Microbial Genomics I

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  1. map-directed sequencing approach (nested shotgun)
    • human genome project
    • 1. chop genome into segments of ever decreasing size and put the segments into rough order
    • 2. blast each segment into small fragments
    • 3. read the DNA sequence of each fragment
    • 4. assemble the sequenced fragments according to their known relative order
  2. shotgun cloning of microbial genomes
    • celera genomics
    • 1. shear DNA to obtain fragments of ~same size
    • 2. clone into plasmid vector, transform, and select colonies containing DNA insert
    • 3. sequence clones, representing 7-10x coverage of the bacterial genome
    • redundancy coverage = LN/G
    • - requires high throughput sequencing
    • 4. assembly by computer, looks for overlapping sequences and puts together genomic sequence
    • - computational power
    • - assembly programs
    • 5. finishing, PCR to get sequences in gaps, assemble, repeat until complete
    • 6. authentication - order of known sequence based markers, in silico restriction enzyme digest
    • annotation
  3. high through-put dideoxy method
    • use fluorescent dyes, one for each base
    • use fluor labelled ddNTPs, can have one reaction and run it on a single lane
    • results are analyzed by computer, output is sequence
  4. 454 pyrosequencing
    • very fast - massively parallel
    • DNA broken into ss segments of ~100 bases and attached to microscopic beads
    • DNA amplified by PCR so each bead carries multiple copies of DNA strand
    • fiber optic plate with wells, each well holds one bead
    • four nucleotides are flowed over the plate in sequential order, when PPi released, coupled enzymatic reaction produces light, the nucleotide that produces the light pulse identifies which base was inserted
  5. illumina sequencing
    • massively parallel
    • uses reversible terminator-based method
    • DNA usually 50-150 bases
    • terminator have fluorescent labels
    • terminator is then cleaved
  6. annotation
    conversion of raw sequence data into a list of the genes present in the genome

    • the process:
    • automated with manual curation
    • required the development of sequence analysis and visualization tools
    • ORF (open reading frame) prediction - shine-dalgarno sequence, in frame start and stop codons, size, codon bias, similarity to known proteins, contains protein domains or motif
    • tRNA and rRNA prediction
Card Set:
Microbial Genomics I
2011-11-09 09:01:54
PMB 112 midterm2

general microbiology midterm 2
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