Topic 10: Microbial Genomics Flashcards
Sanger sequencing
- 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
454 pyrosequencing
- 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
Illumina
- industry standard for short reads (about 150-300 bases)
- most common for genome sequencing
Explain how primer walking can be used for closing microbial genomes
- Primer walking obtains longer sequences
- Using repeated rounds of sequencing with primers complementary to the end of the last segment sequenced
Describe how shotgun sequencing can be used for genome sequencing
- 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)
Explain the roles of bioinformatics in the context of microbial genomics
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
Explain how a microarray is used for transcriptomics analysis
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
Proteome
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)
List the principles by which 2D-PAGE separates proteins for proteomic analyses
- 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
Compare different ways in which protein structure can be determined: 2D-PAGE
Allows separation of proteins on a gel based on
— Isoelectric point (pH where protein has no charge)
— Mass
Compare different ways in which protein structure can be determined: Mass Spectrometry
- Mass spectrometry can identify polypeptides in 2D-PAGE gels
- Comparison to known protein data can help determine identity
Compare different ways in which protein structure can be determined: X-ray Crystallography
- X-ray beam shot at crystallized protein
- Diffraction pattern used to discern protein shape
Compare different ways in which protein structure can be determined: Nuclear Magnetic Resonance
- Measures distances between atomic nuclei
- Can measure proteins in solution
- Limited to relatively small proteins
Describe the advantages of metagenomics and the methods used for metagenomic analyses
- 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
Describe findings from three case studies involving genomics and metagenomics techniques
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.
