Study guide Chapters 14 15 Please also refer to lecture slides notes and homework questions What is WGS The difference between traditional WGS and next generation WGS Determining gene sequence by breaking DNA into small fragments and lining up the fragments based on where they have overlapping sequences These overlapping sequences are known as contigs Traditional methods such as Sanger dideoxy sequencing take place in a bacteria or a living host Next generation WGS uses cell free reactions without cloning in a live host Machines isolate and sequence the dna fragments then using cameras and software they determine the sequence by monitoring reactions in very small volumes The concepts of bioinformatics genomics and other omics Bioinformatics the analysis of the information content of entire genomes Analyzes number of genes gene products location number binding sites Comparative genomics considers the genomes of closely and distantly related species for evolutionary insight and enables conserved sequences to be used as a guide to analyzing gene function Functional genomics using a variety of techniques including reverse genetics to understand gene function and interacting genes and protiens What are the steps in obtaining a genomic sequence First the WGS or whole genome shotgun is performed Once all the contigs are lined up a problem arises as there are repeating sequences and gaps between contigs Scientist use paired end reads that span gaps between contigs and produce scaffolds Compare and contrast contigs and scaffolds Contigs and Scaffolds are terms reffering to overlapping DNA when performing WGS Contigs are DNA fragments that have the same sequence They overlap where their sequences are the same Used to determine the order of genome In whole genome shotgun sequencing first the unique sequence overlaps between sequence reads are used to build contigs Paired end reads are then used to span gaps and to order and orient the contigs into larger units called scaffolds Scaffolds or supercontigs are groups of contigs The contigs are separated by non overlapping DNA Describe how pyrosequencing is performed Pyrosequencing is a sequencing method involving small individual beads and wells 1 Single DNA strands are attached to individual beads 2 Each bead goes through PCR to copy and amplify the DNA 3 Each bead is placed in a separate well with DNA polymerase and primers When a nucleotide binds to the DNA a light is given off Scientist use the emitted light to determine the sequence of DNA What are ESTs What is the relationship between ESTs and cDNAs EST expressed sequence tags they are short cDNA sequences that code for the 5 and 3 ends of the cDNA They are compared to genomic DNA and cDNA and are used to determine the ends of cDNA sequences Give three examples of DNA binding sites for proteins Intron 5 and 3 splice sites Promoter binds RNA polymerase Translation initiation site Ribosome binding site The concepts of orthologs paralogs and homologs Homologs are the same gene in different organisms When comparing genomes of different organisms scientist look at closely related genes called homologs These homologs have similar sequences and are identified by comparing their sequences Orthologs are homologs that evolved from a common ancestor Paralogs are homologs that are related by gene duplication in a genome Reverse genetics vs forward genetics Reverse Genetics An experimental procedure that begins with a cloned segment of DNA or a protein sequence and uses it through directed mutagenesis to introduce programmed mutations back into the genome to investigate function Forward Genetics using a specific phenotype or disease and using cross breeding and statistics to determine which gene is causing this phenotype What is the RNAi technique How does it affect gene expression RNAi technique is using double stranded RNA sequences that match a specific mRNA sequence in the cell and degrade those that match it It stops gene expression for a specific gene What is yeast two hybrid system The CHIP technique Yeast two hybrid system detects physical interactions between two proteins Using the function of the Gal4 scientist have a way of determining if two genes will interact Gal4 is composed of two separate domains and will only express genes if both the domains are together Scientist bind a test protein to each domain of the Gal4 If the proteins interact the Gal4 will be activated and the gene will be expressed If the proteins don t interact the gene will not be expressed Scientist use the gene expression to determine if the two proteins interacted CHIP technique is used to determine if a specific protein will bind to DNA and where it may bind Essentially scientist put proteins and specific chromatin together and analyze their interactions Describe how McClintock discovered transposable elements McClintock discovered that chromosome 9 of the corn she was analyzing broke frequently She found out that there are two factors that determined if the chromosome would break or not There s a dissociation factor located on the chromosome called Ds and an activation factor Ac When both of these factors were present the chromosome would break and cause different phenotypes to be expressed Compare and contrast the transposable elements in prokaryotes and eukaryotes Transposable Elements in Prokaryotes There are two broad types of transposable elements in bacteria 1 Short sequences called Insertion sequence IS elements that can move themselves to new positions but do not carry genes other than those needed for their movement These IS can insert themselves in genes and disrupt there expression 2 Longer sequences called transposons that not only carry the genes they need for their movement but also carry other genes Transposable Elements in Eukaryotes 1 Class 1 retro transposons Much like retroviruses as discussed below These transposons start off as a gene that codes for a specific mRNA sequence This mRNA sequence is then transformed back into DNA via Reverse Transcriptase This newly synthesized DNA strand is exactly the same as the DNA it originally came from except it s a lone segment of DNA called a Ty element This Ty element can insert itself into another chromosome 2 Class 2 DNA transposons Much like the transposons found in prokaryotes these transposons are the actual DNA sequences that break off from their chromosome and insert themselves in a new location These transposons are characterized by P elements
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