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Methods and compositions for whole genome amplification and genotyping

a whole genome and genotyping technology, applied in the field of gene analysis, can solve the problems of inefficiency of current methods for genome-wide interrogation of snps and other markers, rendering the identification of useful diagnostic marker sets impractical, and affecting the accuracy of results

Inactive Publication Date: 2005-03-10
ILLUMINA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides methods for detecting specific parts of a genome, called typable loci, using nucleic acid probes. These methods involve contacting genome fragments with the probes and detecting the probes that hybridize with the fragments. The probes can be at most 125 nucleotides in length. The invention also includes a method for amplifying genomic DNA and a method for inhibiting the extension of probes during a primer extension assay. The technical effects of the invention include improved accuracy and sensitivity in detecting specific parts of a genome, as well as reduced complexity and improved efficiency of the amplification process.

Problems solved by technology

Thus, diagnosis of such diseases based on genetics is considerably more complex as the number of genes to be interrogated increases.
However, current methods for genome-wide interrogation of SNPs and other markers are inefficient, thereby rendering the identification of useful diagnostic marker sets impractical.
A major limitation to whole genome association studies is the lack of a technology to perform highly-multiplexed SNP genotyping.
However, currently available genotyping methods are cumbersome and inefficient for scoring the large numbers of SNPs needed to generate a haplotype map.

Method used

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  • Methods and compositions for whole genome amplification and genotyping
  • Methods and compositions for whole genome amplification and genotyping
  • Methods and compositions for whole genome amplification and genotyping

Examples

Experimental program
Comparison scheme
Effect test

example i

Whole Genome Amplification Using Random-Primed Amplification (RPA)

[0325] This example demonstrates production of an amplified representative population of genome fragments from a yeast genome.

[0326] Yeast genomic DNA, from S. Cerevisiae strain S228C, was prepared using a Qiagen Genomic DNA extraction kit and 10 ng of the genomic DNA was amplified with Klenow polymerase.

[0327] Several parameters were evaluated to determine their effect on the yield of the Klenow (exo−) random-primed amplification reaction. Amplification reactions were carried out under similar conditions with the exception that one parameter was systematically modified. FIG. 3 shows results comparing amplification reactions carried out at different concentrations of deoxynucleotide triphosphates.

[0328] Following each reaction, the amplified DNA was purified on Montage ultrafiltration plates (Millipore), loaded onto an agarose gel and the DNA quantitated by UV260 reading as shown in FIG. 3A. The amplification yiel...

example ii

Detection of Yeast Loci for a Yeast Whole Genome Sample Hybridized to BeadArrays™

[0330] This example demonstrates reproducible detection of yeast loci for a yeast whole genome sample hybridized to a BeadArrays™ and probed with allele-specific primer extension (ASPE).

[0331] Six hundred nanograms of random primer amplified (RPA) yeast gDNA was hybridized to a locus-specific BeadArray™ (Illumina). The BeadArray™ was composed of 96 oligonucleotide probe pairs (PM and MM, 50 bases in length) interrogating different gene-based loci distributed throughout the S. cerevisiae genome. The amplified yeast genomic DNA was hybridized to the BeadArray™ under the following conditions: Overnight hybridization at 48° C. in standard 1× hybridization buffer (1 M NaCl, 100 mM potassium-phosphate buffer (pH 7.5), 0.1% Tween 20, 20% formamide). After hybridization, arrays were washed in 1× hybridization buffer at 48° C. for 5 min. followed by a wash in 0.1× hybridization buffer at room temperature for mi...

example iii

Whole Genome Genotyping (WGG) of Human gDNA Directly Hybridized to BeadArrays™

[0336] This example demonstrates hybridization of a representative population of genome fragments to an array and direct detection of several typable loci of the hybridized genome fragments. This example further demonstrates detection of typable loci on an array using either of two different primer extension assays.

[0337] SBE-Based Detection

[0338] Human placental genomic DNA samples were obtained from Coriell Inst. Camden, N.J. The human placental gDNA sample (150 ug) was hybridized to a BeadArray™ (Illumina) having 4 separate bundles each containing the same set of 24 different non-polymorphic probes (50-mers). The BeadArray™ consisted of 96 probes to human non-polymorphic loci randomly distributed throughout the human genome. The probes were 50 bases long with ˜50% GC content and designed to resequence adjacent A (16 probes), C (16 probes), G (16 probes), or T (16 probes) bases. DNA samples (150 ug hum...

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Abstract

This invention provides methods of amplifying genomic DNA to obtain an amplified representative population of genome fragments. Methods are further provided for obtaining amplified genomic DNA representations of a desired complexity. The invention further provides methods for simultaneously detecting large numbers of typable loci for an amplified representative population of genome fragments. Accordingly the methods can be used to genotype individuals on a genome-wide scale.

Description

FIELD OF THE INVENTION [0001] The present invention relates generally to genetic analysis and more specifically to amplification of whole genomes and genotyping based on pluralities of genetic markers spanning genomes. BACKGROUND OF THE INVENTION [0002] Most of any one person's DNA, some 99.9 percent, is exactly the same as any other person's DNA. The roughly 0.1% difference in the genome sequence accounts for a wide variety of the differences among people, such as eye color and blood group. Genetic variation also plays a role in whether a person is at risk for getting particular diseases or whether a person is likely to have a favorable or adverse response to a particular drug. Single gene differences in individuals have been associated with elevated risk for acquiring a variety of diseases, such as cystic fibrosis and sickle cell disease. More complex interrelationships among multiple genes and the environment are responsible for many traits like risk for some common diseases, suc...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCC12Q1/6816C12Q1/682C12Q1/6827C12Q1/6848C12Q2565/537C12Q2565/549C12Q2565/513C12Q2537/143C12Q2535/125C12Q2537/163C12Q2525/301C12Q2522/101C12Q1/6837
Inventor GUNDERSON, KEVINSTEEMERS, FRANK
Owner ILLUMINA INC
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