Study Guide Ch 14 Ch 15 CHAPTER 14 WGS p 492 slide 4 Whole Genome Shotgun sequencing Strategy for obtaining assembling the sequence of a genome Based on determining sequence of several DNA segments generate by breaking long chromosome into short chromosome Fundamental difference between traditional WGS and next generation WGS Is how the short DNA segments are obtained prepared chemistry used o Traditional WGS used to sequence 1st human genome relied on clones of DNA in microbial cells employed Sanger dideoxy sequencing technique Begins with construction of genomic libraries collection of short segment of DNA representing entire genome Short DNA segment inserted into chromosome to form vector o Next generation WGS DNA molecules prepared for sequencing by cell free method w o cloning in microbial hosts millions of DNA fragments isolated sequenced in parallel during each machine run advanced fluid handling technology camera software allow detection of sequencing reaction production in small reaction volumes high throughput Pyrosequencing p 494 slide 8 Example of next generation system several hundred thousand to more than 1 million reactions can be run simultaneously 1 DNA template library of ssDNA is made ssDNA strands immobilized on individual beads 2 DNA molecule in library amplified into copies using PCR 3 Each bead put into a well 4 Pyrosequencing DNA sequencing technology that is based on the generation and detection of a pyrophosphate group liberated from a nucleotide triphosphate Bioinformatics p 488 slide 2 The analysis of information content of entire genomes Information content Numbers types of genes Gene products number DNA RNA binding site types that allow production of functional products at the correct time place Genetics p 489 slide 2 Study of genome in entirety Comparative genomics p 504 slide 19 study closely and distantly related species for evolutionary insight uses conserved sequences as a guide to study gene function Analysis of the relations of the genome sequences of two or more species o Decide with genomes to compare must understand phylogeny first evolutionary history o Identification of closely related genes homologs bc of similarities in DNA amino acid sequencing Orthologs gene in different species that evolved from a common ancestral gene by speciation Parlogs genes that are related by gene duplication in genome Functional genomics the use of an expanding variety of methods including reverse genetics to understand gene function and to delineate networks of interacting genes and proteins in biological processes Reverse genetics p 516 slide 25 methods to disrupt the function of a specific gene starts with known cloned DNA mRNA or protein then tries to disrupt this molecule to asses the role of the normal gene product o Approaches introduce random mutation target mutagenesis most precise create phenocopies mutant phenotypes Forward starts with genomic analysis to identify a set of genes as candidates for encoding the biological property of interest then induces mutants targeted specifically to those genes and then examines the mutant phenotypes to see if they affect the property under study Steps in obtaining a genomic sequence p Slide 3 1 Cut many genome copies into random fragments 2 Sequence each fragment 3 Overlap sequence reads 4 Overlap contigs for complete sequence Contigs p 493 slide 4 5 11 Sequences of overlapping reads assembled into units sequences that are contagious or touching group of overlapping cloned segments Scaffolds or supercontig collection of joined together contigs in which there may be unsequenced gaps connected by paired end sequence reads ESTs the relationship between ESTs and cDNAs p 499 slide 15 Large data sets of cDNAs for which only the 5 or the 3 ends or both have been sequenced These short cDNA sequence reads are called expressed sequence tags ESTs Alignment of CDNA and ETS reveal exons or gene ends in genome research where CDNA ETS do not align intron in other words their alignment determines the boundaries of the transcript 3 examples of DNA binding sites for proteins p 498 slide 14 1 Regulatory proteins binds DNA to transcription regulatory elements 2 RNA polymerase binds DNA to promoter 3 Operator What is the RNAi technique How does it affect gene expression p 518 slide 27 RNAi has a natural ability to fight foreign DNA RNAi technique is a powerful method for inactivating specific genes with only knowing the genomic sequence A double stranded RNA is made with sequences homologous to part of the gene under study and is introduced into a cell The RNA induced silencing complex or RISC then degrades native mRNA that is complementary to the double stranded RNA Resulting in complete or considerable reduction of mRNA nullifying expression of that gene without changing the DNA sequence Yeast two hybrid system p 514 slide 23 Studies protein protein interaction Detects physical interaction btw proteins basis of test is transcription activator encode by yeast GAL4 gene uses bait protein target protein to restore function of GAL4 protein that activates a reporter gene LacZ The CHIP technique p 515 slide 24 Chromatin immunoprecipitation assay Studies the protein DNA interactome Isolates the DNA and its associated proteins in a specific region of chromatin 1 Cross link proteins to DNA 2 Break chromatic into small pieces 3 Add antibody to target protein purify 4 Reverse cross links to separate DNA protein amplify sequence CHAPTER 15 Describe how McClintock discovered transposable elements p 526 Slide 3 6 While studying the breakage of chromosome of color kernels maize Maize has a total of 10 chromosomes She noticed that in one strain of maiz chromosome 9 broke very frequently at one site or locus This breakage at one locus was due to two gene factors Ds dissociation located at break and an unlinked genetic factor needed to activate the breakage of chromosome 9 at the Ds locus called Ac activator factor Unusual phenotypes are caused by the Ds transposable element insertion of Ds causes colorless kernel while excision of Ds from C gene by Ac allows color again Transposable elements in maize can inactivate a gene in which they reside cause chromosome breaks and transpose to new locations within the genome Compare and contrast the transposable elements in prokaryotes and eukaryotes p 528 Slide 9 Prokaryotes Insertion sequence IS short sequences elements that can move themselves to new positions but don t carry genes other than those
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