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Lecture 2: The code of life Review of the Essentials

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northern. dusky. cod. pollock. Price (US $) Larval identification ... Bought in Pike Place Market. Two molecular methods. Mitochondrial DNA RFLP ... – PowerPoint PPT presentation

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Title: Lecture 2: The code of life Review of the Essentials


1
Lecture 2The code of lifeReview of the
Essentials
2
Reading material
  • Three textbooks
  • Mentioned last time
  • Your introductory biology textbook
  • Reading material
  • Will be put on homepage
  • Selected bibliography
  • Will be used in exams
  • Tuesdays lecture
  • Carvalho GR (1998) Molecular ecology origins and
    approach. In Advances in Molecular Ecology,
    IOSPress, Amsterdam. Pp. 1-23
  • Required sections 1-4 (pp. 1-10)

3
Milestones in DNA history
  • 1859 - Charles Darwin
  • On The Origin of Species by Means of Natural
    Selection or The Preservation of Favoured Races
    in the Struggle for Life (1859)
  • No clear idea of how variability was created
  • Our ignorance of the laws of variation is
    profound
  • 1866 Gregor Mendel (Austrian monk)
  • Hybridization experiment with peas
  • parents provide particles that are passed onto
    offspring.

4
Milestones in DNA history
  • 1900
  • Rediscovery of Mendels work
  • 1949 Erwin Chargraff
  • Composition of DNA
  • GC AT
  • 1952 - Rosalind Franklin
  • X ray crystallography
  • Crystallized DNA
  • Reflects X rays
  • Positions of atoms in 3D

5
Milestones in DNA history
  • 1953 - James Watson (biologist) Francis Crick
    (physicist)
  • Calculated 3D structure of DNA
  • Based on Franklins X-rays
  • Published 50 years ago (almost to the day)
  • Watson, J. D. and F.H.C. Crick. 1953 A Structure
    for Deoxyribose Nucleic Acid. Nature 171 page
    737
  • 1962 Nobel Prize
  • Rosalind Franklin had died

6
The structure of DNA
  • Sugar-phosphate backbone
  • 5 C-atoms in the sugar
  • Chain is directional
  • 3 on one side
  • 5 on the other
  • Two different sugars
  • DNA
  • RNA
  • Nitrogenous base
  • Purines A, G
  • Pyrimidines C, T

Pyrimidines
Purines
7
The structure of DNA
  • Complimentary binding
  • Hydrogen bonds
  • Purine with Pyrimidine
  • A T
  • G C
  • Chain is antiparallel

8
The structure of DNA
  • Each base pair causes a twist
  • 360o turn every 10 base pairs
  • The double helix
  • Human Genome
  • 3billion bases
  • 3,000,000,000
  • About 2.3 m long
  • How does that fit into each small cell?

9
How does lots of DNA fit into small cells?
  • DNA double helix
  • Coiled
  • Wrapped around protein pellets
  • Nucleosomes
  • Pearls on a chain
  • Coiled again
  • Chromatin
  • Wrapped into chromosome
  • Genome consists of many chromosomes
  • 3 several hundred
  • Needs to unwrap for
  • Replication
  • Cell division
  • Transcription
  • Gene expression

10
The Karyotype
  • Chromosome number
  • Often species-specific
  • Even populations
  • Ploidy
  • Number of sets of chromosome
  • 2N diploid
  • N haploid
  • XN polyploid
  • Ploidy change
  • Reproductive isolation
  • Speciation mechanism
  • Kinds of chromosomes
  • Sex chromosomes
  • Heterogametic sex
  • Males XY (mammals)
  • Females WZ (birds)
  • autosomes

11
DNA Replication
It has not escaped our notice that the specific
pairing we have postulated immediately suggests
a possible copying mechanism for the genetic
material. Watson Crick, 1953
12
DNA Replication
  • DNA polymerase
  • Catalyzes the synthesis of new DNA strand from
    free nucleotides
  • Only works 5 ? 3
  • Bits connected by DNA ligase
  • Very fast and very accurate
  • Proofreading enzymes
  • 1 in 1 billion error
  • mutation

13
DNA replication and the cell cycle
  • DNA replication
  • Chromosomes duplicated
  • Chromatids
  • Split up during mitosis
  • Cell cycle
  • DNA replication happens in interphase between
    cell divisions
  • Before and after periods of metabolic activity
  • G1 single chromosomes
  • Cell grows
  • S DNA replication
  • G2 chromosomes with 2 chromatids
  • Cell produces proteins essential to cell division

14
MitosisCell division
centrosome
Mitotic spindle
Interphase
Prophase
Metaphase
Metaphase
Anaphase
Telophase Cytokinesis
15
DNA Replication in the TubePCR
  • Polymerase Chain Reaction
  • Most important recent discovery (1985)
  • Patented all PCR reactions pay royalty
  • Repeated replication of specific DNA sections
  • Small quantities
  • Feathers, hair etc.
  • Specific regions of DNA
  • Target specific sequences
  • Need PCR primers
  • Polymerase can only start synthesizing from
    double stranded DNA
  • Start where primer anneal
  • 5 ? 3
  • Short artificial DNA sequences
  • 15-20 bp
  • Match template DNA
  • Can pick where we want to start PCR

16
PCR
  • How does it work
  • Separate the two strands (94oC)
  • Anneal primers (55oC)
  • Replication start
  • Extension (72oC)
  • replication
  • Repeat 20 30 times
  • Produces billions of copies of template DNA

94
94
72
55
5
3
5
3
5
3
3
5
17
PCR
18
PCR in practice
  • Reaction ingredients
  • Template DNA
  • Primers
  • As a starting point
  • Forward and reverse
  • Nucleotides
  • To synthesize DNA
  • Polymerase
  • Taq polymerase
  • thermostable
  • PCR machines
  • Automatic cycling of temperature
  • Very accurate

19
PCR primers
  • PCR needs sequence information
  • Usually species specific
  • Where do we get primer sequences from?
  • Somebody may have isolated them
  • Check databases
  • Freely available on internet (GenBank)
  • Results not publishable without primer
    information
  • Heterologous primers
  • Isolated from related species
  • Very useful for many applications
  • Problem
  • may not exactly match
  • PCR does not always work
  • Primer isolation
  • Very lengthy and expensive procedure
  • 6 months work

20
PCR primers
  • Annealing temperature
  • Optimal temperature for primers to attach to the
    template DNA
  • Too high
  • Bonds dont work
  • Primer doesnt anneal
  • Too low
  • Primer may attach anywhere
  • Non-specific amplification
  • Depends on strength of bonds
  • Remember
  • G-C three hydrogen bonds
  • A-T two hydrogen bonds
  • Annealing temperature dependson GC content

21
Summary of Key Concepts
  • Milestones of DNA history
  • Darwin, Mendel, Franklin, Watson Crick
  • The structure of DNA
  • Nucleotides
  • Sugar - Phosphate backbone, nitrogenous base
  • Complimentary binding
  • Directionality of DNA
  • 5 end, 3 end, antiparallel strands
  • Coiling of DNA
  • Double helix, histones, nucleosomes, chromatin,
    chromosomes
  • Karyotype
  • Chromosome number, ploidy
  • Autosomes, sex chromosomes

22
Summary of Key Concepts
  • DNA replication
  • Complimentary binding
  • DNA polymerase, ligase
  • Cell cycle phases, mitosis
  • PCR
  • Purpose, principles
  • Produce millions of copies of specified DNA
    region
  • PCR in practice
  • Primers
  • Where can we get them from?
  • Annealing temperatures

23
Lab Project
  • Rockfish (Sebastes sp.)
  • 65 species along the US west coast
  • Long-lived (up to 100 years)
  • Ovoviviparous
  • Slow reproduction
  • Commercially very important

24
Rockfish Fisheries
  • Most are overexploited
  • Some below 10 of the initial population
    abundance
  • Very slow reproduction
  • Maturation 20-30 y
  • Conservation status
  • Almost all overfished
  • Not yet endangered
  • Many considered vulnerable
  • E.g. copper, quillback and brown in Puget Sound

Changes in biomass of yelloweye rockfish (CA)
Decrease in abundance
25
Multi-species Fisheries and Bycatch
  • 65 species categorized as
  • Near-shore, Shelf, Slope
  • Detailed management plans only for 10 species
  • Many caught as by-catch
  • E.g. Halibut fishery (BC)
  • 77,000 t (2003)
  • 21 species
  • 15 30 discarded (AFS)
  • Usually die anyway
  • Species identification
  • Difficult
  • Color, spines, morphology
  • Not possible in
  • Fillets
  • larvae

26
Fillet Identification
  • Some species may become threatened
  • Currently vulnerable
  • Important to identify illegal catches
  • Some rockfish are more equal than others
  • Difference in price
  • 8 c - gt 1
  • Labeling of fillets can be an issue
  • Fair trading
  • Consumer protection

?
27
Larval identification
  • Many species not identifiable morphologically
  • Abundant
  • Most abundant fish larva
  • Important
  • Larval distribution
  • Ecology
  • Stock assessment
  • Recruitment

28
Whos who?
Copper (S. caurinus)
Pacific Ocean Perch (S. alutus)
Shortspine Thornyhead (Sebastolobus alascanus)
Rougheye (S. aleutianus)
Shortraker (S. borealis)
29
How to identify rockfish larvae and fillets
  • Molecular markers
  • Can separate species
  • DNA
  • Proteins (enzymes)
  • We will use
  • Two mystery fillets
  • Bought in Pike Place Market
  • Two molecular methods
  • Mitochondrial DNA RFLP
  • Allozymes (protein marker)
  • More later
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