CHAPTER 21 Genomics PPT Notes 4 24 2014 genome complete DNA sequence of an organism o human genome sequence published in 2001 Human Genome Project humans have 20k genes alternative splicing hypothesis some multicellular eukaryotes do not need large s of genes bc alternative splicing makes different proteins from same gene scientists do not know function of more than half genes found in human genome sequences called transcripts of unknown function TUFs b c role in cell unknown genes for miRNAs more common than thought larger proportion of genome transcribed than thought humans have largest genome sequenced to date human chimp genomes are 98 8 identical level of base sequence hypothesis many structural genes in humans chimps identical genes that code for products regulatory genes differ in both species that code for regulatory transcription factors genomics scientific effort to sequence interpret and compare whole genomes o o most basic task to interpret a gene involves identifying which bases constitute genes shotgun sequencing whole genome sequencing approach genome is broken up into a set of overlapping fragments that are sequenced and then put in order 1 2 sonication high frequency sound waves break genome into pieces 160 kilobases long each piece inserted into plasmid called bacterial artificial chromosome BAC i clone using BACs 3 break cloned 160kb fragments into 1kb fragments 4 5 6 7 1kb segments cloned into plasmid inserted into E coli cells replicated makes shotgun clones fragments sequenced analyzed by computer programs computer orders sequences and reconstructs BACs by matching overlapping ends of fragments reconstruct sequence of original DNA by matching overlapping ends of 160kb fragments pyrosequencing method of DNA sequencing based on sequencing by synthesis principle o o relies on detection of phyrophosphate release on nucleotide incorporation rather than chain termination with dideoxynucleotides single strand of DNA sequenced complementary strand synthesized enzymatically one base pair at a time which base added at each step being detected light produced when nucleotide soln complements first unpaired base of template first genome of an organism sequenced Haemophilus influenza o most sequenced organisms cause disease have other interesting biological properties identifying genes is difficult in eukaryotes bc they have many noncoding sequence in their genomes open reading frames ORFs possible genes long stretches of sequence w o stop codons that are flanked by a start and stop codon identified using computer programs that scan genome sequence in both directions looking for sequences typical of promoters operators regulatory sites confirmed as being a gene if its product is homologous to a known gene eukaryotic genomes larger genes contain introns most of genome does not code for product cannot scan for ORFs have noncoding repetitive sequences genes identified using reverse transcriptase to make cDNA version of mRNA portion of the resulting molecule sequenced to produce an expressed sequence tag EST ESTs represent protein coding genes dominated by repeated DNA sequences that occur btwn genes or inside introns do not code for products used by organism o major source of new genes Is duplication of existing genes o o o o o 4 24 2014 o o o o o o o o o organisms w complex morphology behavior do not have large s of genes bacterial and archaeal genomes prokaryotic genomes of various strains of the same prokaryotic species sequenced compared general correlation exists btwn size of bacterial genome metabolic capacities of organism direct linear function of many bacterial genes unknown genetic diversity among bacteria archaea redundancy of genes is common within a genome multiple chromosomes and plasmids common in many bacterial archaeal species a signficiant portion of genome acquired from another species evidence gene similar to genes in distantly related species than closely species when proportion of GC base pairs to AT base pairs in a gene is different from base composition of rest of genome 90 of prokaryotic genome consists of genes lateral gene transfer movement of DNA from one species to another species very common in bacterial archaean species results bc genes carried on plasmids occurs through transformation o o mutation genetic recombination within species not only sources of genetic variation in bacteria and archaea transposable elements segments of DNA that can move from one location in a genome to another cause repeated sequences in the human genome make up 50 of human genome as opposed to protein coding sequences parent line transcription translation into nucleus cDNA copy insertion daughter LINE long interspersd nuclear elements LINEs type of transposable element contains all sequences required to reproduce itself insert into new location in genome only a few are complete potentially active short tandem repeats STRs small sequences repeated down the length of a chromosome o microsatellites simple sequence repeats repeating units of 2 6 bases o minisatellites variable number terminal repeats VNTRs repeating units of 6 100 bases o individuals have many alleles for STRs because of unequal crossover homologs pair up repeats misalign crossing over recombination occur new repeat s created DNA fingerprinting any technique that identifies individuals by unique features of their genomes o microsatellite minisatellite loci vary among individuals they are markers of choice for DNA fingerprinting o process DNA sample acquired from individual PCR done using primers that flank region containing STR region cloned analyzed to determine of repeats present gene family genes very similar to each other in structure function within a species o o genes comprising gene families hypothesized to come from common ancestral sequence through gene duplication gene duplication extra copy of gene added to genome usually results from unequal crossing over during meiosis redundancy of duplicate genes allows one copy to mutate to make new gene w different function or regulation evolution of novel traits original gene functional produces normal product duplicated genes may retain original function and make more product may create a new important gene from mutation altering a protein beneficially may be a nonfunctional pseudogene that is a remnant of the functional copy of the gene but doesn t make a working product CHAPTER 21 Genomics Blue Thread Questions 4 24 2014 A shotgun blast produces
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