2) Discovery of cSNPs: this will be accomplished through fluorescence labeled dye-terminator and gel-based resequencing of the PCR amplified exons. The PCR amplified exons will be derived from the DNA of 400 individuals drawn from the panel designated as the Resource for the Discovery of SNPs. To maximize the efficiency of single-pass sequencing in SNP detection, the base-calling program PolyPhred will be used to assist in the identification of heterozygous polymorphic sites. This measure will also allow a degree of automation and quality assessment of the SNP detection process at the level of each sequence read.

Institution: UNIVERSITY OF WASHINGTON, 3935 UNIVERSITY WAY, SEATTLE, WA 98195
Department: MEDICINE
Fiscal Year: 1998
Project Start: 30-SEP-98
Project End: 31-AUG-01
ICD: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
IRG: ZHG1

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WEBER, JAMES L.

Grant Number: R01HL62681
Project Title: HUMAN DIALLELIC INSERTION/DELETION OF POLYMORPHISMS

Abstract: Human DNA polymorphisms find application in many areas of research and clinical testing, particularly in the linkage mapping of genes responsible for disease. Short tandem repeat polymorphisms are currently the markers of choice for these applications, but diallelic polymorphisms will likely eventually replace STRPs because of more efficient analysis on miniaturized DNA arrays (DNA chips). Goals of this research project are first, to develop 1,500 new human diallelic polymorphisms of the short insertion/deletion class, second, to investigate new approaches and to enhance existing approaches for the analysis of the insertion/deletion polymorphisms on flow through DNA genosensors, and third, to gradually introduce the chip-based analysis of the diallelic polymorphisms into the genotyping factory at Marshfield. Diallelic short insertion/deletion polymorphisms have a number of advantages over base substitution polymorphisms, especially that of improved hybridization discrimination between alleles. New, putative insertion/deletion polymorphisms will be identified by comparing publicly available overlapping genomic and coding DNA sequences. Putative polymorphisms will be confirmed by laboratory typing. Allele frequencies will be measured in groups of individuals with American, European, African, and Asian ancestry. Drs. Beattie and Doktycz at Oak Ridge National Laboratory have pioneered work on novel, porous DNA chip materials, particularly capillary channel glass. The porous chips support increased hybridization reaction kinetics compared to flat chips. Protocols will be enhanced for production of chips, for hybridization of DNA targets to the chips, and for detection of the hybridized DNA. A number of novel polymorphism analysis methods that bypass the requirement for PCR amplification of hybridization targets will be explored. Instrumentation for chip arraying and reading will be improved. Finally, DNA chips for typing short insertion/deletion polymorphisms will be gradually introduced into the genotyping factory at Marshfield to supplement STRP typing in linkage analysis of disease genes.

Institution: MARSHFIELD CLINIC, 1000 N OAK AVE, MARSHFIELD, WI 54449
Fiscal Year: 1998
Project Start: 30-SEP-98
Project End: 31-AUG-01
ICD: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
IRG: ZHG1

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Last Reviewed: May 21, 2012