Chapter 201. How Are Complete Genomes Sequenceda. genomes range in size from about half a million base pairs-to several billionb. A single sequencing reaction can analyze only about 1000 base pairs2. Shotgun Sequencing Draw figure 20.2a. A genomes is broken up -into a set of overlapping fragments small enough to be sequencedb. Step for Shotgun Sequencing1. High-frequency sound waves randomly break a fenome into pieces-160 kilobases long2. Each 160-kb piece is inserted into a plasmid-called a bacterial artificial chromosome (BAC)-which is then inserted into E. Choli, creating a BAC library-thus isolating large numbers of each 160 kb fragment3. DNA is broken into fragments again that are 1 kb long4. 1 kb fragments inserted into plasmids and placed into bacterial cells-large numbers of 1 kb fragments available for sequencing reactions5. find regions where diff. fragments overlap6. Assemble all the 1-kb fragments from each original 160-kb fragment -by matching overlapping ends7. Assemble all the 160-kb fragments-by matching overlapping endsc. Point of Shotgun strategy consists of -breaking genome into tiny fragments, sequencing them, -and putting the seq. data back into correct order3. Impact of Next Gen. Sequencing Strategiesa. Pyrosequencing takes place on single DNA fragmentb. In pyrosequencing a genome is sheared into many tiny fragments-which are then separated and sequenced directlyc. Only works w. fragments of about 100 base pairs in length4. Bioinformaticsa. a field that fuses computer science and biology-to manage, analyze, and interpret biological information5. Genomes Being Sequenceda. Haemophilus Influenzae-lives in upper respiratory tract-causes earaches, respiratory tract infections in childrenb. Over 800 genomes have been sequencedc. Most organisms chosen for whole-genome sequencing-cause disease or have other interesting properties6. Which Sequences Are Genesa. the most basic task in annotating or interpreting a genome-is to identify which bases constitute genesb. In eukaryotes identifying genes is much more difficult7. Identifying Genes in Bacterial And Archaeal Genomesa. with 3 codons consisting of 3 bases-3 reading frames are possible on each strand-for a total of 6 possible reading framesb. a long stretch of codons that lacks a stop codon-is a good indication of a coding sequencec. computer programs look for sequences typical of promoters-operators or other regulatory sites-DNA segments identified this way are called open reading frames (ORF)d. similarities bet. genes in diff. species are due to homology-genes similar in structure, location and function are homologous genes8. ID Genes in Eukaryotic Genomesa. Impossible to scan for ORFs in eukaryotic genes-bec. coding regions are broken up by intronsb. to ID eukaryotic genes you must isolate mRNA and turn it back into DNA-then seq. a portion of resulting molecule to make an expressed sequence tag (EST)-use the EST to find the matching seq. in genomic DNABacterial and Archaeal Genomes1. History of Prokaryotic Genomesa. genome sequencer must record what is in a genome-number type, organization of genesb. In bacteria there is correlation bet. size of a genome-and the metabolic capabilities of the organismc. There is a lot of genetic diversity among bacteria and archaea-about 15% of genes in each species' genome are uniqued. Many genes are identical in certain bacteriae. Multiple Chromosomes common in bacteria-some bacteria have linear chromosomesf. many species contain plasmids2. Lateral Gene Transfera. Movement of DNA from one species to anotherb. 2 ways to tell ID genes that were acquired via lateral gene transfer1. a gene is more closely related to distant species that those of related species2. base pairing in particular gene is diff from the rest of the genomec. Plasmids appear to be responsible for lateral gene transferd. Clamydia Trachmomatis has genes that resemble eukaryotic genes it parasitizes -incorporated genes from eukaryotes into itself3. Environmental Sequencing (Metagenomics)a. cataloging all of the genes present in a community of bacteria and archaeab. Research group iin Bahamas collected cells from diff. water depths-and locations and isolated DNA from the samples and sequenced themc. Found alleles that code for proteins -similar to rhodopsin found in cells of human retina-bacterial cells use the same protein to absorb light-to help produce ATP1. Eukaryotic Genomesa. Problems w. sequencing eukaryotic genomes-enormous size of genomes of eukaryotes-coping w. noncoding sequences that are repeated many timesb. Repeated Sequences-repeated DNA seqs that occur bet. genes or inside introns that dont code for products-why do these exist?2. Parasitic and Repeated Sequencesa. 50% of ave. eukaryotic genome -consists of repeated seqs that dont code for product used by cellb. Many repeated seqs derived from Transposable Elements-segments of DNA that are capable of being inserted into new locations-or transposing in a genomec. Transposable elements, unlike viruses, never leave host celld. transposable elements and viruses are parasitic-bec. it takes time and resources to copy them-and they disrupt gene function when they insert in a new location-so they decrease their host's fitness3. How Transposable Elements Work Figure 20.5 Drawa. There are distinct types of transposable elementsb. well studied type is a long interspersed nuclear element (LINE)-found in humans and other eukaryotesc. hypothesized that LINEs evolved from retro-virusesd. Most LINEs in human genome dont function-bec. they dont have promoter or genes for reverse transcriptase or integrasee. Many genomes riddled w. parasitic seqs4. Repeated Seqs and DNA Fingerprintinga. Eukaryotes have several thousand loci called Short Tandem Repeats-small seqs repeated one after another contiguously along part of a chromosomeb. Two classes of STR1. repeating units that are just 1 to 5 bases long-are known as microsatellites of simple sequence repeats2. repeating units that are 6 to 500 bases long-known as minisatellites or variable number tandem repeatsc. microsatellite seqs originated-when DNA polymerase skips or mistakenly adds extra bases during replicationd. minisatellites and microsatellites have so many diff alleles-because of unequal crossover at meiosis 1 ---draw from figure 20.6-when crossover occurs resulting chromosomes have diff numbers of repeats5. Fingerprintinga. Genome of every individual has at least 1 new alleleb. the variation in repeat
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