Genomics Review Midterm #2 From topic 5 (Assembling Info from Clones Into Contigs) to the first few slides of topic 9 (eukaryotic genome II, up to pseudogenes). All the material that was covered for BPS 3101 second midterm for 2010.
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GENOMICS REVIEW MIDTERM IITOPIC 5 PART II Assembling Info from Clones Into Contigs 1Chromosome Walking by hybridizationsequence from one clone is used as a probe to screen library of clones to find overlapping onesex Use Clone A1 as a probehybridization on plate of clones A1 F6 and E7On next grid use Clone F6 as probehybridization on plate of clones A1 F6 and B12 thereforeA1B12 E7 F6 A1 overlaps with E7 and F6 and F6 but not A1 overlaps with B12 and E7 overlaps with A1 but not F6NOTE if there are repetitive sequences on one probe that hybridize to many clones instead use short unique sequences mapping close to the end of the clone as a probeprehybridize the repeated sequencesuse DNA complementary to repetitive DNA to hybridize so that only unique sequence can hybr2Chromosome Walking by PCRdesign primers for PCR that are based on the sequence at the end of a clone similar to question 6 on the assignmentuse other clones in library for template DNA will get PCR amplicons with any new clones with that sequencereactions can be carried out as pools for more rapid screening 3Clone fingerprintingto find overlapping clones look for features they have in common example restriction profile fingerprint run clones on gel after restriction digestoverlapping clones are ones with shared restriction fragmentsEXAMPLE Haemophilus genome projectDNA was sonicated and put into plasmid vectorsshotgun sequencing created 140 contigs analyzed sequencing and physical gaps in genomescreened for overlapping clonesreduced to 42 contigsassumed gaps represented genome regions unstable in plasmid vector therefore changed to lambda vectorthe lambda library was probed with oligomers from the contig ends of the plasmid library or used PCR using primer pairs of the first libraryNext generation sequencing technologies 1 Pyrosequencing Pyrosequencing strategy4 enzymes used 1 DNA polymerase 2 apyrase 3 ATP sulfurylase 4 luciferaseSteps dNTPs added sequentiallyIf the right base is added it is integrated into the growing sequence by DNApolymerase After dNTP is added pyrophosphate is releasedATP sulfurylase converts PPi into ATPATP helps luciferase to convert luciferin into oxylucerinlight any other extra ATPs or dNTPs degraded by apyraseMassiveparallel pyrosequencing on beads or chips 1 DNA is sheared2 Adapters are ligated onto the DNA fragmentsAdaptors are two kinds of primers 1 annealing primers and 2 generic primers 3 Beads are added to the fragments which bind to the annealing primer 4PCR amplification is done to have multiple fragments on the same bead 5Each bead is added to a well on a plate 6Pyrosequencing done in each wellAverage read is 250 bp 2 Illumina sequencing parallel microchip 1 DNA is sheared 2Adaptors with sequencing primers are ligated onto the DNA 3The DNA fragments are attached to a chip 4PCR is done via bridge amplification 5The clusters are denatured for singlestranded templates anchored to the substrate 6Sequential sequencing is done using 4 fluorophonelabelled nucleotidesaverage read is 40100 bp3 Single molecule realtime sequencingHelicos Pacific Biosciencesthere is a high cost and long development time to do thisTOPIC 6 How to find a gene within a DNA sequence 1 BACTERIAL GENOMEthere are no introns and genes are tightly packedScan for longest ORF of the 6 possible reading frames to find the longest one between a start and stop codonoften computer program such as Open Reading Frame Finder on NCBI used Problems1 initiation codon used in the organism may not be ATG relatively rare2 deviations from the standard code ex mitochondria use stop codon as Trp but we can change default3 if gene contains introns but if bacterial this is rare4 if there are overlapping genes rare5 fortuitously long stretch before a stop codon and after ATG note usually the longer a reading frame is the less likely it occurred by chance2EUKARYOTIC GENOMElong introns short exons may cause a problem if there is a stop codon within an intronALGORITHMS TO LOOK FOR 1 Exonintron boundary Usually at the boundary of an exon and intron there is an AGGT exonintron but not always the case 2 Codon bias some organisms will use one codon for an amino acid over the other codons available 3 Regulatory sequences look for upstream promoters downstream polyA addition signals though usually very short 4 Homologous sequences in the databank