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1:​ ​DNA,​ ​CHROMOSOMES,​ ​AND​ ​GENETICS

2

2:​ ​TRANSCRIPTION

4

2:​ ​POST-TRANSCRIPTIONAL​ ​MODIFICATION

4

2:​ ​RNA​ ​VS
...
​ ​all​ ​of​ ​the​ ​DNA​ ​found​ ​in​ ​a
body​ ​cell)
● Genomics:​ ​the​ ​genome-wide​ ​analysis​ ​of​ ​gene​ ​structure​ ​and​ ​expression
○ Genomes​ ​can​ ​be​ ​entirely​ ​sequenced​ ​(eg
...
​ ​the​ ​human​ ​genome​ ​is:
○ 21%​ ​LINEs
○ 13%​ ​SINEs
○ 8%​ ​retroviral-like​ ​elements
○ 3%​ ​DNA-only​ ​transposon​ ​“fossils”
○ 3%​ ​segmental​ ​duplications
○ 5%​ ​simple​ ​sequence​ ​repeats
○ 37
...
5%​ ​exons​ ​(protein-coding​ ​regions)
○ 8%​ ​heterochromatin
● The​ ​size​ ​of​ ​a​ ​genome​ ​is​ ​not​ ​directly​ ​related​ ​to​ ​its​ ​biological​ ​complexity
○ The​ ​bulk​ ​of​ ​genomes​ ​are​ ​heterochromatin​ ​(not​ ​sequenced)​ ​and​ ​introns/other
non-coding​ ​parts​ ​of​ ​genes​ ​from​ ​euchromatin
○

Joanna​ ​Griffith​ ​(2017)

Comparisons​ ​of​ ​genomes​ ​among​ ​species​ ​can​ ​suggest​ ​evolutionary​ ​and​ ​functional
relationships​ ​among​ ​genes
--------------------------------------------------------------------------------------------------------------------------●

2:​ ​TRANSCRIPTION
Genetic​ ​information​ ​must​ ​be​ ​stable​ ​for​ ​storage,​ ​but​ ​also​ ​available​ ​to​ ​direct​ ​cellular
processes
○ The​ ​genetic​ ​instructions​ ​carried​ ​by​ ​the​ ​DNA​ ​must​ ​be​ ​transcribed​ ​into​ ​RNA
■ Messenger​ ​RNA​ ​(mRNA)​ ​acts​ ​as​ ​a​ ​“messenger”​ ​to​ ​direct​ ​the
production​ ​of​ ​proteins
● Transcription​ ​produces​ ​an​ ​RNA​ ​molecule​ ​that​ ​is​ ​complementary​ ​to​ ​one​ ​strand​ ​of
DNA
● RNA​ ​is​ ​synthesised​ ​in​ ​a​ ​5’​ ​to​ ​3’​ ​direction​ ​from​ ​a​ ​DNA​ ​template​ ​by​ ​RNA​ ​polymerase
● How​ ​does​ ​transcriptional​ ​machinery​ ​know​ ​where​ ​to​ ​start​ ​and​ ​stop?
○ ATG​ ​start​ ​codon
○ Stop​ ​codons​ ​can​ ​differ
● Initiation​ ​of​ ​transcription​ ​(general​ ​principles):
○ RNA​ ​polymerase​ ​interacts​ ​with​ ​transcription​ ​factors​ ​when​ ​it​ ​binds​ ​to​ ​the
promoter​ ​region​ ​of​ ​the​ ​DNA
○ A​ ​basic​ ​promoter​ ​is​ ​required​ ​for​ ​RNA​ ​polymerase​ ​to​ ​bind​ ​and​ ​initiate
transcription​ ​at​ ​the​ ​appropriate​ ​site
■ Additional​ ​control​ ​sequences​ ​can​ ​determine​ ​when​ ​a​ ​gene​ ​is
transcribed
What​ ​types​ ​of​ ​RNA​ ​are​ ​transcribed​ ​from​ ​DNA?
● Ribosomal​ ​RNA​ ​(rRNA)
○ Forms​ ​part​ ​of​ ​the​ ​ribosome,​ ​catalyses​ ​protein​ ​synthesis
● Messenger​ ​RNA​ ​(mRNA)
○ Encodes​ ​proteins
● Transfer​ ​RNA​ ​(tRNA)
○ Acts​ ​in​ ​protein​ ​synthesis​ ​as​ ​adaptors​ ​between​ ​specific​ ​codon​ ​sequences​ ​on
the​ ​mRNA​ ​and​ ​amino​ ​acids
● Small​ ​RNA​ ​(small​ ​nuclear​ ​RNA,​ ​snRNA)
○ Used​ ​in​ ​pre-mRNA​ ​processing,​ ​transport​ ​of​ ​proteins​ ​to​ ​the​ ​endoplasmic
reticulum,​ ​and​ ​other​ ​cellular​ ​processes
● Micro​ ​RNA​ ​(miRNA)
○ Act​ ​in​ ​regulation​ ​of​ ​gene​ ​expression,​ ​eg
...
​ ​DNA
RNA​ ​has​ ​a​ ​ribose​ ​sugar,​ ​DNA​ ​has​ ​deoxyribose
RNA​ ​has​ ​uracil,​ ​DNA​ ​has​ ​thymine
RNA​ ​is​ ​chemically​ ​more​ ​reactive​ ​than​ ​DNA
○ Ribose​ ​has​ ​two​ ​OH​ ​groups
● RNA​ ​is​ ​less​ ​stable​ ​than​ ​DNA
○ Does​ ​not​ ​last​ ​as​ ​long
■ Can​ ​only​ ​be​ ​used​ ​to​ ​synthesise​ ​proteins​ ​for​ ​a​ ​short​ ​amount​ ​of​ ​time
○ DNA​ ​can​ ​be​ ​stored​ ​and​ ​used​ ​for​ ​many​ ​years
● RNA​ ​is​ ​more​ ​prone​ ​to​ ​mutate​ ​than​ ​DNA
○ Cytosine​ ​deamination​ ​to​ ​uracil​ ​cannot​ ​be​ ​detected​ ​and​ ​repaired​ ​in​ ​RNA
● RNA​ ​is​ ​single-stranded,​ ​DNA​ ​is​ ​double-stranded
● RNA​ ​can​ ​base​ ​pair​ ​to​ ​form​ ​3D​ ​shapes​ ​(such​ ​as​ ​enzymes​ ​with​ ​enzymatic​ ​functions),
DNA​ ​cannot
--------------------------------------------------------------------------------------------------------------------------●
●
●

3:​ ​CRACKING​ ​THE​ ​GENETIC​ ​CODE
The​ ​genetic​ ​code​ ​is​ ​degenerate​ ​and​ ​a​ ​triplet​ ​code
● Degenerate:​ ​a​ ​single​ ​amino​ ​acid​ ​can​ ​be​ ​coded​ ​for​ ​by​ ​a​ ​number​ ​of​ ​different​ ​variations
of​ ​bases
● How​ ​many​ ​bases​ ​correspond​ ​to​ ​one​ ​amino​ ​acid?
○ Four​ ​different​ ​types​ ​of​ ​base​ ​in​ ​nucleic​ ​acids​ ​(A,​ ​C,​ ​T,​ ​G)
○ Twenty​ ​different​ ​types​ ​of​ ​amino​ ​acid​ ​in​ ​proteins
○ 1​ ​base​ ​per​ ​amino​ ​acid​ ​=​ ​only​ ​4​ ​possible​ ​amino​ ​acids
○ 2​ ​bases​ ​per​ ​amino​ ​acid​ ​=​ ​only​ ​16​ ​possible​ ​amino​ ​acids
○ 3​ ​bases​ ​per​ ​amino​ ​acid​ ​=​ ​64​ ​possible​ ​amino​ ​acids
■ Therefore,​ ​there​ ​must​ ​be​ ​3​ ​bases​ ​per​ ​amino​ ​acid​ ​(triplet​ ​code)​ ​and
more​ ​than​ ​one​ ​combination​ ​of​ ​three​ ​can​ ​code​ ​for​ ​one​ ​amino​ ​acid
(degenerate​ ​code)
● Crick,​ ​Brenner​ ​et​ ​al
○ Demonstrated​ ​the​ ​triplet​ ​code
○ Used​ ​the​ ​T4​ ​bacteriophage​ ​which​ ​infects​ ​E
...

coli​ ​that​ ​could​ ​be​ ​identified)
● Proflavin​ ​is​ ​a​ ​planar​ ​molecule​ ​that​ ​inserts​ ​between​ ​base​ ​pairs
in​ ​DNA​ ​and​ ​causes​ ​“frameshift”​ ​mutations​ ​(eg
...

● Non-overlapping:​ ​ABC,​ ​DEF,​ ​GHI,​ ​etc
...
​ ​control​ ​of​ ​iron​ ​import​ ​into​ ​human​ ​cells
○ Iron​ ​(Fe)​ ​binds​ ​to​ ​an​ ​extracellular​ ​protein​ ​called​ ​transferrin,​ ​and​ ​the
Fe-transferrin​ ​complex​ ​then​ ​enters​ ​the​ ​cell​ ​via​ ​the​ ​transferrin​ ​receptor
■ An​ ​‘Iron​ ​Response​ ​Element’​ ​on​ ​mRNA​ ​is​ ​recognised​ ​and​ ​bound​ ​by​ ​an
IRE​ ​binding​ ​protein
Methylation
● Methylating​ ​the​ ​5’​ ​end​ ​of​ ​a​ ​gene​ ​changes​ ​the​ ​levels​ ​of​ ​expression​ ​(on/off)
○ Methylating​ ​a​ ​mammal​ ​gene​ ​turns​ ​off​ ​the​ ​gene
○ Methylating​ ​an​ ​insect​ ​gene​ ​turns​ ​on​ ​the​ ​gene
--------------------------------------------------------------------------------------------------------------------------■

5:​ ​DNA​ ​REPLICATION
Semiconservative​ ​(Meselson​ ​and​ ​Stahl,​ ​1958)
DNA​ ​is​ ​replicated​ ​through​ ​complementary​ ​base​ ​pairing
Replication​ ​takes​ ​place​ ​5’​ ​to​ ​3’
DNA​ ​polymerase​ ​must​ ​have​ ​3’-OH​ ​residue​ ​to​ ​extend​ ​from
The​ ​breakage​ ​of​ ​phosphoanhydride​ ​bonds​ ​of​ ​dNTPs​ ​provides​ ​energy​ ​for
polymerisation
DNA​ ​replication​ ​is​ ​bidirectional​ ​from​ ​origins​ ​of​ ​replication
● Forms​ ​replication​ ​bubbles
● Replication​ ​fork
○ both​ ​strands​ ​(lagging​ ​and​ ​leading)​ ​are​ ​copied​ ​at​ ​replication​ ​forks,​ ​in​ ​a​ ​5’​ ​to​ ​3’
direction
■ Synthesis​ ​of​ ​the​ ​leading​ ​strand​ ​is​ ​continuous
■ Synthesis​ ​of​ ​the​ ​lagging​ ​strand​ ​is​ ​discontinuous,​ ​leaving​ ​Okazaki
fragments​ ​that​ ​are​ ​later​ ​joined​ ​by​ ​DNA​ ​ligases
● Enzymes​ ​at​ ​the​ ​replication​ ​fork
○ Helicase,​ ​unwinds​ ​the​ ​double​ ​helix​ ​of​ ​DNA
○ Ssbinding​ ​protein,​ ​stabilises​ ​ssDNA
○ Primase​ ​(RNA​ ​polymerase),​ ​synthesises​ ​RNA​ ​primers
○ Initiating​ ​DNA​ ​polymerase,​ ​synthesis​ ​the​ ​new​ ​DNA​ ​strand
○ Progressive​ ​DNA​ ​polymerase,​ ​proofreads​ ​the​ ​replicated​ ​strand
●
●
●
●
●

Joanna​ ​Griffith​ ​(2017)

○ Sliding​ ​clamp,​ ​keeps​ ​DNA​ ​polymerase​ ​on​ ​the​ ​DNA
○ Clamp​ ​loader,​ ​loads​ ​the​ ​sliding​ ​clamp​ ​and​ ​DNA​ ​polymerase​ ​onto​ ​the​ ​DNA
○ Nucleases,​ ​trim​ ​the​ ​Okazaki​ ​fragments
○ DNA​ ​ligases,​ ​join​ ​the​ ​Okazaki​ ​fragments
○ Replisome,​ ​other​ ​factors
● Speed​ ​of​ ​DNA​ ​replication
○ About​ ​50​ ​base​ ​pairs/second​ ​at​ ​every​ ​fork​ ​in​ ​eukaryotes
○ About​ ​1000​ ​base​ ​pairs/second​ ​at​ ​each​ ​fork​ ​in​ ​prokaryotes
■ High​ ​processivity​ ​because​ ​DNA​ ​polymerase​ ​attaches​ ​to​ ​a​ ​sliding
clamp
DNA​ ​replication​ ​through​ ​PCR
● Needed:
○ Enzyme​ ​(DNA​ ​polymerase)​ ​and​ ​Mg​2+
○ dNTPs​ ​(dATP,​ ​dCTP,​ ​dGTP,​ ​dTTP),​ ​provide​ ​energy​ ​for​ ​polymerisation
○ Single-stranded​ ​template​ ​DNA
○ 3’-OH​ ​primer
● Process:
○ Initial​ ​denaturation​ ​of​ ​DNA​ ​at​ ​95​o​C​ ​for​ ​5​ ​minutes
■ Separates​ ​double-stranded​ ​DNA​ ​into​ ​single​ ​strands​ ​and​ ​detaches​ ​any
existing​ ​primers​ ​bound​ ​to​ ​the​ ​DNA
○ Primer​ ​annealing​ ​stage​ ​at​ ​55​o​C​ ​for​ ​1​ ​minute
○ Extension​ ​stage​ ​at​ ​72​o​C​ ​for​ ​1​ ​minute
■ Creation​ ​of​ ​new​ ​DNA​ ​complementary​ ​strands
○ Steps​ ​3-4​ ​are​ ​repeated​ ​for​ ​34​ ​cycles
○ Final​ ​extension​ ​stage​ ​at​ ​72​o​C​ ​for​ ​10​ ​minutes
○ Gel​ ​electrophoresis​ ​is​ ​used​ ​to​ ​separate​ ​the​ ​amplified​ ​DNA​ ​into​ ​size​ ​bands
--------------------------------------------------------------------------------------------------------------------------5:​ ​THE​ ​EUKARYOTIC​ ​CELL​ ​CYCLE
● S​ ​phase​ ​=​ ​synthesis​ ​(DNA​ ​replication)
● M​ ​phase​ ​=​ ​mitosis
● G1​ ​and​ ​G2​ ​=​ ​growth​ ​phases
Mitosis
● Prophase​ ​(early)
○ Chromosomes​ ​start​ ​to​ ​condense​ ​into​ ​chromatin
○ Mitotic​ ​spindle​ ​begins​ ​to​ ​form​ ​at​ ​the​ ​centromeres
○ The​ ​nucleolus​ ​disappears
● Prophase​ ​(late)
○ Chromosomes​ ​finish​ ​condensing
○ The​ ​nuclear​ ​envelope​ ​breaks​ ​down,​ ​releasing​ ​the​ ​chromosomes
○ More​ ​mitotic​ ​spindle​ ​forms,​ ​and​ ​some​ ​of​ ​the​ ​microtubules​ ​start​ ​to​ ​“capture”
chromosomes
● Metaphase
○ Chromosomes​ ​align​ ​along​ ​the​ ​equator
● Anaphase
Joanna​ ​Griffith​ ​(2017)

○ The​ ​spindle​ ​fibres​ ​pull​ ​the​ ​sister​ ​chromatids​ ​apart
● Telophase
○ Mitotic​ ​spindle​ ​is​ ​broken​ ​down
○ Two​ ​new​ ​nuclei​ ​form,​ ​along​ ​with​ ​nuclear​ ​membranes​ ​and​ ​nucleoli
○ Chromosomes​ ​decondense
● Cytokinesis
○ Division​ ​of​ ​the​ ​cytoplasm​ ​to​ ​form​ ​two​ ​new​ ​daughter​ ​cells
--------------------------------------------------------------------------------------------------------------------------5&6:​ ​BACTERIAL​ ​GENETICS
Genetic​ ​material
● Single​ ​circular​ ​double-stranded​ ​DNA​ ​molecule​ ​(chromosome/nucleoid)
○ No​ ​histone​ ​proteins
○ Associated​ ​with​ ​Mg​2+​​ ​and​ ​polyamines
■ Spermine
■ Spermidine
■ Putrescine
● Bacterial​ ​cells​ ​may​ ​also​ ​contain​ ​plasmids​ ​(smaller​ ​circles​ ​of​ ​DNA)
○ Can​ ​be​ ​passed​ ​between​ ​cells​ ​by​ ​conjugation
Replication
● Prokaryotes​ ​only​ ​have​ ​one​ ​origin​ ​of​ ​replication
● Lagging​ ​strand​ ​synthesis:
○ Primase​ ​synthesises​ ​short​ ​RNA​ ​oligonucleotides​ ​(primers)​ ​copied​ ​from​ ​DNA
○ DNA​ ​polymerase​ ​III​ ​elongates​ ​RNA​ ​primers​ ​with​ ​new​ ​DNA
○ DNA​ ​polymerase​ ​I​ ​removes​ ​RNA​ ​at​ ​the​ ​5’​ ​end​ ​of​ ​the​ ​neighbouring​ ​fragment
and​ ​fills​ ​the​ ​gap
○ DNA​ ​ligase​ ​connects​ ​adjacent​ ​fragments
Gene​ ​expression
● Prokaryotes​ ​do​ ​not​ ​have​ ​a​ ​nuclear​ ​membrane​ ​and​ ​only​ ​have​ ​one​ ​cytoplasmic
compartment,​ ​so​ ​they​ ​undergo​ ​coupled​ ​transcription​ ​and​ ​translation
○ Transcription​ ​and​ ​translation​ ​occur​ ​simultaneously​ ​in​ ​the​ ​cytoplasm
● Genes​ ​of​ ​related​ ​function​ ​are​ ​often​ ​clustered​ ​into​ ​operons
○ An​ ​operon​ ​has​ ​one​ ​promoter,​ ​and​ ​all​ ​genes​ ​in​ ​an​ ​operon​ ​are​ ​transcribed
together
--------------------------------------------------------------------------------------------------------------------------7:​ ​BIOTECHNOLOGY
Gene​ ​sequencing
● Genomics:​ ​everything​ ​from​ ​sequencing​ ​genomes,​ ​ascribing​ ​functions​ ​to​ ​genes,​ ​and
studying​ ​the​ ​structure​ ​of​ ​genes​ ​(gene​ ​architecture)
○ By​ ​studying​ ​an​ ​individual’s​ ​entire​ ​genome,​ ​we​ ​can​ ​see​ ​which​ ​genes​ ​are​ ​active
at​ ​particular​ ​times​ ​and​ ​under​ ​different​ ​environmental​ ​conditions,​ ​and​ ​see​ ​how
these​ ​affect​ ​outward​ ​characteristics
● Genome:​ ​all​ ​of​ ​the​ ​genes​ ​contained​ ​within​ ​an​ ​organism
● DNA​ ​sequencing
Joanna​ ​Griffith​ ​(2017)

Techniques​ ​used​ ​to​ ​produce​ ​millions​ ​of​ ​copies​ ​of​ ​short​ ​pieces​ ​of​ ​DNA
■ Nucleotides​ ​have​ ​fluorescent​ ​dyes​ ​attached,​ ​which​ ​are​ ​detected​ ​by
the​ ​sequencer
○ The​ ​ultimate​ ​goal​ ​is​ ​to​ ​determine​ ​the​ ​order​ ​of​ ​nucleotides​ ​throughout​ ​all​ ​of​ ​the
genetic​ ​material​ ​of​ ​an​ ​organism
○ Whole​ ​genome​ ​shotgun​ ​sequencing​ ​(allows​ ​entire​ ​genomes​ ​to​ ​be​ ​sequenced)
■ Breaks​ ​the​ ​genomic​ ​DNA​ ​into​ ​many​ ​small​ ​fragments
● Fragments​ ​are​ ​cloned​ ​into​ ​vectors
● The​ ​collection​ ​of​ ​fragments​ ​is​ ​called​ ​a​ ​genomic​ ​library
● Fragments​ ​can​ ​then​ ​be​ ​sequenced​ ​using​ ​a​ ​process​ ​called
chain​ ​termination​ ​DNA​ ​sequencing
○ Sanger​ ​sequencing
■ Depends​ ​on​ ​DNA​ ​replication​ ​machinery
■ Requires​ ​a​ ​3’​ ​OH​ ​group
○ Cycle​ ​sequencing
○ Chain​ ​termination​ ​sequencing
○ Whole​ ​genome​ ​shotgun​ ​sequencing
● How​ ​do​ ​we​ ​deal​ ​with​ ​genomic​ ​data?
○ Bioinformatics:​ ​the​ ​application​ ​of​ ​information​ ​technology​ ​to​ ​molecular​ ​biology
■ Primary​ ​goal​ ​is​ ​to​ ​increase​ ​our​ ​understanding​ ​of​ ​biological​ ​processes
■ Focus​ ​on​ ​developing​ ​and​ ​applying​ ​computationally​ ​intensive
techniques
● What​ ​sorts​ ​of​ ​questions​ ​can​ ​we​ ​ask​ ​of​ ​collected​ ​data?
○ Sequence​ ​alignment
○ Gene​ ​discovery
○ Gene​ ​assembly
○ Protein​ ​structure​ ​prediction
○ Prediction​ ​of​ ​gene​ ​expression
○ Modelling​ ​evolutionary​ ​relationships​ ​(eg
...
​ ​BamHI​ ​is​ ​from​ ​Bacillus​ ​amyloliquefaciens
● Recognises​ ​and​ ​cuts​ ​specific​ ​gene​ ​sequence
○

Joanna​ ​Griffith​ ​(2017)

○

●

●

●

●

This​ ​sequence​ ​occurs​ ​every​ ​4096​ ​base​ ​pairs
(therefore,​ ​an​ ​E
...
​ ​cloning​ ​DNA-recombinant​ ​E
...
​ ​DNA/RNA​ ​hybridisation
(measuring​ ​the​ ​degree​ ​of​ ​genetic​ ​similarity​ ​between​ ​pools​ ​of​ ​DNA
sequences)
■ Can​ ​be​ ​used​ ​to​ ​search​ ​for​ ​an​ ​expressed​ ​protein​ ​with​ ​an​ ​antibody
The​ ​power​ ​of​ ​recombinant​ ​DNA​ ​technology
○ Can​ ​clone​ ​genes​ ​from​ ​any​ ​organism
○ Can​ ​study​ ​any​ ​individual​ ​gene
■ Eg
...
​ ​microarrays)
○ Used​ ​in​ ​reverse​ ​genetics
■ The​ ​role​ ​of​ ​genes​ ​is​ ​investigated​ ​by​ ​measuring​ ​its​ ​effect​ ​on​ ​phenotype
○ Used​ ​in​ ​gene​ ​targeting
■ In​ ​vivo​ ​gene​ ​disruptions​ ​or​ ​deletions
○ Used​ ​in​ ​in​ ​vitro​ ​mutagenesis
■ Gene​ ​tagging​ ​(adding​ ​GFP​ ​(green​ ​fluorescent​ ​protein)​ ​or​ ​RFP​ ​(red
fluorescent​ ​protein))
■ Can​ ​reintroduce​ ​mutated​ ​genes​ ​in​ ​many​ ​cases​ ​(eg
...
​ ​Hepatitis​ ​B
--------------------------------------------------------------------------------------------------------------------------●

7:​ ​PCR​ ​AND​ ​GEL​ ​ELECTROPHORESIS
Polymerase​ ​Chain​ ​Reaction​ ​(PCR)
● Individual​ ​gene​ ​replication​ ​without​ ​cloning
● In​ ​vitro​ ​DNA​ ​synthesis​ ​reaction
○ Uses​ ​DNA​ ​+​ ​DNA​ ​polymerase​ ​+​ ​primers
○ Repeated​ ​many​ ​times,​ ​“chain​ ​reaction”
● Process:
○ Initial​ ​denaturation​ ​of​ ​DNA​ ​at​ ​95​°C​ ​for​ ​5​ ​minutes
■ Separates​ ​double-stranded​ ​DNA​ ​into​ ​single​ ​strands​ ​and​ ​detaches​ ​any
existing​ ​primers​ ​bound​ ​to​ ​the​ ​DNA
○ Primer​ ​annealing​ ​stage​ ​at​ ​55°C​ ​for​ ​1​ ​minute
○ Extension​ ​stage​ ​at​ ​72°C​ ​for​ ​1​ ​minute
■ Creation​ ​of​ ​new​ ​DNA​ ​complementary​ ​strands
○ Steps​ ​1-3​ ​are​ ​repeated​ ​for​ ​34​ ​cycles
○ Final​ ​extension​ ​stage​ ​at​ ​72°C​ ​for​ ​10​ ​minutes
○ Gel​ ​electrophoresis​ ​is​ ​used​ ​to​ ​separate​ ​the​ ​amplified​ ​DNA​ ​into​ ​size​ ​bands
● 30​ ​cycles​ ​of​ ​PCR​ ​produce​ ​about​ ​a​ ​10​6​-fold​ ​amplification
○ 1pg​ ​to​ ​1µg​ ​of​ ​DNA,​ ​enough​ ​to​ ​analyse​ ​on​ ​gel
● Only​ ​the​ ​DNA​ ​between​ ​primers​ ​is​ ​amplified
○ Specific​ ​sequences​ ​can​ ​be​ ​amplified​ ​from​ ​a​ ​complex​ ​mixture​ ​of​ ​DNA
○ The​ ​ends​ ​of​ ​the​ ​amplified​ ​fragment​ ​are​ ​defined​ ​by​ ​two​ ​primers
● Very​ ​powerful​ ​tool,​ ​with​ ​many​ ​research​ ​and​ ​applied​ ​uses
○ Detection​ ​of​ ​pathogens​ ​in​ ​water/blood
○ Genetic​ ​fingerprinting
○ Forensic​ ​analysis
Joanna​ ​Griffith​ ​(2017)

○ Diagnosis​ ​of​ ​genetic​ ​disorders
○ Prenatal​ ​diagnosis
○ Analysis​ ​of​ ​ancient​ ​DNA
○ Detection​ ​of​ ​insecticide​ ​resistance
● Limitations
○ Sequence​ ​information​ ​is​ ​required​ ​in​ ​order​ ​to​ ​design​ ​the​ ​primers
○ Limit​ ​on​ ​length​ ​of​ ​amplified​ ​fragments
○ Significant​ ​in​ ​vitro​ ​mutation​ ​rate
○ Very​ ​sensitive​ ​to​ ​exact​ ​reaction​ ​conditions
--------------------------------------------------------------------------------------------------------------------------8:​ ​REVISION
--------------------------------------------------------------------------------------------------------------------------9:​ ​PATTERNS​ ​AND​ ​PRINCIPLES​ ​OF​ ​HEREDITY:​ ​HOW​ ​ARE​ ​TRAITS​ ​TRANSMITTED?
The​ ​particulate​ ​theory​ ​of​ ​inheritance
● Characters​ ​are​ ​distinct​ ​and​ ​hereditary​ ​determinants​ ​(genes)​ ​are​ ​particulate​ ​in​ ​nature
● Each​ ​adult​ ​has​ ​two​ ​genes​ ​for​ ​each​ ​character
○ Different​ ​forms​ ​of​ ​the​ ​genes​ ​are​ ​called​ ​alleles
● Members​ ​of​ ​the​ ​gene​ ​pair​ ​segregate​ ​equally​ ​into​ ​gametes
○ Different​ ​genes​ ​assort​ ​independently​ ​in​ ​gametes
● Fusion​ ​of​ ​gametes​ ​at​ ​fertilisation​ ​restores​ ​the​ ​pair​ ​of​ ​genes​ ​and​ ​is​ ​random
Monohybrid​ ​crosses​ ​and​ ​law​ ​of​ ​segregation
● Eg
...
​ ​YY)
● Heterozygote:​ ​different​ ​alleles​ ​at​ ​a​ ​locus​ ​(eg
...
​ ​seed​ ​colour:​ ​Y​ ​=​ ​yellow,​ ​y​ ​=​ ​green​ ​and​ ​seed​ ​shape:​ ​R​ ​=​ ​round,​ ​r​ ​=​ ​wrinkly
■ Formation​ ​of​ ​gametes
● Rr​ ​x​ ​Yy
○ RY​ ​=​ ​¼
○ Ry​ ​=​ ​¼
○ rY​ ​=​ ​¼
○ ry​ ​=​ ​¼
■ The​ ​two​ ​traits​ ​are​ ​independent
● The​ ​9​ ​:​ ​3​ ​:​ ​3​ ​:​ ​1​ ​ratio​ ​is​ ​a​ ​random​ ​combination​ ​of​ ​two
independent​ ​3​ ​:​ ​1​ ​ratios
--------------------------------------------------------------------------------------------------------------------------●
●

10:​ ​RNA​ ​INTERFERENCE​ ​(RNAi):​ ​A​ ​MECHANISM​ ​FOR​ ​SILENCING​ ​GENE
EXPRESSION
Major​ ​scientific​ ​discoveries​ ​often​ ​begin​ ​with​ ​a​ ​surprising​ ​(unexpected)​ ​result
● Control​ ​of​ ​gene​ ​expression
○ Around​ ​1990,​ ​molecular​ ​biologists​ ​obtained​ ​a​ ​number​ ​of​ ​unexpected​ ​results
that​ ​were​ ​difficult​ ​to​ ​explain,​ ​given​ ​what​ ​was​ ​understood​ ​about​ ​control​ ​of​ ​gene
expression​ ​at​ ​the​ ​time
■ The​ ​most​ ​striking​ ​results​ ​were​ ​observed​ ​by​ ​plant​ ​biologists​ ​trying​ ​to
increase​ ​colour​ ​intensity​ ​of​ ​petunias​ ​by​ ​introducing​ ​a​ ​gene​ ​that​ ​leads
to​ ​the​ ​formation​ ​of​ ​red​ ​pigment
● Hypothesis:​ ​extra​ ​copies​ ​of​ ​a​ ​gene​ ​will​ ​result​ ​in​ ​more​ ​pigment,
leading​ ​to​ ​more​ ​intensely-coloured​ ​flowers
○ Actually​ ​found​ ​that​ ​flowers​ ​got​ ​less​ ​pigmented
■ How​ ​did​ ​adding​ ​extra​ ​copies​ ​of​ ​a​ ​gene​ ​that
enhances​ ​pigment​ ​production​ ​result​ ​in​ ​less
pigment?
● Control​ ​injection​ ​flowers​ ​had​ ​more
mRNA​ ​than​ ​the​ ​experimental​ ​injection
flowers
○ Reduced​ ​mRNA​ ​will​ ​lead​ ​to​ ​less
enzyme​ ​and​ ​therefore​ ​less
pigment​ ​production,​ ​so​ ​there​ ​will
be​ ​less​ ​pigment,​ ​but​ ​how​ ​does
increasing​ ​the​ ​number​ ​of​ ​copies
of​ ​the​ ​gene​ ​lead​ ​to​ ​reduced
mRNA?
dsRNA​ ​reduces​ ​levels​ ​of​ ​mRNA​ ​with​ ​matching​ ​nucleotide​ ​sequences,​ ​resulting​ ​in
gene​ ​silencing
● Andrew​ ​Fire​ ​and​ ​Craig​ ​Mello​ ​(1998)
○ Working​ ​on​ ​gene​ ​expression​ ​in​ ​the​ ​nematode​ ​C
...
​ ​p19)​ ​that
suppress​ ​gene​ ​silencing
○ microRNAs,​ ​which​ ​act​ ​like​ ​RNAi,​ ​have​ ​been​ ​shown​ ​to​ ​be​ ​key​ ​regulators​ ​in
many​ ​biological​ ​processes,​ ​such​ ​as​ ​development,​ ​cell​ ​birth​ ​and​ ​death,​ ​and
cancer
Is​ ​there​ ​clinical​ ​potential​ ​for​ ​RNAi?
● RNAi​ ​has​ ​the​ ​potential​ ​to​ ​specifically​ ​target​ ​gene​ ​expression,​ ​so​ ​has​ ​the​ ​potential​ ​to
fight​ ​almost​ ​every​ ​disease
● How​ ​close​ ​are​ ​we​ ​to​ ​seeing​ ​RNAi​ ​transform​ ​medicine?
○ Eg
...
​ ​hepatitis​ ​C
■ In​ ​2002,​ ​researchers​ ​at​ ​Stanford​ ​University​ ​announced​ ​that​ ​their​ ​RNAi
treatment​ ​had​ ​controlled​ ​the​ ​hepatitis​ ​C​ ​virus​ ​in​ ​lab​ ​mice
○ Eg
...
​ ​genetic​ ​contraceptives​ ​(not​ ​hormonal)
○ Currently,​ ​stem​ ​cells​ ​seem​ ​to​ ​be​ ​more​ ​useful​ ​than​ ​RNAi
--------------------------------------------------------------------------------------------------------------------------11:​ ​PATTERNS​ ​AND​ ​PRINCIPLES​ ​OF​ ​HEREDITY:​ ​WHAT​ ​COMPLEXITIES​ ​CAN​ ​BE
ENCOUNTERED​ ​IN​ ​RELATING​ ​GENOTYPE​ ​TO​ ​PHENOTYPE?
Interactions​ ​between​ ​alleles​ ​of​ ​a​ ​gene
● Dominance​ ​is​ ​not​ ​always​ ​complete
○ Incomplete​ ​dominance:​ ​heterozygotes​ ​show​ ​an​ ​intermediate​ ​(eg
...
​ ​in​ ​butterflies,​ ​different​ ​colours​ ​are​ ​more​ ​or​ ​less​ ​present​ ​in​ ​wing​ ​colouration
due​ ​to​ ​differences​ ​in​ ​dominance​ ​of​ ​colouration​ ​genes
○ Co-dominance​ ​:​ ​heterozygotes​ ​show​ ​phenotypes​ ​of​ ​both​ ​alleles
○ Eg
...
​ ​agouti​ ​-​ ​‘a’​ ​black​ ​and​ ​‘at’
black/yellow)
○ Eg
...
​ ​the​ ​gene​ ​involved​ ​in​ ​cilia​ ​and​ ​flagella​ ​production,​ ​if​ ​mutant,​ ​causes
respiratory​ ​problems​ ​(failure​ ​to​ ​clear​ ​airways)​ ​and​ ​sterility​ ​(sperm​ ​don’t​ ​have
normal​ ​motility)
○ Lethal​ ​alleles​ ​can​ ​cause​ ​skewed​ ​phenotypic​ ​ratios​ ​(missing​ ​from​ ​progeny)​ ​as
carriers​ ​of​ ​these​ ​alleles​ ​do​ ​not​ ​survive​ ​to​ ​be​ ​born
○ Eg
...
​ ​coat​ ​colour​ ​in​ ​mammals
■ Determined​ ​by​ ​at​ ​least​ ​5​ ​major​ ​genes

Joanna​ ​Griffith​ ​(2017)

A​ ​gene:​ ​determines​ ​the​ ​distribution​ ​of​ ​pigment​ ​in​ ​the​ ​hair​ ​(A​ ​=
agouti,​ ​a​ ​=​ ​solid)
● B​ ​gene:​ ​determines​ ​the​ ​colour​ ​of​ ​pigment​ ​in​ ​the​ ​hair​ ​(B​ ​=
black,​ ​b​ ​=​ ​brown)
● C​ ​gene:​ ​permits​ ​colour​ ​expression​ ​(C​ ​=​ ​colour​ ​expressed,​ ​c​ ​=
no​ ​colour)
● D​ ​gene:​ ​controls​ ​intensity​ ​of​ ​pigment​ ​specific​ ​by​ ​other​ ​genes
(D​ ​=​ ​full​ ​expression,​ ​d​ ​=​ ​dilute)
● S​ ​gene:​ ​controls​ ​distribution​ ​of​ ​pigment​ ​(S​ ​=​ ​solid​ ​colour,​ ​s​ ​=
spotted​ ​(piebald))
● Alleles​ ​of​ ​one​ ​gene​ ​can​ ​mask​ ​the​ ​effects​ ​of​ ​alleles​ ​at​ ​another​ ​gene
○ Epistasis:​ ​a​ ​gene​ ​interaction​ ​in​ ​which​ ​the​ ​effects​ ​of​ ​an​ ​allele​ ​at​ ​one​ ​gene​ ​hide
the​ ​effects​ ​of​ ​alleles​ ​at​ ​another​ ​gene
● Eg
...
​ ​AaBb​ ​x​ ​AaBb
■ Gametes:
● AB​ ​=​ ​¼
● Ab​ ​=​ ​¼
● aB​ ​=​ ​¼
● ab​ ​=​ ​¼
● But​ ​during​ ​meiosis​ ​I,​ ​chromatids​ ​from​ ​homologous​ ​pairs​ ​can
exchange​ ​strands​ ​in​ ​a​ ​process​ ​known​ ​as​ ​recombination
○ If​ ​a​ ​geneticist​ ​were​ ​to​ ​closely​ ​examine​ ​the​ ​genetic​ ​makeup​ ​of​ ​a​ ​single,
autosomal​ ​chromosome​ ​from​ ​one​ ​of​ ​your​ ​cells,​ ​that​ ​chromosome​ ​would​ ​be
found​ ​to​ ​be​ ​a​ ​mosaic​ ​of​ ​genes​ ​derived​ ​from​ ​just​ ​two​ ​of​ ​your​ ​grand-parents​ ​either​ ​your​ ​maternal​ ​grandparents​ ​or​ ​your​ ​paternal​ ​grandparents
--------------------------------------------------------------------------------------------------------------------------13:​ ​VARIATION​ ​IN​ ​CHROMOSOME​ ​NUMBER​ ​AND​ ​STRUCTURE:​ ​LARGE-SCALE
CHROMOSOMAL​ ​CHANGES
Changes​ ​in​ ​chromosome​ ​number
● Organisms​ ​with​ ​multiples​ ​of​ ​the​ ​basic​ ​chromosome​ ​set​ ​(genome)​ ​are​ ​referred​ ​to​ ​as
euploid
○ Chromosome​ ​number​ ​can​ ​vary​ ​among​ ​closely​ ​related​ ​species
■ Eg
...

■ An​ ​individual​ ​of​ ​a​ ​typically​ ​diploid​ ​species​ ​that​ ​has​ ​only​ ​one​ ​set​ ​of
chromosomes​ ​is​ ​called​ ​a​ ​monoploid​ ​(rather​ ​than​ ​a​ ​haploid,​ ​which​ ​is
the​ ​normal​ ​condition​ ​for​ ​some​ ​species)
■ May​ ​result​ ​in​ ​abnormal​ ​development,​ ​but​ ​n
absence​ ​of​ ​wild-type​ ​counterpart)
■ Having​ ​only​ ​one​ ​gene​ ​copy​ ​(monosomic)​ ​is​ ​worse​ ​than​ ​having​ ​three
(trisomic)
Changes​ ​in​ ​chromosome​ ​structure
● Chromosomes​ ​can​ ​have​ ​missing​ ​pieces:​ ​deletions
○ A​ ​deletion​ ​is​ ​the​ ​loss​ ​of​ ​part​ ​of​ ​one​ ​chromosome​ ​arm
○ Deletions​ ​can​ ​be​ ​small,​ ​only​ ​covering​ ​a​ ​part​ ​of​ ​one​ ​gene,​ ​or​ ​large,​ ​with
chromosomes​ ​missing​ ​pieces​ ​large​ ​enough​ ​to​ ​be​ ​visualised​ ​on​ ​a​ ​karyotype
●

Joanna​ ​Griffith​ ​(2017)

Eg
...
3​ ​=​ ​cat-like​ ​cry
○ Sp15
...
​ ​Williams​ ​syndrome
● FISH​ ​allows​ ​identification​ ​of​ ​the​ ​deletion​ ​of​ ​one​ ​elastin​ ​gene
● More​ ​than​ ​25​ ​genes​ ​deleted​ ​on​ ​chromosome​ ​7
○ ELN​ ​(elastin)​ ​gene​ ​=​ ​connective​ ​tissue​ ​abnormalities
and​ ​cardiovascular​ ​disease
○ CLIP2,​ ​GTF21,​ ​GTF21RD1,​ ​LIMK1​ ​=​ ​problems​ ​with
visual​ ​spatial​ ​tasks
○ NCF1​ ​(neutrophil​ ​cytosolic​ ​factor​ ​1)​ ​=​ ​related​ ​to​ ​risk​ ​of
developing​ ​hypertension​ ​if​ ​NOT​ ​deleted​ ​(part​ ​of
NADPH​ ​oxidase​ ​which​ ​increases​ ​reactive​ ​oxygen
species​ ​and​ ​blood​ ​vessel​ ​changes)
○ Most​ ​deletions​ ​are​ ​not​ ​inherited​ ​(no​ ​family​ ​history)
■ Usually​ ​a​ ​result​ ​of​ ​random​ ​events​ ​during​ ​the
production​ ​of​ ​eggs​ ​and​ ​sperm
Chromosomes​ ​can​ ​have​ ​extra​ ​pieces:​ ​duplications
○ Duplications​ ​play​ ​an​ ​important​ ​role​ ​in​ ​evolution​ ​of​ ​the​ ​genome​ ​(eg

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