Module 3

DNA

Nucleotide polymer

ACGT

Genes

DNA regions encoding for proteins

Codons (3 nucleotides) code for amino acids

RNA

Nucleotide polymer

ACGU

Bacterial Chromosome

Large

Double-stranded

Circular

In nucleoid

Code for growth, metabolism, replication functions

Chromosome Operon

Grouped genes coding for proteins with related functions

Transcribe together on mRNA

Transcription regulation by 1 promoter and operator

Chromosome: Vertical Transfer

Offspring inherit 1 chromosome copy

Binary fission

Chromosome: Horizontal Transfer

Acquire external DNA

Bacterial Plasmid

Mobile genetic material (transfer between cells)

Smaller

Double-stranded

Circular

In cytoplasm

Code non-essential genes (selective pressure from environment)

Fertility Factor Plasmid

Conjugation functions

Horizontal plasmid copy transfer between cells

Resistance Plasmid

Confer antibiotic resistance

Virulence Plasmid

Pathogenicity

Degradative Plasmid

Expand metabolic scope

Catabolize more substances

Col Plasmid

Produce bacteriocins

Peptides killing bacteria

Bacterial Transposons

Mobile genetic material (between DNA strands)

Short

Contain 1 gene

Code for transposase enzyme and antibiotic resistance

Natural Selection Changes

Improve survival

Pass onto offspring

Evolution: Change DNA

Mutation

Recombination

Evolution: New DNA

Conjugation

Transformation

Transduction

Horizontal gene transfer

Mutations

Permanent DNA changes

Caused by mutagens (chemicals, radiation, replication error)

Mutation: Base Substitution

Nucleotide substitution

Impact protein structure and function

Base Substitution: Missense

Codon code for different amino acid

Base Substitution: Nonsense

Codon code for stop codon

Premature end in protein synthesis (truncated)

Base Substitution: Silent

Codon code for same amino acid

No protein change

Mutation: Frameshift

Gain/lose nucleotide

Affect ribosome reading frame

Change amino acids

Deleterious Mutation: Lethal

Prevent replication and mutation propagation

Deleterious Mutation: Unfavourable

Disfavour growth

Deleterious Mutation: Neutral

No impact on growth

Beneficial Mutation

Survival advantage

Mutation propagation to offspring

Ex: Antibiotic resistance

Recombination

Nucleotide sequence exchange between DNA

Foreign DNA incorporate into bacterial chromosome

Beneficial, neutral, and deleterious effects

Horizontal Gene Transfer Methods

Conjugation

Transformation

Transduction

Conjugation

Genetic material between cells

Fertility factor code sex pilus channels in F+ cells

Conjugation 1: Cell Attachment

Cell pilus attach F+ to F-

Pilus contraction forms channel

Conjugation 2: DNA Transfer

Single-stranded fertility factor plasmid DNA transmit through pilus

Conjugation 3: DNA Synthesis

Complementary plasmid strands synthesized in both cells

Fertility factor in both cells

Transformation

DNA uptake from environment

Naturally and artificially competent

Transformation Plasmid DNA 1

Plasmid pass through bacterial envelope into cytoplasm

Transformation Plasmid DNA 2

Enzymes replicate plasmid

Transformation Non-Plasmid DNA 1

Foreign DNA bind bacteria surface

Transformation Non-Plasmid DNA 2

Enzymes digest DNA in cytoplasm

Transformation Non-Plasmid DNA 3

Recombination

Non-digested DNA into chromosome

Transformation Non-Plasmid DNA 4

Chromosome replicate with integrated DNA

Pass DNA to offspring

Transduction

Acquire DNA from viruses (bacteriophages)

Transduction 1: Infection

Bacteriophage inject genetic material into bacteria

Transduction 2: Genome Replication

Viral genetic material replicate and damage bacteria

Produce proteins

Transduction 3: Capsid Assembly and Mispackaging

Viral proteins form capsid

Pack genetic material inside (new bacteriophage)

Transduction 4: Cell Lysis

Release new bacteriophages

Transduction 5: Transduction

Bacteriophage infect new cell

Transduction 6: Recombination

Foreign DNA insert into chromosome

DNA transfer with bacteriophage intermediate

DNA Purification: Cell Lysis

Break open cells

Release DNA and cell contents into solution

Use: detergents, sonication, alkaline solution

Phenol-Chloroform Extraction

Liquid-liquid extraction

Phenol-Chloroform Extraction 1

Form organic layer (phenol and chloroform) and aqueous layer (water)

Phenol-Chloroform Extraction 2

Add DNA mixture

Macromolecule polarity determine layer

Polar DNA in aqueous layer

Phenol-Chloroform Extraction 3

Collect aqueous layer with purified DNA

Ethanol Precipitation

Purify DNA from solution

Based on poor DNA solubility in organic solvent

Ethanol Precipitation 1

Ethanol and sodium acetate added to DNA

Na+ neutralize negative DNA backbone (less soluble)

Ethanol Precipitation 2

Centrifuge and pellet DNA

Separate solution contents

Ethanol Precipitation 3

Remove supernatant with contaminants

Ethanol Precipitation 4

Remove and wash DNA pellet

Solid-Phase Extraction

Spin columns

Based on chemical DNA properties

Selective DNA binding to solid material (wash other molecules)

Solid-Phase Extraction 1

Bind DNA to silica

Add ethanol (decrease solubility) and choatropic salts (prevent H bond between DNA and water)

Solid-Phase Extraction 2

Pass sample through spin column and centrifuge

DNA stick to silica membrane

Solid-Phase Extraction 3

Elute purified DNA with water/buffer

Solid-Phase: Purify Plasmid 1

Resuspension to pellet cells

Solid-Phase: Purify Plasmid 2

Lysis with detergent

Denature DNA

Solid-Phase: Purify Plasmid 3

Neutralization to anneal shorter plasmid DNA

Longer chromosomal DNA cannot renature

Solid-Phase: Purify Plasmid 4

Centrifugation to pellet insoluble precipitate

Solid-Phase: Purify Plasmid 5

Load spin column

Plasmid DNA binds silica membrane

Other components pass through

Solid-Phase: Purify Plasmid 6

Spin column washing with ethanol and buffer

Wash away contaminants

Solid-Phase: Purify Plasmid 7

Elute DNA from spin column with water/buffer

Gel Extraction

Separate molecules by charge and size (electrophoresis)

Gel Extraction 1

Cut DNA section from agarose gel

Gel Extraction 2

Add buffer and heat

Dissolve gel

Gel Extraction 3

Centrifuge solution in spin column

DNA bind to silica membrane

Gel Extraction 4

Wash and elute DNA

UV-Vis Absorbance

Turbidimetry

Measure light absorbance of liquid sample

Concentration Absorbance

Measure DNA amount

Max absorbance at 260 nm

Based on Beer-Lambert law (A=Elc)

Concentration Absorbance Limitations

Cannot measure number of DNA molecules

Cannot determine DNA identity

Purity Absorbance

Max DNA absorbance at 260 nm

Max protein absorbance at 280 nm

Determine DNA and protein amount

260/280 = 1.8 for minimal protein contamination

Volume Absorbance

Measure dilute sample absorbance

Extrapolate concentration of initial sample

Fluorescence-based assays quantify small DNA amounts

Electrophoresis

Separate molecules by charge

Agarose Gel: Nucleic acids

SDS-PAGE: Proteins

Agarose Gel Electrophoresis Process

1. Load DNA into gel with running buffer
2. Apply electric field
3. DNA separate by size
4. Compare to DNA ladder (densitometry)

Electrophoresis: DNA Migration

Electrostatic force pull negative DNA backbone to positive anode

Smaller DNA migrate further

Electrophoresis: DNA Visualization

Stain agarose gel

DNA hydrophobic core become fluorescent

Gel imager produce visualization

Fluorescence intensity proportional to DNA amount

Electrophoresis: DNA Fingerprinting

Restriction enzymes shorten chromosome DNA for electrophoresis

Produce unique band pattern from rescripted DNA

Electrophoresis: Southern Blotting

Short nucleotide probes complementary to DNA

Bind sequence of interest

Southern Blotting Process

1. Separate DNA with agarose gel electrophoresis
2. Transfer and cross-link (UV) DNA to membrane
3. Wash membrane with probe
4. Detect probes

Sanger Sequencing

Determine sequences 500-1000 bp long

Reactions cover different DNA section

Use oligonucleotide primer

FASTA format

Next-Generation Sequencing

Determine all sequences

Read and assemble many short sequences

FASTA format

Next-Generation: Coverage

Amount of overlapping reads

Determine assembled DNA quality

Next-Generation: Contig

Separate non-assembled DNA sequences

Software cannot read

ExPASy Translate Tool

Identify genes in DNA

Identify possible start and stop codons

FASTA format

ExPASy: Frames

Different amino acid sequences

Start at different codons

BLAST

Compare DNA sequences to database

Measure similarity

BLAST: Sequence Alignment

Score sequence match

BLAST: Expect Value

Level of similarity

Low E = high similarity

BLASTn

For nucleotide sequences

Analyze 16S rRNA

BLASTp

For amino acid sequences (proteins)

BLASTp: Identities

Total - blank spaces - positives - gaps

% match

BLASTp: Positive

Amino acids with similar properties

Total - blank spaces - gaps

% match

BLASTp: Blank

Non-similar amino acids