Module 1

Nucleotide

Structural unit for DNA and RNA

Nucleotide: Nitrogenous Base

Purines
Pyrimidines

Nitrogenous Base: Purines

PURE AGriculture
Adenine
Guanine

Nitrogenous Base: Pyrimidines

PYRamids can CUT you
Cytosine
Thymine (Uracil)

Nucleotide: 5C Sugar

deoxyribose
ribose

Nucleotide: Phosphate Group

attached to sugar
1 phosphate backbone

Mononucleotide

Max 3 phosphate groups

Nucleoside

nucleotide with no phosphate group

ATP

energy source
2 high energy phosphate bonds
consumed in endergonic processes
generated in exergonic processes
GTP: similar

DNA Structure: Double Helix

2 antiparallel strands (opposite directions)
Nitrogenous bases inside
Sugar phosphate backbone outside

DNA Structure: H Bonds

Between nitrogenous bases
A and T: 2
C and G: 3

DNA Structure: Phosphate Backbone

phosphodiester bonds
phosphate + hydroxyl on 3'

RNA Structure

single stranded
U instead of T
ribose (extra OH, more polar)

Central Dogma

Convert DNA to RNA to functional protein

Central Dogma 1: Replication

DNA helicase unwinds DNA
Single strand binding proteins keep DNA unwound
RNA primase adds RNA primers to DNA
DNA polymerase replicates leading strand from 5' to 3'
Read parent 3' to 5'
DNA polymerase replicates lagging strand from 3' to 5'
Okazaki fragments
Produce 2 strands of DNA

Central Dogma 2: Transcription

RNA polymerase transcribe DNA into RNA
mRNA for protein template
RNA polymerase synthesize from 5' to 3'
Read DNA from 3' to 5'
mRNA transported out of nucleus into cytosol

Central Dogma 3: Translation

Synthesize proteins
Function depends on amino acid sequence
3 nucleotide codon for 1 amino acid
tRNA with anticodon transport amino acid
rRNA reads codons
Small subunit binds mRNA
Large subunit contains tRNA sites

Translation 1: Initiation

Initiator tRNA recognizes start codon AUG
Enter P site

Translation 2: Elongation

Aminoacyl-tRNA enters A site
Amino acid added to COOH end of peptide (bond)
Ribosome moves from 5' to 3'
tRNA with peptide chain enters P site
Spent tRNA ejected from E site

Translation 3: Termination

Reach stop codon (UGA, UAG, UAA)
Protein released

Mutations

Permanent changes in nucleotide sequence

Polymorphisms

Non-harmful mutations
Allele variations

Point Mutation

Single nucleotide change

Silent Point Mutation

No amino acid change
No protein sequence change

Missense Point Mutation

Change amino acid

Nonsense Point Mutation

Premature stop codon
Shorten protein

Frameshift Point Mutation

Insert/delete nucleotide
Change amino acids beyond mutation location

Gain Functional Changes

increase protein function

Lose Functional Changes

decrease protein function

Dominant Negative Functional Changes

reduce function of non-mutated allele proteins

Electrophoresis

Separate molecules by size in gel
Larger fragment = shorter travel distance
Transferred to membrane in blots

Blotting

Probe for DNA/RNA and antibodies for proteins
Detect presence of specific sequence/protein

Electrophoresis and Blotting

Determine size and semi-quantitative presence of molecule

Electrophoresis and Blotting 1

Separate fragments on agarose gel

Electrophoresis and Blotting 2

Transfer onto nylon/PVDF membrane

Electrophoresis and Blotting 3

Wash with prehybridization solution containing blocking agent

Electrophoresis and Blotting 4

Wash with probes
Bind to sequence of interest

Electrophoresis and Blotting 5

Detect labels
Band intensity proportional to concentration

SNOW DROP

Southern Blot: DNA
Northern Blot: RNA
Western Blot: Protein

DNA: Restriction Endonucleases

Enzyme recognize and cleave at palindromic sequences
Allow DNA cloning and elimination of foreign DNA

Restriction Endonucleases Sticky End

Staggered cuts
Complementary single stranded sequences
H bonds

Restriction Endonucleases Blunt End

Middle cuts
No H bonds

DNA: DNA Cloning

Amplify specific DNA sequence
Allow DNA mutations and manipulation

DNA Cloning 1

Cleave DNA with restriction endonuclease
Produce sticky ends

DNA Cloning 2

DNA ligase joins DNA and vector with phosphodiester bond

DNA Cloning 3

Insert DNA into vector (plasmid)
Transmit vector into cell

DNA: ASO/DNA Probes

Short labeled nucleotide sequences
Bind complementary DNA to detect single nucleotide differences
Detect sequence of interest in southern blot and PCR

ASO

Allele Specific Oligonucleotides
Probe binds 1 allele version
Detect polymorphisms

DNA: Southern Blots

Determine size and presence of DNA sequence
Detect large changes in DNA (Point mutations creating/destroying restriction sites)

DNA: PCR

Rapid amplification of specific sequence
Cycle in thermocycler
Genotyping for mutations
Allow DNA cloning and DNA microarray

PCR 1: Denature DNA

Separate into single strands

PCR 2: Anneal Primers

Complementary primers attach to DNA

PCR 3: Extend Chain

Taq DNA polymerase synthesize complementary strands
Add dNTPs to 3' of DNA primer

DNA: Genomic Microarray

Compare many genomic DNA samples
Identify DNA sequence changes
Determine mutation/polymorphism location
Cancer diagnosis
Determine presence of genes and alleles

Genomic Microarray 1

Probe genes with fluorescent compounds

Genomic Microarray 2

Expose genes to gene chip
Fluorescence in spot = DNA amount

DNA: CRIPSR-Cas9

Edit genomic DNA in living cells
DNA fragments integrated in CRISPR loci

CRISPR: Cas9

Endonuclease enzyme
Identify and cleave foreign DNA complementary to CRISPR sequence

RNA Techniques

Measure gene expression (mRNA)

RNA: Northern Blot

Detect presence and size of mRNA

RNA: Quantitative PCR (qPCR)

Probe for amount of gene expression
More sensitive and quantitative
Faster (no gel) and safer (no radioactivity)

qPCR 1

Reverse transcription
Make cDNA from mRNA sequence

qPCR 2

Compare cDNA to DNA template with fluorescence
Determine mRNA present

RNA: cDNA Microarray

Measure relative expression of many mRNA gene transcripts

cDNA Microarray 1

Make cDNA from mRNA

cDNA Microarray 2

Probe cDNA with fluorescence

cDNA Microarray 3

Expose cDNA to gene chip
Band colour and intensity = DNA and mRNA concentration

Protein: SDS-PAGE

Detect size and relative amounts of denatured proteins (monomers)

SDS-PAGE 1

Polyacrylamide gel with SDS (anionic detergent) separate proteins
Proteins remain linear and unfolded
Smaller proteins migrate further

SDS-PAGE 2

Compare to DNA ladder
Determine relative protein size

Protein: Antibodies

Probe for specific proteins

Antibodies 1

Attach enzymes/fluorescent tags
Detect specific proteins/macromolecules

Protein: Western Blot

Determine size and relative amount of protein
Combine SDS-PAGE and antibodies

Western Blot 1

Generate band on membrane at antibody
Confirm presence and relative size

Protein: ELISA

Determine quantitative amount of protein (antigens) or antibodies

Direct ELISA 1

Coat wells with antigen

Direct ELISA 2

Test blood for antibodies (immunity)

Sandwich ELISA 1

Coat assay with capture antibody

Sandwich ELISA 2

Add samples with antigen to wells

Sandwich ELISA 3

Detect using enzyme labeled antibody

Western Blot + ELISA

Western: Protein presence and size
ELISA: Protein quantity

Activity Assays

Measure amount of enzyme activity (not just presence)

Continuous Activity Assay

Signal increases over time

Discontinuous Activity Assay

Measure radioisotope labelled products