The nature of mutation

1
(No Transcript)
2
The nature of mutation

• Change in DNA sequence
• Genetic code
• 64 codon possibilities
• 20 amino acids stop
• Redundancy, mostly at 3rd position
• Substitution
• Replace 1 nucleotide with another
• Synonymous (silent) or non-synonymous

3
The nature of mutation

• Non-synonymous substitution causes change in
  protein shape or function
• Synonymous substitution is neutral

4
The nature of mutation

• Insertions deletions
• Addition or subtraction of 1 or more nucleotides
  in a sequence
• Indel
• Insertions separating coding regions (exons) are
  introns

5
The nature of mutation

• Repetitive DNA
• Enough DNA in mammalian genomes for 300,000 genes
• Human Genome Project estimates between
  25,000-30,000 genes
• Most DNA is non-coding
• Most DNA is repetitive
• DNA-DNA hybridization

6
The nature of mutation

• Highly repetitive DNA
• VNTR (variable number tandem repeat)
• Microsatellites
• One repeat unit (2-4 nucleotides) repeated
  multiple times within a sequence
• Alleles defined by length differences
• Synonyms
• Short tandem repeats (STRs)
• Simple sequence repeats (SSRs)

7
(No Transcript)
8
Allelic Variation at a Microsatellite Locus
GCCATGACACACACAGTAACGT
Allele A
Allele B
GCCATGACACACACACACACACACAGTAACGT
9
Polymorphism at Microsatellite Loci
10
Measures of Genetic Diversity

• Terms
• Locus portion of DNA (plural loci)
• Not always a gene
• Allele form that a locus can take Mendelian
• Subjective depending on resolution of measurement
• Nucleotide state difference (sequencing)
• Length difference (microsatellite)
• Functional difference (ABO blood group)
• Electrophoretic difference (allozyme)

AGGCGTTCGCTTATGATAA AGGCGTACGCTTATGATAA
AGGCGTTCACACACACACGCTTATGATAA AGGCGTTCACACACACA
CACGCTTATGATAA
11
S
M
F
Direction of travel
12
Measures of diversity

• Diploid genomes
• Allelic diversity (A)
• Mean number of alleles per locus
• Heterozygosity (H)
• Sum of proportions of heterozygotes at all loci
  divided by number of loci
• 0.1 0.2 0.25 0.05 0.1 / 5 0.14
• Percent loci polymorphic (P)
• Percent of loci with 1 allele

13
Types of markers

• Co-dominant
• Allozymes
• Enzymes (proteins), not DNA
• Cheap and fast
• Requires tissue where enzyme is active
• Evolutionary relationships among alleles not
  apparent
• Not necessarily neutral
• American oyster

14
Types of markers

• Co-dominant
• RFLP
• Restriction enzymes cut DNA at specific places
• EcoRV GATATC
• AluI AGTC
• DNA is cut into pieces by one or more enzymes
• Animation
• The number of pieces and their length can be
  considered alleles
• First used on circular, haploid DNA (mtDNA,
  cpDNA)
• Now used with PCRPCR-RFLP, allows DNA to be
  characterized without large amount of tissue
• Animation

15
Restriction enzyme recognition sites
GAATTC CTTAAG
G AATTC CTTAA G

• EcoRI
• EcoRV
• MboI
• AvaI

GATATC CTATAG
GAT ATC CTA TAG
GATC CTAG
GATC CTAG
CYCGRG GRGCYC
C YCGRG GRGCY C
Y T or C R A or G
16
(No Transcript)
17
PCR

• In vitro creation of DNA
• Thermochemical xerox machine
• 3 steps denature, anneal, extend
• Chain-reaction aspect invented by Kary Mullis
  (Nobel Prize)
• Thermus aquaticusbacteria in hot springs
• Thermostable DNA polymerase
• Animation

18
Types of markers

• Co-dominant
• SNPs (pronounced snips)
• Single nucleotide polymorphisms
• A site in a DNA sequence that is variable for 2
  nucleotides (alleles)
• The site becomes the locus

19
Types of markers

• Co-dominant
• Microsatellites
• Length mutations
• Highly variable
• Evolutionary model not known
• Infinite allele model
• Stepwise mutation model
• Two-phase model

20
Mechanisms of mutation(slipped-strand mispairing)
21
Types of markers

• Dominant
• RAPD (Random Amplified Polymorphic DNA)
• 1 10-bp primer used in PCR
• Multiple sites amplified
• Electrophoresis produces banding patterns
• Problems with reproducibility
• Some journals no longer accept RAPD studies

22
Types of markers

• Dominant
• AFLP (Amplified Fragment Length Polymorphism)
• Cut DNA to produce fragments with sticky ends
• Attach known sequence of DNA to sticky end
• Design primer to anneal to known section
• Amplify via PCR
• Electrophorese PCR product to get banding pattern

23
DNA sequences

• Co-dominant
• Ultimate level of resolution
• Neutral or functional variation
• Population-level to deep phylogeny

24
Wildlife Forensics

• Molecular genetic techniques can identify
  products of illegal harvest of protected species
• Example Detection of Illegal hunting and sale of
  meat from protected whales by Japan and Norway
  (Baker and Palumbi 1996)
• IWC instituted a global moratorium on commercial
  whaling in 1985
• Japan and Norway continued to hunt few species
  (minke) for scientific purposes - meat sold
  commercially
• Issue Were protected species also being
  illegally harvested and meat sold as from species
  that could be hunted?

25
Genetic Analysis of Whale Meat

• Undercover purchase of supposedly-legally
  hunted fresh whale meat (i.e. minke) in Japanese
  markets
• Illegal to transport tissues from protected
  species
• PCR-amplification of whale mtDNA control region
  sequence in hotel room in Tokyo
• Sequenced in US labs

26
Analysis of 16 Whale Meat Samples
Samples 19b, 41, 3 11, WS4 from Protected
species 19b - Humpback 41, 3, 11, WS4 - Fin
whales 16, 13, 28 - Porpoise and dolphin
27
Conclusions

• Results of analysis stricter controls over sale
  of scientifically harvested whale meat
• Meat harvested prior to harvest bans genotyped to
  monitor distribution
• Other applications species identity of caviar
  and seal penises, population origin of poached
  chimps

28
Use of Genetic Markers to Identify Units of
Conservation in Natural Populations

• e.g. Daugherty et al.1990. Neglected taxonomy and
  continuing extinctions of tuatara (Sphenodon).
  Nature 347177-179.
• Ancient reptilian lineage - restricted to 12
  island groups off of New Zealand
• For conservation purposes all populations
  classified as belonging to one species (S.
  punctatus) despite earlier work suggesting
  multiple species
• Analysis of phylogenetic relationships among
  individuals using allozyme loci

29
3 phylogenetically distinct lineages identified
1 previously unrecognized species restricted to
single island - no special status Bad
Taxonomy Can Kill