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Perspectives on the Gene
30 September 2014

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13:36

Mendelian genetics
Microbial genetics
Sex-linked genetics
Molecular genetics
Population genetics
Applied genetics

Gregor Mendel: The Friar who grew peas

Mendelian (classical) Genetics
• Particles of inheritance
○ Alleles
○ Dominance
• Research methods: crosses and pedigrees

Complicating factors
○ Physical genetic structure
○ Chromosomes
 Linkage
 Random assortment
○ Epistasis and Pleiotropy
 Several genes affecting one trait
 One gene affecting many traits

Population Genetics
• Gene concept
○ Cause of inheritable variation
 Within populations
 Between populations
 Between species
• Research methods: Population sampling and
statistical analysis
Uses
○ Evolutionary biology
 Natural selection
 Genetic drift
○ Conservation biology
 Inbreeding
 Resilience

Genetics Page 1

Molecular Genetics
• Instructions for protein synthesis
○ Replication
○ Mutation
○ Transcription
○ Translation
• Research methods: DNA sequencing and recombinant DNA
technology
Uses
○ Population
 Genetic markers
○ Plant and animal breeding
 Trait loci
○ Development genetics
○ Forensics
○ Medicine
 Screening
 Therapy
 Artificial hormones
○ Genetically modified crops and livestock

Practical
1
...
Recombination and centromere mapping in
Sordaria brevicolis
3
...
ac
...

Red x red = red

RW

RW
If ti genes then 9:3:1 ?

White cows breed true
White x white = white

W

R

R

RW

RR

W

Red x white produced mottled roan cows

WW

WR

Incomplete codominance
Red plants x white = pink
Pink x pink = 1 red, 2 pink, 1 white

1:2:1

Lethal genes
Lethal white syndrome

It's even more complex with full organism
Lots of genes interact

Leghorn and Wyandotte chicken
F1 generation is all white
F2 is 13 white and 3 coloured

13:1

At one locus there is an allele for white and one for colour, white is dominant
...
BBLL
The Wyandotte has both recessive alleles
...

LLBB
LlBB
LlbB

Inhibitor
Parents both white
F1 white
F2 13 white, 2 blue, 1 black
13:2:1
At locus 1 an allele prevents colour
At locus 2 partially dominant white-black alleles give blue
as the heterozygous phenotype
...

The normal allele is dominant
...

Aabb

Cats
Black and albino
F1 is black
F2 is 9 black, 3 brown, 4 albino

Locus has coloured/albino
...

Other has black-brown alleles, black is dominant
...

aa = albino
Aa/AA = coloured
AaBb = black
Aabb = brown

Ratios
3:1
• Single locus
• 1 dominant allele

More complex
There can be several potential alleles available for
each locus (though only two per individual)

Genetics Page 2

3:1
• Single locus
• 1 dominant allele

More complex
There can be several potential alleles available for
each locus (though only two per individual)

1:2:1
• Single locus
• Codominant alleles/incomplete dominance
• e
...
red, roan, white, cows
9:3:3:1
• 2 independent loci
• Or 2 unrelated traits
○ Texture and colour
3:6:3:1:2:1
• 2 independently segregating loci
• 1 dominant/recessive
• The other codominant
15:1
• 2 loci
• Both for the same phenotype
○ Longhorn and wyandotte
12:3:1
• 2 loci
• One affects the other
○ White overrides 2 colours
13:3
• 2 loci
• One affects the other
○ Same phenotype dominant at one, recessive at the other

9:7
•
•
•
•

2 loci
Either can give the same phenotype
Both recessive
Any which is homozygous is expressed
○ Rex

9:3:4
• 2 loci
• One means the other ignored
○ Albinism
1:2:1:2:4:2:1:2:1
• 2 independent loci
• Both codominant

Genetics Page 3

e
...
Cats
Cats can be black, siamese, or albino
...

2
...

4
...


Remove 1 seedling
Cut off stained tip
Drop of aceto-carmine stain
Apply cover slip
X10 then x40

C-Metaphase
• 0
...
Males have just 1
This often leads to X inactivation - only one X expressed - in females
This can lead to carried diseases
Colour blindness
Haemophilia
Duchenne muscular dystrophy

Genetics Page 5

Sex-linked genes
• The X chromosomes also explain why tortoise-shell cats are always females
• The allele on one X chromosome is black, the other is ginger
...

Hence you have the same mitochondria as your mother
...

The only exceptions are the genes found on the mitochondria
...
Enters by fusion between spikes and cell
receptors
2
...
Reverse transcriptase
3
...
Transcription gives new viral RNA
5
...
Mature retrovirus buds out, taking
membrane and attachment spikes

• Viral DNA or protein?
○ Herschey-Chase experiment

Uses of viruses
• Bacteriophage lambda
○ 48,502 base pairs
• Anneal COS sites DNA ligase
• Replication
• Cut at COS sites
○ Every 50kb
• Replace genes with ones we want
• Genes can be cloned
○ Lots of copies
• Can make those genes be expressed
○ Produce loads of proteins
• Lambda is simple, so desired proteins can be
identified
• Pharmaceutical



Genetics Page 7

Microbial Genetics - Bacteria
21 October 2014

17:08

Generalities
• Single chromosome
○ Genes - structural components and metabolism
○ Only 1 copy of each gene
• Plasmids
○ Antibiotic resistance, virulence, conjugation
○ Often several copies

Historical studies
• Depend on the ability to recognise changes in the bacteria
• Change are usually due to mutations
Modern Studies
• Generally rely on analysing DNA sequence data

Detecting Changes
• Plate out colonies on an agar plate with all nutrients
• Have an exact replica with one nutrient missing
• Look for differences
○ Mutants might lack nutrient producing gene and not grow

○

Regulation of Genes
• Gene expression is carefully controlled to respond
to changes
• Some genes are always expressed
○ Constitutive
• Some genes are expressed when needed
○ Inducible
○ Particular nutrient sources
• Some genes are switched off when no longer
needed
○ Repressible
○ e
...
enzymes involved in biosynthesis
• To allow genes with similar roles, or those which
interact, to work together, they are switched on/off
together
○ This is an operon

Linked Genes
• Genes sit next to each other
• At meiosis chromosomes cross over
• The closer genes are, the less likely they are to cross
over
How can changes occur?
• Transforming free DNA gets incorporates
○ Can replace equivalent gene - not always good
○ E
...
Griffith's heat killed cells
• Conjugation
○ 1 strand of plasmid transferred via pilus

Mutations
• Chemical
○ Nitrous acid
○ Alkylating agents
○ 5-bromouracil
○ Benzpyrene
○ Ethidiume bromide
• Radiation
○ X-rays
○ UV
• Ames Test for mutagens
○ 1 chromosome - mutations always expressed

•

•

○

• It is possible to map the genes by measuring the
proportion which recombine (cM)

Genetics Page 8

Microbial Genetics - Sequencing
23 October 2014

14:22

• Currently we find out which genes are present through DNA sequencing
• However, we used to have to go through identifiable markers and map the distances between them

Identification of Organisms
• All bacteria have a gene known as the 16S rDNA
• There are thousands of these genes which have been characterised and sequenced

Identifying Sequences
• Just looking at a DNA sequence is not helpful
• However, it can be compared to a database
• Therefore, we can identify the source organism for this DNA
• Sometime multiple genes can be detected
○ This can identify different strains of the organism

Genetics Page 9

Populations and Species
28 October 2014

13:17

Population
• A group of individuals that share a common gene pool
• A gene pool comprises all the alleles found within the individuals of a population
Species
• "Groups of interbreeding natural populations that are reproductively isolated from other such groups"
• Individuals of the same species may interbreed
• Individuals of different species do not normally interbreed
○ Biological species concept

Hybridisation
• Occasionally individuals of different species do interbreed
• Allows alleles to cross from one gene pool to another
○ Introgression
• May occur due to introduction of a new species
○ May erode the genetic identity of native species
 Scottish wildcat
○ May produce dangerous offspring
 Wolf/dog hybrids in Italy

Asexual Organisms
• Many organisms reproduce without sex
○ Microbes
○ Fungi
○ Parthenogenetic animals
 Whiptail lizards
○ Biological species concept cannot be applied

Variations within species
• Populations
○ May differ morphologically
 Different geographical locations
 Different ecological conditions
• Polytypic species
○ Different forms grade into one another when they meet
• Subspecies
○ Distinct forms that don't intergrade
• Can still interbreed
• Hybrid Zone
○ Narrow areas of overlap between subspecies
○ Some hybrid individuals
○ No continuous gradation
○ Selection against hybrids
 Reproduce less
Ring Species
• Adjacent populations interbreed
○ Except at the ends of the ring
○ Too different
• E
...
Ensatina salamanders
○ Form ring around California's central valley
○ Adjacent species interbreed
○ E
...
Croceater cannot
Extinct Species
• Known only from preserved specimens
○ Fossils
 Interbreeding cannot be assessed
○ Specimens containing DNA
 Hair, skin, bone, leaves, seeds
 Inferred from polymorphism
Other species concepts
• Morphological
○ Species are varieties with no intermediate forms
• Genotypic cluster criterion
○ Species are clusters of similar genotypes with intermediates between gene clusters
• Ecological
○ Each species occupies an ecological niche different from any other species
• Cladistic
○ Species form different unbranched sections of the tree of life

Genetics Page 10

Population genetics and evolution
• Darwin - 1859
○ Speciation occurs through natural selection
○ Heritable variation in the ability to survive and
reproduce
• Mendel - 1850-60s
○ Heredity occurs through the transmission of genes
 Particulate units of inheritance
○ Diversity caused by gene variants - alleles
• The Modern Synthesis - 1900s-60s
○ Natural selection acts within populations by
changing allele frequencies
Evidence for evolution within populations
• Demonstrated through:
○ Laboratory experiments
○ Plant/animal breeding
○ Introduction
Evidence for speciation
• Difficult to observe directly in nature
○ Takes a long time
• Logical extension of observed processes
• Lab experiments
○ Fly populations fed different diets become
reproductively isolated

The Gene Pool
• Parents
○ Contribute equally to a pool of gametes
• Gametes
○ Fertilise one another
• Zygotes
○ Become parents of next generation

Equilibrium
30 October 2014

13:55

What to expect when nothing is happening
Calculating allele frequency from genotype frequency
• Each diploid individual has 2 copies of each gene
• An individual can be homozygous or heterozygous
Genotype AA

Aa

aa

Total

Individuals 2

36

162

200

Frequency 0
...
18

0
...
01 + 0
...
1
○ a= aa + Aa/2
=0
...
18/2
= 0
...
1)

a (0
...
1)

AA (0
...
09)

a (0
...
09) aa (0
...
01 Aa = 0
...
81
p= 0
...
9
A (o
...
9)

A (0
...
04) Aa (0/16

a (0
...
16) aa (0
...
2, q = 0
...
g
...
5
• Genetic Drift
• Small breeding population
○ Sampling error
○ Random small samples will have biased ratio
○ Fewer breeders = greater deviation
• Founder effect
○ Small group of colonists, biased distribution of alleles
• Bottleneck
○ Near extinction
○ Population can recover, but not with the same allele ratios
• E
...
Pingalap atoll
Small, low lying
Genetics Page 13

○ Small, low lying
○ 30 survivors of 1780s typhoon
 Bottleneck
○ One survivor carried rare allele for total colour blindness
 Sampling error
○ 10% of population now carries that allele
 Allele frequency of 0
...
g
...
g
...
g
...
g
...
g
...
g
...
bbc
...
g
...

• Loss of genetic diversity
○ Genetic diversity is
 Measured as
Genetics Page 15

 Measured as
□ Proportion of polymorphic loci
□ Number of alleles at a locus
□ Proportion of heterozygotes
 Raw material for adaptation
□ To environmental damage
□ To disease
 Decreases with reduced population size
□ Due to genetic drift
○ E
...
Cheetahs
• Accumulation of deleterious mutations
○ Genetic drift
 Increases in small populations
 Becomes stronger relative to purifying selection
 May cause increase in prevalence of deleterious alleles
○ E
...
Koalas
 Endangered on mainland
 Population translocated to French Island
 Then to Kangaroo Island
□ Double founder effect
□ High numbers
□ Low genetic diversity
 High incidence of testicular abnormalities

• Adaptation to captivity
○ Natural and artificial selection
 Adapts to captive environment
○ Directed artificial selection
 Intentional selection for traits
□ Handling, breeding
○ Unconscious artificial selection
 Unintentional
 Unintentionally selecting desirable traits
○ Incidental artificial selection
 Properties of captive environment
 Abundant water
 Lack of predators
○ Prevention
 Minimise generations in captivity
□ Reintroduction
□ Delayed reproduction
□ Cryopreservation of sperm and oocytes
 Minimise selection
□ Use systematic breeding
□ No bias
□ Mimic natural environment
 Fragment populations
□ Selection is less effective in small populations
□ Translocate between populations to avoid inbreeding
Immigration

□ Continue to bring wild individuals in

Genetics Page 16

Quantitative traits and heritability
11 November 2014

13:17

Quantitative Traits
• Many traits vary continuously within population
○ normal distribution of trait values
○ described by mean and standard deviation
• Mendelian traits
○ dry and wet earwax
• Quantitative traits
○ shades of eye colour
○ shades of skin colour
○ height
• The environment
○ Not all variation is genetically determined
■ weight is influenced by diet
■ skin colour is influenced by UV exposure
• Genotype and phenotype
○ Genotypic component
■ genetic
○ phenotypic component
■ environmental
○ pure-bred lines
■ no genetic variation
■ used to distinguish genotypic from phenotypic variation
Gene-environmental interactions
• Single genotype
○ expressed differently depending upon conditions
• norm of reaction
○ describes variation of a trait across a range of environmental conditions
• E
...
Phenylketonuria
○ Hereditary
■ mutation of a single gene
○ carriers cannot metabolise phenylamine
■ accumulates as phenylpyruvate
■ mental retardation, brain damage and seizures result
○ Treated by restricting phenylamine in diet
○ only expresses if diet includes phenylamine
■ gene-environment interaction

Heritability
• 3 components of phenotypic variance (Vp)
○ Genetic variance (Vg)
○ Environmental effects (Ve)
○ Genotype-environment interactions (Vge
Vp=Vg+Vg+Vge
• Heritability (H)
○ Proportion of phenotype variance due to genetic factors
H=Vg/Vp
• Importance
Genetics Page 17

• Importance
○ If heritability is high
■ Trait can be modified by breeding
○ If heritability is low
■ Trait can be modified most successfully by changing the environment
Additive genetic variation
• Several genes contribute to same trait
• E
...
two genes influence coat colour
○ Alleles A and B code for pigment
○ Alleles a and b code for no pigment
○ ADD PICTURE
• As the number of genes for the trait increases
○ Number of genotypes increases
○ With n genes there are 3n genotypes
○ Distribution of trait values approaches normal distribution

Quantitative trait loci (QTL)
• QTL mapping
○ Score members of population for trait
○ Determine genotype with respect to a marker gene
○ Look for association between genotype and trait
■ Does genotype explain the variance?
○ This shows that the marker is linked to a QTL
■ This doesn't necessarily mean that the marker is the QTL
○ Make linkage map to see how close the marker is to the QTL
Selection on Quantitative Traits
• Evolutionary biologists often assume genetic variation is continuous
○ Assume many genes influence a trait
○ Assume that genes contribute additively
• Selection
○ Acts upon the mean and variance of a trait
○ It is implied that this action reflects changes in the frequency of alleles
○ Directional, stabilising and diversifying selection
○ E
...
Peacocks
■ Long tail
□ More mates
□ Directional selection
■ Too long
□ Easy prey
□ Stabilising selection

Genetics Page 18

DNA and Chromosomes
13 November 2014

15:00

Genetic material
• Must store complex information
○ All traits and function
○ Must mutate occasionally
• Must replicate faithfully
○ Billions of cell divisions
• Must be translated into protein to produce phenotype
Nucleotides
• Building blocks of DNA polymer
Polynucleotide strands
• Phosphodiesterase
○ 5'-phosphate group
○ 3' carbon of next
• Strand
○ Backbone of alternating sugars and phosphates
○ 5' -> 3'
3D structure
• Double helix
• 2 polynucleotide strands
• Bases form rungs
Properties
• Base sequence
○ Varies freely
○ Complex coding
• Complementary strands
○ Synthesis templates
○ Complementary RNA
 Production of phenotype

Genetics Page 19

Packaging
• Human cell
○ 1
...
4 billion base pairs in humans
○ Replication must be rapid
• Development of a multicellular organism
○ Requires millions of cell divisions
 Potential for error
 Replication must be very accurate
Semi-conservative replication
• Double helix
○ 2 complementary strands
• Replication
○ Original strands separate
○ Each is template
• New DNA molecule
○ One new strand
○ One old
○ Confirmed through labelling with 15N
Eukaryotic division
• Linear chromosomes
• 100 million base pairs
• Rate of replication
○ 500-5,000 nucleotides per minute
 7 days for a single chromosome
○ Multiple origins of replication
 20-300 thousand base pair units
 Entire genome in minutes
• Replication bubbles
○ At each point of origin
 DNA unwinds
 Forms replication bubble
○ DNA synthesis on both strands in both directions
○ Replication forks
 Move outwards in both directions
 Where strands separate
 Run into each other and merge
• dNTPs
○ AT, CG
○ Added to 3'OH group of new strand
○ Phosphodiester bond
• Direction of replication
○ DNA polymerase adds nucleotides to 3' end
○ Strands are antiparallel
○ Leading strand
 Same direction and replication
○ Lagging strand
 Opposite to replication
 Discontinuously replicated
 Okazaki fragments bind as every few hundred bases are exposed
• Licensing
○ Ensures 1 replication per origin per cycle
○ Origin recognition complex (ORC)
 Binds to every origin
○ Replication licensing factor (MCM)
 Attaches to origins
 Contains helicase
 Unwinds short section of DNA
□ Replicator can begin
○ Geminin
 Blocks MCM from reattaching while replication is in progress
• Unwinding
○ DNA helicase breaks H bonds
○ Single strand binding proteins
 Prevent strands from reannealing
○ Topoisomerases
 Make double strand breaks
 Removes coils
• Primers
○ DNA polymerase
 Can only add to existing strand
○ Primers
 Synthesised by primase
□ Short strands of complementary RNA
□ Single stranded
□ 30-40 nucleotides
 Anneal to unwound DNA strand
□ 3' end
□ Initiate synthesis
Replication of Chromosome Ends
• Primers
○ Composed of RNA
○ Lack 3'OH group
○ Removed after replication
○ Replaced by adjacent fragments
 Neighbouring strand just runs in
○ Terminal fragments
 No adjacent fragments
 Chromosomes shorten each cycle
• Telomeres

Genetics Page 21

PCR
• Specific gene is tiny part of DNA
○ Must be amplified
• Artificial replication
• Billions of copies
• Ingredients
○ DNA
○ Primers
○ dNTPs
○ Magnesium ions
 Affect specificity
○ Buffer
 Correct pH
○ Thermostable polymerase
 E
...
f
...

• Holliday junction
Migrates along chromosome
Genetics Page 24

○
○
○
○

Migrates along chromosome
Rotates
Forms cross-shaped Holliday intermediate
Cleaved in vertical or horizontal plane

○

• Gene conversion
○ Occurs in heterozygous non-crossover chromosomes
• vertical : crossing over
• horizontal: no recombination
• Homologous repair of double strand breaks
○ Can be caused by
 Radiation
 Mutagen
○ Homologous chromosome used as template for repair

○

○ Homologous chromosome used as template for repair
○ Potentially the origin of crossing over
Recombination and evolution
• Shuffles alleles at different loci
• Genotypic diversity
○ Adaptive response to environmental change
• Decouple beneficial alleles from harmless ones
Prevents genetic hitchhiking
Genetics Page 25

○ Prevents genetic hitchhiking
○ If selection for whole genome, with no recombination, then bad and good all inherited
Recombination between sex chromosomes

•

• Suppression of recombination
○ Y only slightly recombines with X
○ Y and Y never meet
 Females never carry Y
○ Genes on Y can never recombine
○ X recombines in females
○ Genetic integrity of Y no longer maintained, except at small area
...

It is ideal for studying
• Many progeny
• Products of early meiosis can be observers and are maintained in linear
order
• Genes can be mapped
• Mutants are available

Reproduction involves the meeting of hyphae, which form binucleate
cells
...

Meiosis create 4 1n cells, which then mitose into 8 1n spores
...
Drop of lactic acid/glycerol in middle of slide
2
...
Coverslip
4
...
40x and count

1/2 because only half ascus is recombinant

Results
Beige Ab

4,4

100

2,2,2,2

107

Yellow S27 4,4
2,2,2,2

1/2x100/207 x 100 = 39% (of the time, crosslink is between
centromere and the gene
...

This results in beige, yellow, and black
...


Method
Using F2 flies, produced from F1 gametes --->
1
...
Knock F2 flies to bottom then pour into
bottle
3
...
Spot 10 µm of FlyNap on paper strip, and
close the lid on it
5
...
Tip onto filter paper
7
...
Classify with respect to characters
9
...
3503%

The gap between the two is 29cM
If other mutations are tested then a full
map can be assembled

1
...
Immobilise
3
...

Gene expansion is quantified by penetrance and expressivity
Penetrance

Genetics Page 30

Penetrance
The proportion of genotypes that show curled wings

Penetrance = no
...
354% (have curled wings)
Expressivity
The degree to which a particular genotype is expressed in the phenotype
Expressivity = no
...
C) / 2(no
...
6851%

The expression of the gene is affected by the environment
As it's temperature involved it's likely to be due to a protein denaturing

Genetics Page 31

Mutation
25 November 2014

12:48

• An hereditary change in
○ DNA sequence
 Gene mutation
○ Chromosome number, form, or structure
 (DNA & Chromosomes)
○ Caused by
 Errors in replication
 Spontaneous damage
□ Radiation
□ Chemicals

Genetic diversity
• Polymorphic DNA
○ Several different alleles present at the same time
• Increased by
○ Mutation
○ Immigration
• Reduced by
○ Natural selection
○ Genetic drift
Types of gene mutation
• Substitution
○ Substitutes 1 nucleotide for another
○ Transition
 A <--> G
 C <--> T
○ Transversion
 A <-> C, A <-> T, G <-> C, G <-> T
○ More possible transversions
 But transitions more likely
□ Molecular mechanisms make them more likely to occur
□ More likely to persist
 Less likely to change any amino acids
The genetic code
• DNA to mRNA to protein
• Codon
○ 3 base pairs
○ 1 amino acid
• Sequence of amino acids determines protein structure and function
• Reading frame
○ Describes how continuous DNA is divided into codons
○ 6 frames

○

○ Frameshift mutation
 Substitution
□ Can change 1 codon
 Amino acid changes
 Slightly different protein
□ Stop codon
 Truncated protein
 TAC to TAA, TGC to TGA
 Insertion or deletion
□ In multiple of 3 base pairs
 Loss or gain of amino acid
□ Not in multiple of 3
 Knock on effect down entire protein
 Radical effect
□

Genetics Page 32

Classes of mutation
• Beneficial
○ Increase fitness
○ Occur rarely
 Random changes unlikely to make improvements
○ Constantly fixed into genome by directional selection
 Accumulate into evolution
 Lost through genetic drift in small population
○ Rarely polymorphic
 Pushed straight to fixation
○ E
...
deer mouse
 Dark soil = dark fur
...

□ Mutation occurred 4000 years ago
• Deleterious
○ Reduce fitness
○ Frequent
 Random changes much more likely to cause damage
○ Constantly removed
 Purifying selection
 Mutation/selection balance
○ Usually polymorphic and rare
 But can be fixed into genome by drift in small populations
□ Pingalap atoll
○ Often recessive
 Not exposed to purifying selection in heterozygotes
 Lurks in heterozygotes
□ Isn't expressed
□ Isn't selected against
 If dominant normally selected against
□ Unless presented in old age
○ E
...
Over 4000 human diseases caused by single gene mutation
• Neutral
○ Occur frequently
○ Non-coding mutation
 In untranslated DNA
□ Introns
 Intergentic regions
Synonymous mutations

○ Synonymous mutations
 In exons
 No change in amino acid sequence
 Redundancy
□ Degenerate code
□ GCA, GC, GCU, GCG = Alanine
○ Often polymorphic
 Not affected by selection
 May be fixed into genome or lost through genetic drift
 Eye/hair colour?
○ Nearly neutral
 Slightly beneficial/deleterious
 Small selective coefficient
□ Not enough for fixation or loss
□ Determined by drift
Neutral Theory of Molecular Evolution
• Motoo Kimura (1988)
• Most DNA polymorphisms
○ Neutral or nearly neutral
○ Retained by balance between mutation and genetic drift
• Most evolution at a molecular level are due to drift of mutant alleles

Genetics Page 33

PCR Uses
27 November 2014

14:51

• Developed as a tool for molecular genetic analysis
• Now used in a wide range of biological disciplines
• Polymerase chain reaction
○ DNA replication in vitro
○ Capable of producing billions of copies of DNA within hours
○ Very small quantities of template needed
○ Disadvantages
 Require knowledge of sequence to find primers
□ Need to find common sequence
 Very susceptible to contamination
○ Is there a common sequence to all life?]
 No
 But
□ 16S RNA gene in all prokaryotes
□ 18S RNA gene in almost all eukaryotes
□ Much of DNA sequence is common in each of these groups
• What can we do with the genes?
• Think of ecosystems with lots of microbes
○ Soil, gut, water, food
• Within these niches different microbes play different roles
• What effects do environmental changes have on the systems' dynamics?
○ Isolate ALL DNA from ALL organism
○ Perform PCR with 16S
○ Gives bands in electrophoresis gel
 Not very helpful
 Lots the same size
○ Other approaches
 DGGE, TRFLP, SSCP, TGGE
 Distinguish between PCR products
 E
...
different bacteria in guts of animals eating different foods

•
•
•

•
•
•

Real-time PCR
Quantifies amount of specific sequence present in a sample
This is based on detection of a fluorescent tag which is introduced during PCR
The more 'source' molecules present, the sooner the pre-determined detection threshold
• e
...
food contamination
DNA fingerprinting
Detecting differences between people at DNA level
More similar = closer relation
Microsatellites
• Short repeats of DNA
• Highly variable
• Usually non-coding
○ Selectively neutral
• Issues
○ Have to be identified for a new species
 Cost and time
• Applications
○ Paternal testing
○ Strain detection for pathogenic bacteria
 E
...
coli
○ Strain detection for viral outbreak
 Foot and mouth
 Identifying strains for tracing and vaccination

Genetics Page 35

Cloning and Recombinant DNA Technology
02 December 2014

12:06

DNA technology
• Restriction enzymes
○ Cut NA at specific sequences
○ Different enezymes cut different sequences
 Leave sticky ends
○ This makes a wide variety of gene fragments possible
• Cut DNA strands can be recombined in different ways
○ Naturally recombine for stability
○ Recombinant DNA molecules
• Plasmids can be used to replicate recombinant DNA
○ e
...
Human insulin
 Insulin gene isolated
 Same restriction enzymesused to cut it out, and to cut a plasmid open
 Join plasmid and human fragment
 Return plasmid to bacteria
• However, human DNA contains introns, bacteria have the bare minimum DNA, so cannot
splice exons together
○ cDNA is reverse transcribed from RNA
○ Reverse transcriptase and free nucleotides with RNA
 Then polymerase to make cDNA double stranded
• Gene for antibiotic resistance often included
○ Easy identification
• PCR products can be used
○ A overhang inserted
○ Plasmid with T overhand

Screening
• Checking for uptake of correct DNA
1
...
Pressed onto nitrocellulose filter
3
...
Labelled probe annealed, washed and dried
5
...
Detected colonies contain desired DNA
Protein pharmaceutical
• Insulin - cow/pig pancreas
• Albumin - human blood
• Factor VIII - human blood
• Calcitonin - salmon
• Anti-venom - horse or goat blood

Genetics Page 36

Cytogenetics
04 December 2014

14:39

• The study of the genetics of the cell
• Tend to deal with the genetics of chromosomes
Karyotypes
• Humans have 22 autosomes, and 2 sex chromosomes
○ 2n = 46
• Other species vary
○ Cattle 2n=60
○ Pig 2n=38
○ Mouse 2n=40
• Extremes
○ Jack jumper ant
 2n = 2
○ Adders tongue (plant)
 2n = 1260
• At times this goes wrong
○ Turner's syndrome
 45 chromosomes
 X0
 Stunted growth
 Puberty fails
○ Supermale syndrome
 XYY
 Taller than average
 Above average aggression
 Possibly lower IQ
○ Klinefelter's Syndrome
 XXY
 Taller than average
 Poorer coordination
 Less muscular
○ Downs Syndrome
 Trisomy 21
○ Edward's Syndrome
 Trisomy 18
 Growth problems
 Heart defects
○ Patau syndrome
 Trisomy 13
 Fucked
 Nervous, muscular, urogenital systems broken

○ Aneuploidy
 Gain or loss of individual chromosomes
○ Monosomy
 1 chromosome of a pair is missing
 2n-1
○ Trisomy
 1 extra chromosome
 2n+1
○ Robertsonian translocation
 Chromosomes swap with the wrong ones



○ In some organism it's much less important
 Plants e
...
Brassica

□

 Ploidy issues in plants can be complex
□ Cultivated potatoes
 4n
□ Wild potatoes
 2n
□ Wheat
 6n
□ Some bananas
 3n

Genetics Page 37

Chromosome structure
• R-banding
○ Heat metaphase chromosomes
○ Denature partially
○ Giemsa stain
○ View with fluorescent microscope
• C-banding
○ Chemically treat metaphase chromosomes
○ Extract DNA from arms
○ Giemsa stain centromeric parts
• Chromosome identification
○ Size
○ Centromere position
○ Banding pattern
○ Chromosome painting
 Fluorescent dyes
 Unique for each chromosome
○ Chromosome regions
 Based on banding
 'Addresses'
□ Chromosome
□ Arms
 P smaller
 Q
□ Region
□ Band

Sex determination - chromosomes
• Mammals XX = female
○ X0 =female
○ XY = male
○ XXY = male
○ XYY = male
• Birds
○ WZ = female
○ ZZ = male
• Drosophila
○ 3 autosome pairs
○ 2 sex chromosomes, X Y
○ Female:
 3 autosome pairs, and XX
 3 autosome pairs and XXY
 3:2 autosome:X
○ Male
 3 autosome pairs and XY
 3 autosome pairs and X0
 3:1 autosome:X
○ Control is autosome:X ratio
• Temperature control
○ Map turtles
 28oC male
 30oC female
○ Alligators
 28oC female
 30oC male
○ Crocodiles
 ~29OC male
 Extremes <20, >35oC female

Transposable Elements and Insertion Sequences
• Pieces of DNA which can move around the genome
• Originally described in maize
○ In many organism
• Mutations arise due to elements inserting into genes
• Sequence can then retranspose and revert to the original phenotype
• Can show gene funtion
○ What the transposable element breaks = what codes for the altered phenotype

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