Sequence variation and sequence detection

1
Sequence variation and sequence detection

• Why interesting?
• Sequence variation
• Mutations
• Somatic vs. germline
• Kinds of mutations
• Mutation rates
• Sequence detection
• Direct sequencing
• Indirect
• RFLP
• Will do in lab

2
Assignment

• 10 of course marks
• Due next Thursday before class
• Aims
• Identify the most important results and their
  significance
• Communicate the findings to a non-specialist
  audience
• Identify possible problems and their solutions
• Paper
• Garnier et al. (2001) Mating system and
  reproductive skew in black rhinoceros
• To do
• Write popular article (National Geographic)
• 1000 words
• Tell story, explain microsatellites
• Data evaluation
• Plug microsatellite data into spreadsheet
• H HO
• Use 6 most variable and 6 least variable loci
• Test assignment success and compare with paper
• Identify potential problems
• Have they dealt with them?

3
Direct and indirect sequence detection

• DNA sequence
• Ultimate code of life
• What we are after?
• Other methods used to detect it
• Allozymes
• Microsatellites
• Indirect methods detect only small part
• Question
• How do sequences mutate?
• How can we detect variation?

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Mutations

• Somatic mutations
• Occur in soma (tissue)
• Not heritable
• May cause disease
• E.g. cancer
• Germline mutations
• Heritable
• Introduce genetic variability
• One of the main driving forcesof evolution
• Random process
• Blind watchmaker

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Kinds of mutation

• Substitutions
• Synonymous / nonsynonymous
• Transitions / transversions
• Indels
• Insertions / Deletions
• Rearrangements
• Pieces of chromosomes switched around
• Transposable elements
• Move around in genome
• Gene duplications
• May free up genes from selection
• May acquire new function
• Ploidy changes

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Mutation mechanism

• Base substitutions
• Wrong bases
• Wobble pairing
• G-T, A-C
• A-G, C-T

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Substitution
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Types and effects of substitutions
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Types and effects of substitutions
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Insertion - deletion
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Mutation rates

• Depend on sequence and site
• DNA repair mechanisms
• Selection
• MtDNA gt nuclear
• Less efficient repair
• Coding gt non-coding
• Repair more efficient
• Selective removal of mutations
• Variation among coding genes
• Transversions gt transitions
• Less interuption to 3D structure
• Less likely to be repaired
• Codon position
• Related to amino acid change
• 3rd gt 1st gt 2nd

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Mutation rates
A

• Some substitutions more likely than others
• Transitions gt Transversions (101)
• Transition (Ts) purine ? purine (A,G),
  pyrimidine ? pyrimidine (C, T)
• Transversion (Tv) purine ? pyrimidine
• But repeated mutations make apparent
  transversions more common
• Deep phylogenies only use transversions

C
T
G
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Mutation rates

• Example
• Cichlid fishes
• Transversion / transition 0.1
• More divergent species
• Ratio approaches 0.5

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Mutation rates - DNA sequences Substitution
saturation

• Coding sequences
• 3 nucleotides per amino acid (AA)
• Redundant code
• Change in 3rd position usually doesnt change the
  AA
• Synonymous mutation
• Relaxed selection higher mutation rates
• Sites with multiple hits
• Observed rate of substitution lower than real
• May have to discard from analysis

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Substitution saturationribulose 1,5 biphosphate
carboxylase in algae
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Sequencing

• PCR
• dNTP as ingredients
• Building blocks for DNA
• Sanger method
• ddNTP
• Stops DNA extension
• Bad nucleotide
• 4 reactions
• A,C,G,T ddA
• A,C,G,T ddC
• A,C,G,T ddG
• A,C,G,T ddT

Deoxynucleotide triphosphate (dNTP)
Dideoxynucleotide triphosphate (ddNTP)
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Sequence Reaction
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Sequencing
nucleotides
bad
good
G A G C T A G C G A T T C A G
G A G A G C T A G A G C T A G C G A G A G C T A
G C G A T T C A
G A G C G A G C T A G C G A G C T A G C G A T T C
G G A G G A G C T A G G A G C T A G C G G A G C
T A G C G A T T C A G
G A G C T G A G C T A G C G A T G A G C T A G C G
A T T
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Electrophoresissort by size
A C G T
-
G A G C T A G C G A T T C A G G A G C T A G C G
A T T C A G A G C T A G C G A T T C G A G C T A
G C G A T T G A G C T A G C G A T G A G C T A G
C G A G A G C T A G C G G A G C T A G C G A G
C T A G G A G C T A G A G C T G A G C G A G
G A G

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Sequencing
nucleotides
bad
good
G A G C T A G C G A T T C A G
G A G A G C T A G A G C T A G C G A G A G C T A
G C G A T T C A
G A G C G A G C T A G C G A G C T A G C G A T T C
G G A G G A G C T A G G A G C T A G C G G A G C
T A G C G A T T C A G
G A G C T G A G C T A G C G A T G A G C T A G C G
A T T
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Electrophoresissort by size
-
G A G C T A G C G A T T C A G G A G C T A G C G
A T T C A G A G C T A G C G A T T C G A G C T A
G C G A T T G A G C T A G C G A T G A G C T A G
C G A G A G C T A G C G G A G C T A G C G A G
C T A G G A G C T A G A G C T G A G C G A G
G A G

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Automated sequencing
Peaks instead of bands
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Key Concepts

• Distinction of types of DNA
• Only some amenable to indirect detection
• Quest for sequence data
• Types of mutations
• Substitutions
• Synonymous / non-synonymous
• Codon position
• Transition / transversion
• Mutation rates
• Accumulation of mutations
• Multiple hits
• Transitions
• 3rd codon positions
• Direct sequencing