Topic 10 - Microbial Genomics

Question 1

genome meaning

Answer 1

• entire complement of genetic info, incl genes, regulatory seq, non-coding seq

Question 2

genomics meaning

Answer 2

• discipline involving mapping, sequencing, analyzing, and comparing genomes

Question 3

functional genomics meaning

Answer 3

• assigning function to unknown genes by analyzing biochemical and physiological effects of mutants

Question 4

sanger/dideoxy DNA sequencing 3 main steps, explanation

Answer 4

cloning a gene fragment of interest
DNA synthesis
gel electrophoresis

DNA pol needs free 3’ OH to continue adding bases
- adding dideoxynucleotides terminates synthesis w/ a labeled end-point nucleotide
- gel electrophoresis separates fragments and detects which labeled nucleotide is on end of fragment, providing a seq!

Question 5

automated methods using fluorescent labels instead of radioactive labels are safer, cheaper, and easier; how long seq are obtained in hours? how are longer seq obtained?

Answer 5

seq of 700-1000 bases obtained in hours

longer seq obtained by primer walking (using repeated rounds of sequencing with primers complementary to the end of last sequences segment)

Question 6

shotgun sequencing (explanation, how are gaps closed?

Answer 6

• attempting to sequence entire genome in one set up
• DNA fragments are sheared, cloned possibly, then sequenced
  – software puts seq together (may need 10x total genome coverage to be successful)
• gaps closed by primer walking

Question 7

high-throughput sequencing (5 types)

Answer 7

• AKA “next-generation sequencing”

incl:
pyrosequencing
- detects nucleotide additions to end of a DNA strand by production of light by luciferase (SHORT READS), obsolete

Ion Torrent
- like pyrosequencing but detects nucleotide additions to end by pH change (protein released (SHORT READS)

Illumina
- industry standard (SHORT READS), most common for genome sequencing.
- fluorophores added to template strands on glass array (high resolution fluorescence scan)

Pacific Bioscience (“PacBio”)
- LONG READS, recording fluorophores incorporation in real time, high error rates

Oxford Nanopore (nanopore sequencing)
- passes DNA strands through massively paralleled “nanopores”, recording current disruptions that are specific to each DNA base
- LONG READS, high error rates

Question 8

bioinformatics

Answer 8

• use of computational tools to analyze, compare, assemble, and store DNA & protein seq
• annotation of genomes helps researchers identify OPEN READING FRAMES (ORFs)
  – ORFs allow us to better determine start and stop for a gene
• sequencing speed and cost are good but only gives raw info -> need functional genomics

Question 9

transcriptomics

Answer 9

transcriptome: collection of transcribed mRNA molecules in a cell
- a library of expressed mRNA of a cell can be formed as a cDNA library using reverse transcriptase
- can be sequenced directly or analyzed by microarrays

• RNA sequencing
  – performed by conversion of mRNA -> cDNA through reverse transcriptase
  – cDNA then sequenced v quickly

Question 10

microarrays

Answer 10

• a method for examining transcriptional activity of all genes in a cell simultaneously
• probe DNA fragments are amplified by PCR and placed on a glass slide in a known pattern
• total cell mRNA converted to cDNA by reverse transcriptase, labeled with fluorescent molecule, and passed over the microarray slide
• more intense the light, the more cDNA is present (quantitative measure)
• ssDNA hybridize with ss probes (which represents a gene on microarray)

Question 11

microarray uses (5)

Answer 11

• global gene expression
• expression of certain gene classes under diff conditions - REGULONS
  – incl mutant strains
• expression of genes with unknown function
  – gives clues to function
• comparison of gene content in related organisms
• species identification

Question 12

proteomics

Answer 12

proteome analysis
- proteome: collection of expressed proteins in a cell
- can be studied by multiple methods:

2D-polyacrylamide gel electrophoresis (PAGE_
Mass spectrometry
X-ray crystallography
Nuclear magnetic resonance (NMR)

Question 13

2D-PAGE

Answer 13

allows separation of proteins on a gel based on:

• isoelectric point (charge on surface); pH where protein has no charge)
  -> 1D differentiation across
• mass
  -> 2D differentiation down

Question 14

mass spectrometry

Answer 14

• can determine AA seq of polypeptides from 2D-PAGE

(isolate protein from 2D gel, digest with protease to smaller peptides, determine molecular weight by mass spectrometry, search database)

Question 15

3D shape of proteins (2 methods)

Answer 15

X-ray crystallography
- X-ray beam shot at crystallized protein
- diffraction pattern used to discern protein shape

Nuclear magnetic resonance (NMR)
- measures distances between atomic nuclei
- can measure proteins in solution
- limited to proteins of ~30kDa

Question 16

metagenomics

Answer 16

• involves construction and analysis of gene libraries from DNA extracted directly from complex microbial communities (and analyzing it all together)
• e.g., in acid mine drainage, deep sea thermal vents, wastewater treatment)

Question 17

metagenomics steps (general)

Answer 17

• obtain DNA from area of interest
• sequence
• analyze
  (eliminate known seq to find new genes, potentially from new organisms)