In Silico Mapping of Complex Disease-Related Traits in MiceCrosses of inbred strains of miceMapping genes for complex traits in crosses of inbred mouse strainsChromosome 4 Congenic StrainSingle Nucleotide Polymorphisms: An Introductionhttp://mouseSNP.roche.comGenotyping SNPs on a large scale is a challenge.PowerPoint PresentationSlide 9A demonstration of the utility of SNPs: Two approaches to gene localizationSlide 11The in silico approach: no new genotypingSlide 13Slide 14Comments on the Grupe et al methodWhy does it work? Linkage disequilibriumWhy it works, continued.How might we refine the method?Slide 19Slide 20In Silico Mapping of Complex Disease-Related Traits in Mice Stat 246, Spring 2002, Week 6, Lecture 2Based on the paper with the same title by A. Grupe et al, Science 292 2001: 1915-1918Crosses of inbred strains of miceHere the grey scale denotes a quantitative trait.Mapping genes for complex traits in crosses of inbred mouse strainsI will refer you to the notes from Weeks 3 and 4 of my Stat 260, Spring 1998. There I describe mouse crosses and and linkage mapping methods for analyzing genome scans for localizing quantitative trait loci (genes) to regions of chromosomes. We take up the problem from there.The next step is quite time-consuming, and usually involve creating so-called congenic strains, which can take 1-2 years. After this still more time-consuming work is required: physical mapping and genomic sequencing. Of course you could get lucky sequencing candidate genes, but this cannot be relied upon. We now consider alternatives: in silico and microarrays.Chromosome 4 Congenic Strain1 2 3 419...Strain 11 2 3 4 19...Strain 2Achieved by repeated backcrossing following selection for the region to be retained.Single Nucleotide Polymorphisms: An Introduction “Some people have blue eyes, some are great artists or athletes, and others are afflicted with a major disease before they are old. Many of these kinds of differences among people have a genetic basis--alterations in the DNA that change the way important proteins are made. Sometimes the alterations involve a single base pair (the smallest building block of DNA) and are shared by many people. Such single base pair differences are called "single nucleotide polymorphisms", or SNPs for short. Nonetheless many SNPs, perhaps the majority, do not produce physical changes in people with affected DNA. Why then are genetic scientists eager to identify as many SNPs as they can, distributed on all 23 human chromosomes? Two reasons: Even SNPs that do not themselves change protein expression and cause disease may be close on the chromosome to deleterious mutations.Because of this proximity, SNPs may be shared among groups of people with harmful but unknown mutations and serve as markers for them. Such markers help unearth the mutations and accelerate efforts to find therapeutic drugs. Analyzing shifts in SNPs among different groups of people will help population geneticists to trace the evolution of the human race down through the millenia and to unravel the connections between widely dispersed ethnic groups and races.” From http://snp.cshl.org/about/introduction.shtmlhttp://mouseSNP.roche.com In a variant on the two reasons for collecting SNPs, we will see how databases of mouse SNPs can help us map complex and quantitative traits in mouse. The site above is “A web-accessible database …which contains information across 15 inbred strains and specifics genotyping assays for over 500 SNPs at defined locations on the mouse genome. ….The oligonucleotide primer sequences and conditions for performing allele-specific kinetic PCR genotyping assays are also provided….” We digress to learn a little about the genotyping method, as it is quite similar to so-called Real-Time PCR (RT-PCR), which is an important method of quantifying mRNA. If you don’t know how PCR works, find out: Week 9 of Stat 260, 1998 has some notes on this important technique.Genotyping SNPs on a large scale is a challenge. “We have developed an accurate, yet inexpensive and high-throughput, method of determining the allele frequency of biallelic polymorphisms in pools of DNA samples. The assay combines kinetic (real-time quantitative) PCR with allele-specific amplification and requires no post-PCR processing. The relative amounts of each allele in a sample are quantified. This is performed by dividing equal aliquots of the pooled DNA between two separate PCR reactions, each of which contains a primer pair specific to one or the other allelic SNP variant. For pools with equal amounts of the two alleles, the two amplifications should reach a detectable level of fluorescence at the same cycle number. For pools that contain unequal ratios of the two alleles, the difference in cycle number between the two amplification reactions can be used to calculate the relative allele amounts. We demonstrate the accuracy and reliability of the assay on samples with known predetermined SNP allele frequencies from 5% to 95%, including pools of both human and mouse DNAs using eight different SNPs altogether. The accuracy of measuring known allele frequencies is very high, with the strength of correlation between measured and known frequencies having an r2 = 0.997. The loss of sensitivity as a result of measurement error is typically minimal, compared with that due to sampling error alone, for population samples up to 1000. We believe that by providing a means for SNP genotyping up to thousands of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.” Abstract of S. Germer et al, Genome Res. 10 258 (2000).The basis of allele frequency measurement using kinetic PCR. Shown are amplification growth curves of PCR reactions performed for the ApoB71 polymorphism. A sample was constructed from two DNAs each homozygous for the different alleles of the ApoB71 SNP and contains 5% of allele 1. Equal aliquots of the pool (20 ng of DNA each) were put into PCRs containing either of the two allele-specific primer sets. Four replicate reactions were performed with each primer set (eight PCRs total). The relative allele frequency is determined on the basis of the Ct using freq of allele 1 = 1/(2^Ct +1). Figure 1 of Germer et alThe relationship between Ct and allele
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