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BIO235 - molecular ecology > molecular markers > Flashcards

molecular markers Flashcards

1
Q

what are molecular markers

A

Molecular markers = specific DNA sequences with a known location in the genome that let us see variation (polymorphism) between individuals, populations or species
- refer to genetic markers (DNA)
- can have many different types e.g. allozymes, RFLPs, Microsatellites, RAPDs, AFLPs, SNPs

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2
Q

locus meaning

A

the fixed position on a chromosome - the variant forms of an allele

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3
Q

kinds of molecular markers

A
  1. single base differences - single base pairs differ
  2. repeat regions - certain regions within the genome repeat themselves
    - many different ways of measuring them - techniques have evolved
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4
Q

5 reasons for using molecular markers

A
  1. Conservation biology
    - Genetic diversity
    - Measure inbreeding
    - Detecting invasive species
  2. Agriculture & aquaculture
    - Trait selection
    - Pedigrees + breeding
    - Domestication
  3. Forensic science
    - DNA fingerprinting
    - Parental analysis
    - Archaeology
  4. Disease
    - Diagnosis + heritability
    - Functional genomics
    - Personalised medicine
  5. Evolutionary biology
    - Response to environmental change
    - Speciation
    - Historical patterns of dispersal
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5
Q

what is the first molecular marker

A

proteins
- Changes in DNA sequence may result in changes to amino acid sequence + protein structure

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6
Q

explain allozymes as a molecular marker

A

allozymes = variant proteins (enzymes) encoded by different alleles
- vary structurally (i.e. different mass, charge or shape)
- can be separated by gel electrophoresis based on size/charge
- different allozymes indicate genetic variation in a population
- Allozyme analysis provides information about genetic diversity at the protein level

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7
Q

what are the early DNA molecular markers

A

Restriction Fragment Length Polymorphisms (RFLPs)
- e.g. sickle cell disease (β-globin gene)

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8
Q

explain RFLPs

A
  • Restriction enzymes - recognise & cut short, specific stretches of DNA sequence
  • Mutations cause loss or addition of cut sites
  • Separate DNA fragments with a gel based on size
  • Hybridise fragments of interest using specific DNA probes
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9
Q

what are the 2 Advances in molecular biology

A
  1. PCR
  2. DNA sequencing (1st, 2nd & 3rd gen)
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10
Q

what are 1st gen markers

A

sequence data (highly accurate) and Microsatellites dominate - still in use

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11
Q

what are 2nd & 3rd gen markers

A
  • more powerful + sensitive molecular markers
  • Typical markers = SNP’s but still microsatellites and sequence data
  • greater genome coverage- Whole Genome Sequencing
  • high-throughput - lots of sequences at once
  • cheaper overall
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12
Q

explain the evolution of molecular ecology

A
  1. dominance of DNA based methods
  2. microsatellites + emergence of DNA sequencing
  3. emergence of NGS (2nd gen)
  4. emergence of 3rd gen sequencing (long reads)
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13
Q

explain macrosatellites (STRs)

A
  • Repetitive DNA = widespread in eukaryotic genomes
  • Microsatellites (or STRs) = DNA loci containing variable numbers of short repeated nucleotide units
  • Typically ~2-6bp units, repeated ~5-100 times in a locus
  • First markers to take full advantage of PCR
  • Abundant & evenly distributed in eukaryotic genomes
  • Higher mutation rates than in non-repetitive regions
  • Most are in non-coding (or regulatory) regions - accumulate & can become highly polymorphic
  • Excellent neutral markers (i.e. gene flow & population structuring) e.g. Herring
  • Extensively used and very versatile
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14
Q

name some ways we can apply macrosatellites (STRs)

A

-genome mapping
- forensic science
- hybridisation + breeding
- taxonomic + phylogenetic studies
- population genetics + conservation biology

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15
Q

technique used with microsattelites

A
  1. template DNA (allele 1 and 2)
  2. PCR with florescent primers
  3. gel electrophoresis : Tells us number of base pairs in each individual
  4. sanger sequencing : Produces different colours – means when it goes through sanger sequencing we can use different markers at same time
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16
Q

how are micro satellites used in DNA fingerprinting

A
  • power of DNA fingerprinting comes from sampling multiple markers simultaneously
  • Each microsatellite allele is shared by ~5-20% of the population
  • Early DNA fingerprinting used only 4 loci = not that accurate
  • Today 17 microsatellite markers are used simultaneously in the UK - chances of matching an unrelated individual are < 1 in a billion (but cannot distinguish between monozygotic twins)
17
Q

technique used to use micro satellites in DNA fingerprinting

A
  1. Collect DNA sample e.g. hair, sweat, blood – only need a tiny amount
  2. PCR using a specific set of primers designed to amplify microsatellite regions
  3. Separate DNA fragments using gel or capillary electrophoresis
  4. Match the unique set of DNA fragments to a database or family members
18
Q

what are the Workflow (bioformatics) steps

A
  1. DNA extraction
  2. PCR -> amplify specific sequences (Each cycle increases the copy number exponentially)
  3. Gel electrophoresis -> visualise DNA fragments
  4. Sanger sequencing OR NGS -> Identify/confirm specific sequences
19
Q

what enzyme is used in PCR amplification

A

Taq polymerase
- Very heat-stable, active at 70°C - human or E.coli DNA polymerase would denature
- High temps used to denature DNA in the process of PCR

20
Q

what are PCR primers

A
  • Primers = 18-20 bp oligonucleotides
  • 2 primers flank the section of DNA to be copied
  • Taq polymerase can only make DNA if a primer is present
21
Q

what is the PCR process

A
  1. Denaturation (96 °C): Heat denatures DNA strands > single stranded template (separated both strands)
  2. Annealing (45-65 °C): cooling allows primers to bind to complementary sequences
  3. Extension (72 °C): Optimal temperature for Taq polymerase to synthesise new strands of DNA
22
Q

what is Sanger sequencing and what are its pros and cons

A

1st gen sequencing
pros:
- low cost
- highly accurate
- 100-1500bp length
- Excellent for mtDNA (one copy)
cons:
- Only does single genes (i.e. single PCR product)
- Struggles with diploid genome- heterozygous sites

23
Q

how does Sanger sequencing work

A
  • Dye -labelled dideoxy nucleotides (ddNTP)
  • PCR Denaturing, annealing and extension
  • dNTPs are added until a ddNTP is added
24
Q

what is sequence data

A
  • ‘Neutral’ markers = gene sequences where variants are considered to confer no fitness advantage e.g. CO1
  • Often Mitochondrial - multiple copies
  • Nuclear markers too (cloning resolve heterozygous sites)
  • Traditionally Sanger sequenced but also NGS
25
Q

what is DNA barcoding

A
  • Taxonomic identification based on sequence variance – identify species level
  • Target regions (e.g. 16S, COI) are variable between taxa
26
Q

what is Next generation sequencing (NGS ) and its pros and cons

A
  • Thousands of markers are now available throughout the genome
  • SNP’s = most used molecular markers today
    pros:
  • Markers under selection
  • Whole Genome sequencing
  • NGS = Massively parallel sequencing of millions of DNA templates simultaneously (but short reads)
  • Very high-throughput -> much faster & cheaper (per base)
  • Rapid improvement in technology & reduction in cost
    cons:
  • Requires high performance computing
27
Q

what are Single Nucleotide Polymorphisms (SNPs)

A
  • Usually biallelic
  • > 1% occurrence in a population (otherwise ‘a mutation’)
  • A single base change in DNA sequence
  • Account for most variation in the genome - human genome ~ 1 SNP every 1000bp
  • Most are in non-coding (or regulatory) regions - but some are functional
  • main advantage of SNPs = both neutral & selected markers
  • Lots of different methods for analysing SNPs
  • Availability of sequenced genomes, improved sequencing technologies & bioinformatics have made it much easier
  • Allows automated, high-throughput analysis at moderate cost -can screen 10s of thousands of SNPs in hundreds of individuals
  • SNP Chips = high throughput genotyping - Can include up to ~500k probes
  • If you don’t have a sequenced genome or SNP chip available for your species = whole genome sequencing (or reduced representation sequencing)
  • Align short reads to each other -> identify SNPs (and other sequence variants) for barcoded individuals
28
Q

how was SNPs used to analyse The Mary Rose that sunk in 1545

A
  • contained thousands of artefacts and well preserved skeletons
    >Facial reconstruction
    >Isotope analysis
    >Genetic analysis
    1.Specialist DNA extraction for old, degraded bones
    2.Genotyping based on 1000s SNPs (SNP chip)
    3.Compare genotypes to databases of known traits (e.g. height, weight, eye colour, hair colour, diseases) + regional SNP frequencies to identify origin
29
Q

what did they find using First Gen Sequencing compared to NGS

A

First Gen Sequencing:
- 9 Microsatellite loci
- Fragment length analysis
- Structure analyses K=2 - identifies two genetic clusters
Next Gen Sequencing:
- 38 microsatellite loci
- ~ 570 individuals genotyped
- Genotyping by Sequencing
- Structure (K=4) - identifies four genetic clusters

30
Q

compare SNP’S v Microsatellites

A

SNP’s:
- 45 SNP markers-derived whole genome sequencing (WGS)
- Many associated or even in genes controlling reproduction
- SNP’s (mostly) outperform microsatellite markers - clearer pop delimitation, more pop’s ID’d - tend to identify more genetic clusters (K=)

  • BUT it’s likely the future of research for molecular Ecology will be whole genome approaches – hence Genomic era
31
Q

name Other molecular techniques

A
  • Transcriptomics (gene expression)
    e.g. genotype-dependent transcriptomic response to drought
    1. Extract mRNA & convert to cDNA
    2. High throughput sequencing
    3. Align reads to the genome
    4. Quantify expression for each gene (x 30,000 genes) e.g. 1 : 2
  • Proteomics (& Metabolomics)
    e.g. response to temperature stress in sea squirt
    1. Extract proteins (or metabolites)
    2. Digestion & peptide enrichment
    3. HPLC separation
    4. Mass spectrometry detection

BIO235 - molecular ecology (11 decks)

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