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Introduction to microbiology (Multi-locus Sequence Typing (MLST) (Methods,…
Introduction to microbiology
Bacterial Replication
Binary fission
Doubling times can be < 20mins
DNA Replication
Nucleotide base pairing
Adenine to Thymine
Cytosine to Guanine
DNA double helix
DNA replication = semi-conservative
5'-3' extension mediated by DNA polymerase
Process Bacterial Transcription - Gene expression
DNA, via Transcription, to RNA, via Translation, to Protein
Transcription and Translation occur in same compartment in prokaryotes; therefore almost simultaneously.
Polymerase chain reaction PCR
Start = Genomic DNA + Polymerase + Buffer + Pirmers
Denature, Anneal, Extend then Repeat
End = PCR Product
Applications
Pre-requisite for Sanger sequencing
+sequencing = for Gene/ Protein Discovery
16S rDNA + Sequencing = for taxonomy (See intro Lecture)
Housekeeping genes + Sequencing = For MLST
Diagnostics (See diagnostics lecture) - Primers located on unique pathogen specific sequences = +/-
Cloning = for recombinant protein production
DNA Sequencing
Maxam and Gilbert sequencing
Enzymic cleavage after certain bases
Not very user friendly (Use Radioactive)
Developed on to X-ray film to call nucleotide sequence form
Sanger (chain termination) sequencing
Chemically terminate after certain bases
More user friendly. Fluorescence linked produces easy to read traces.
Still used for small sequencing e.g. single genes
No longer used for whole genomes
Requires PCR and primer extension
Machines
Qualities
High throughput/ next generation sequencing
Name is Company/Machine not Inventor/Chemistry
Vastly reduces price of sequencing whole genomes
Examples
454 Pyrosequencing
Discontinued
Uses
sequencing by synthesis
and measure light emission
Illumina sequencing
uses a different
Sequencing by synthesis
and measure fluorescence emission
Pacific Biosciences
Single molecule, Real-Time sequencing uses another
Sequencing by synthesis
and measure fluorescence emission. Single sequence reads are much longer than others
Bacterial Taxonomy and Classification
Genotypic
DNA based: Genomic content (G+C)
DNA-DNA hybridisation
Phylogenetic analyses
Ribosomal rRNA sequences
MLST
Whole genome sequence analyses
Multi-locus Sequence Typing (MLST)
6-8 housekeeping genes - e.g. recA, groEL
PCR amplify bacterial gene and sequence
Deposit sequence data in database
Construct dendrograms of genetic relationships
Mostly 'within' species diversity
Allows pathogen tracking (through typing) as wells phylogenetic analyses
Methods
DNA isolation, then PCR each gene, DNA sequencing, Define Alleles
Whole genome sequence analyses
A range of applications for bacterial classification
WGS now routine used for Diagnostics in Public Health
Allows monitoring transmission - can use a greater part of the genome for MLST-like studies e.g. all ribosomes
For new pathogens enables vaccine design
Extract and compare genes encoding cell surface membranes