7.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/031Lecture 28: Polymorphisms in Human DNA Sequences•SNPs•SSRs7.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/032The methods of genetic analysis that you have been learning are applicable to mammals — even to humans. However, we need to combine these genetic principles with an understanding of the physical realities of the human genome. To genetics we will add genomics.Eukaryotic Genes and Genomes= DNA content of a gamete (sperm or egg)genome = DNA content of a complete haploid set of chromosomesH. sapiensM. musculusD. melanogasterC. elegansS. cerevisiaeE. coligenes/ haploidyear sequence completedDNA content/ haploid(Mb)cMChromosomesSpecies116642023N/A4000300280170033005121001803000300019971997199820004,2005,80019,00014,00030,000?30,000?Mb = megabase = 1 million base-pairs of DNAKb = kilobase = 1 thousand base-pairs of DNANote: cM = centi Morgan = 1% recombination2002 draft2001 draft2005 finished?2003 finished7.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/03330003300H. sapiens30001700M. musculus180280D. melanogaster100300C. elegans124000S. cerevisiae5N/AE. colitrue breeding strains?design crosses?generation timeDNA content/ haploid (Mb)cMSpecies30 min90 min4 d2 wk3 mo20 yryes yesyes yesyes yesyes yesyes yesno noLet's add some columns to a table we constructed several lectures back:You might add a column indicating the number of offspring per adult. What are the implications of this table for human genetic studies? Obviously they're difficult.7.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/034• Human genetics is retrospective (vs prospective). Human geneticists cannot test hypotheses prospectively. The mouse provides a prospective surrogate. • Can’t do selections• Meager amounts of data Human geneticists typically rely upon statistical arguments as opposed to overwhelming amounts of data in drawing connections between genotype and phenotype. • Highly dependent on DNA-based maps and DNA-based analysis The unique advantages of human genetics:• A large population which is self-screening to a considerable degree• Phenotypic subtlety is not lost on the observer• The self interest of our species More specifically:7.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/035Let's consider the types and frequency of polymorphisms at the DNA level in the human genome. DNA polymorphisms are of many types, including substitutions, duplications, deletions, etc. Two types of DNA polymorphisms are of particular importance in human genetics today:This means that, on average, at a randomly selected locus, two randomly selected human alleles (chromosomes) differ at about 1 nucleotide per 1000. This implies that your maternal genome (the haploid genome that you inherited from your mother) differs from your paternal genome at about 1 nucleotide per 1000.Similarities and differences: This also implies that the genomes of any two individuals are 99.9% identical. Conversely, the genomes of two randomly selected individuals will differ at several million nucleotides. (Identical twins are a notable exception.)1) SNPs = single nucleotide polymorphisms = single nucleotide substitutionsHnuc=A locus is said to be polymorphic if two or more alleles are each present at a frequency of at least 1% in a population of animals.In human populations:average heterozygosity per nucleotide site = 0.0017.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/036The great majority (probably 99%) of SNPs are selectively “neutral” changesof little or no functional consequence: • outside coding or gene regulatory regions (>97% of human genome) • silent substitutions in coding sequences • some amino acid substitutions do not affect protein stability or function A small minority of SNPs are of functional consequence and are selectively advantageous or disadvantageous.• disadvantageous SNPs selected against --> further underrepresentation7.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/037Affymetrix chip to identify SNPs6000 datapoints, tabular and visual views of the data.Note that only 1500 showing in image on left, a few hundred at most on right.Following slides show…how we visualize dataImage removed due to copyright considerations.7.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/0387.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/039PEDIGREE: DOMINANT TRAIT WITH SUPPRESSOR SEGREGATINGIt looks like we've been lucky. Allele A at SSR37 appears to segregate with HD. But can you be confident that the HD gene is in close proximity to the SSR37 locus, or even that it is on chromosome 4?7.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/0310All normal. C57blackAKRX13/16 normal:: 3/16 tumorsTUMORSNON-TUMORSAAbbaaBBAaBbA-B-aaB-aabbaaB-AKR HAS A GENE THAT SUPPRESSES TUMORSIt looks like we've been lucky. Allele A at SSR37 appears to segregate with HD. But can you be confident that the HD gene is in close proximity to the SSR37 locus, or even that it is on chromosome 4?7.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/03117.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/03127.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/03137.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/03147.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/0315OOHHOHOHOOLactoseHβ(1,4)-Glycoside Linkage1OOHHOHOOH4HgalactoseresidueOOHHOHOHOOCellobioseHβ(1,4)-Glycoside Linkage1OOHHOHOOH4Hglucoseresidueglucose residueCANDIDATE GENELACTOSE7.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/0316LACTOSE TOLERANCELACTASE GENESNP7.03 Lecture 28-30 11/17/03, 11/19/03, 11/21/03172) SSRs = simple sequence repeat polymorphisms = "microsatellites"Most common type in mammalian genomes is16F15E14D13C12B11Anallelesprimer #1primer #2PCRgel electrophoresisGenotype⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯nCA repeat:(CA)n(GT)nAB CD EF AD CFFEDCBA16151413
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