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Iden6fying genes their regulatory regions and

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Unformatted text preview: cm generally) that binds some reagents and lets other reagents flow over it, modifying the bound reagents •  Flowing reagents can then be washed off and a new reac6on cycle can occur –  Don’t worry about ABI Solid squencing – too complicated •  One or more ques6ons on the final exam will be from content in this video accuracy ma1ers •  sequencing errors look like rare muta6ons •  in a large dataset, many rare single nucleo6de variants are not real SNPs –  “singletons” •  need repeat sequencing (oien with Sanger) to confirm Genomic Annota6on • The first task postsequencing in any genome project is annota6on. Iden6fying genes, their regulatory regions and transcripts. • Essen6ally, this is iden6fica6on of the func6onal units of the genome. • This can be done empirically (ESTs) or using computer algorithms. Varia6on in Genomes • Human beings are, for example 99.9% gene6cally iden6cal. • The remaining 0.1% corresponds to ~ 3 million base pairs. • Muta6ons are rare – the majority of differences are from polymorphisms, alleles, segrega6ng in the popula6on. Varia6on in Genomes • 95% of varia6on is in the form of Single nucleo6de polymorphisms (SNPs). • The vast majority of SNPs have no effect. A rare few have overt phenotypic effect, some others act addi6vely influencing disease suscep6bility or drug response. • The remaining 5% are simple sequence repeats – “microsatelites”. Varia6on in Genomes • A map of the BRCA1 gene from humans showing all known SNPs • muta6ons in this gene, and another gene BRCA2, are associated with a higher than average risk of breast cancer • Studies using yeast indicate that BRCA1 and 2 are involved in DNA break repair. cDNA Microarrays • PCR amplified cDNA (ESTs) are spo1ed at high density (10- 50 spots per mm2) on a glass slide. • Current arrays include ~10,000 ESTs. • Differen6ally Labeled cDNA from treatment groups is then hybridized to the plate. • The color of the spots indicates the group in which that transcript is expressed. • The intensity of the signal at a given spot reflects the amount of that transcript in the sample(s). Cell type 1 - red label Cell type 2 - green label Yellow = bo...
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