Topic 10: Microbial Genomics Flashcards

1
Q

Sanger sequencing

A
  • Automated methods using fluorescent labels instead of radioactive labels are safer, cheaper, and easier
  • Sequences of 700–1,000 bases obtained in hours

Method:
1. Cloning gene fragments of interest
2. DNA synthesis
— DNA polymerase requires a free 3′ OH to continue synthesis
— Placing dideoxynucleotides into the DNA synthesis mixture terminates the process with a labeled end-point nucleotide
3. Electrophoresis
— Gel electrophoresis separates the fragments and detects which labeled nucleotide is on the end of each fragment, providing a sequence

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2
Q

454 pyrosequencing

A
  • Pyrosequencing detects addition of a nucleotide to the end of a strand of DNA by production of light.
  • Faster and cheaper than Sanger method
  • DNA doesn’t need to be cloned, only fragmented
  • Many sequencing reactions carried out at once in a well format
  • Software analysis evaluates the reaction results
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3
Q

Illumina

A
  • industry standard for short reads (about 150-300 bases)
  • most common for genome sequencing
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4
Q

Explain how primer walking can be used for closing microbial genomes

A
  • Primer walking obtains longer sequences
  • Using repeated rounds of sequencing with primers complementary to the end of the last segment sequenced
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5
Q

Describe how shotgun sequencing can be used for genome sequencing

A
  • Attempting to sequence entire genome in one setup
  • DNA fragments are sheared, possibly cloned, then sequenced
  • Software aligns/assembles sequences (may need 10 × total genome length to do so successfully)
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6
Q

Explain the roles of bioinformatics in the context of microbial genomics

A

Bioinformatics: use of computational tools to analyze, compare, assemble, and store DNA & protein sequences

  • Annotation of genomes helps researchers identify open reading frames (ORFs)
  • ORFs allow us to better determine the start and stop points for a given gene
  • Sequencing speed and cost is incredible today, but it only gives us raw information
  • Functions of genes need to be determined, not just sequences — this is the goal of functional genomics
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7
Q

Explain how a microarray is used for transcriptomics analysis

A

Transcriptome: collection of transcribed mRNA molecules in a cell

A library of the expressed mRNA molecules of a cell can be formed as a cDNA library using reverse transcriptase

Microarrays are a method for examining transcriptional activity of all genes in a cell simultaneously

  • Probe DNA fragments placed on a glass slide in a known pattern
  • Total cell mRNA is converted to cDNA by reverse transcriptase, labelled with a fluorescent molecule, and passed over the microarray slide
  • The more intensely a “spot” on the microarray lights up, the more cDNA is present

Microarray uses
- Global gene expression
- Expression of specific gene classes under different conditions
- Expression of genes with unknown function

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8
Q

Proteome

A

Proteome: the collection of expressed proteins in a cell

  • Can be studied by multiple methods
    – 2D-polyacrylamide gel electrophoresis (2D-PAGE)
    – Mass spectrometry
    – X-ray crystallography
    – Nuclear magnetic resonance (NMR)
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9
Q

List the principles by which 2D-PAGE separates proteins for proteomic analyses

A
  • Extract proteins and run them on a gel
  • allows the separation of proteins on a gel based on:
  • -Surface Charge: migration of proteins in the electric field to the isoelectric point where the net charges on the surface of the protein are neutral
    • Size: large things are near the top, smaller things move down quickly
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10
Q

Compare different ways in which protein structure can be determined: 2D-PAGE

A

Allows separation of proteins on a gel based on
— Isoelectric point (pH where protein has no charge)
— Mass

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11
Q

Compare different ways in which protein structure can be determined: Mass Spectrometry

A
  • Mass spectrometry can identify polypeptides in 2D-PAGE gels
  • Comparison to known protein data can help determine identity
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12
Q

Compare different ways in which protein structure can be determined: X-ray Crystallography

A
  • X-ray beam shot at crystallized protein
  • Diffraction pattern used to discern protein shape
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13
Q

Compare different ways in which protein structure can be determined: Nuclear Magnetic Resonance

A
  • Measures distances between atomic nuclei
  • Can measure proteins in solution
  • Limited to relatively small proteins
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14
Q

Describe the advantages of metagenomics and the methods used for metagenomic analyses

A
  • Involves construction and analysis of gene libraries from DNA extracted directly from complex microbial communities
  • This field is changing our understanding of life on Earth, finding evidence for newly discovered organisms in very diverse and challenging locations
    – acid mine drainage
    – deep sea thermal vents
    – wastewater treatment areas
  • Some of the genes discovered have possible applications in biotechnology and medicine
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15
Q

Describe findings from three case studies involving genomics and metagenomics techniques

A

Case Study 1: Marine Vitamin B12 Producers
- Metagenomic sequencing of aquarium filter DNA revealed a role for the archaea in global production of vitamin B12.
- Mutual exclusion between some marine vitamin B12-producing phyla.

Case Study 2: A New View of the Tree of Life
- Genomic sequences demonstrate the phylogenetic connections between all life on the planet.
- Many uncultivated phyla radiations remain.
- Two-domain tree suggested by ribosomal protein sequences.

Case Study 3: CRISPER
-CRISPR-Cas system serves as a viral defence strategy.
- Cas surveillance complex proteins (i.e., Cas9) cleave viral DNA.
-Other Cas proteins then add cut DNA to chain of spacers, between palindromic repeats.
- CRISPR region transcribed to generate crRNAs that assist Cas9 in defeating future viral infections.
-CRISPR has enormous implications for genetic engineering. This is big, and entirely discovered through genomics.

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