molecular markers Flashcards
(31 cards)
what are molecular markers
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
locus meaning
the fixed position on a chromosome - the variant forms of an allele
kinds of molecular markers
- single base differences - single base pairs differ
- repeat regions - certain regions within the genome repeat themselves
- many different ways of measuring them - techniques have evolved
5 reasons for using molecular markers
- Conservation biology
- Genetic diversity
- Measure inbreeding
- Detecting invasive species - Agriculture & aquaculture
- Trait selection
- Pedigrees + breeding
- Domestication - Forensic science
- DNA fingerprinting
- Parental analysis
- Archaeology - Disease
- Diagnosis + heritability
- Functional genomics
- Personalised medicine - Evolutionary biology
- Response to environmental change
- Speciation
- Historical patterns of dispersal
what is the first molecular marker
proteins
- Changes in DNA sequence may result in changes to amino acid sequence + protein structure
explain allozymes as a molecular marker
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
what are the early DNA molecular markers
Restriction Fragment Length Polymorphisms (RFLPs)
- e.g. sickle cell disease (β-globin gene)
explain RFLPs
- 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
what are the 2 Advances in molecular biology
- PCR
- DNA sequencing (1st, 2nd & 3rd gen)
what are 1st gen markers
sequence data (highly accurate) and Microsatellites dominate - still in use
what are 2nd & 3rd gen markers
- 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
explain the evolution of molecular ecology
- dominance of DNA based methods
- microsatellites + emergence of DNA sequencing
- emergence of NGS (2nd gen)
- emergence of 3rd gen sequencing (long reads)
explain macrosatellites (STRs)
- 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
name some ways we can apply macrosatellites (STRs)
-genome mapping
- forensic science
- hybridisation + breeding
- taxonomic + phylogenetic studies
- population genetics + conservation biology
technique used with microsattelites
- template DNA (allele 1 and 2)
- PCR with florescent primers
- gel electrophoresis : Tells us number of base pairs in each individual
- sanger sequencing : Produces different colours – means when it goes through sanger sequencing we can use different markers at same time
how are micro satellites used in DNA fingerprinting
- 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)
technique used to use micro satellites in DNA fingerprinting
- Collect DNA sample e.g. hair, sweat, blood – only need a tiny amount
- PCR using a specific set of primers designed to amplify microsatellite regions
- Separate DNA fragments using gel or capillary electrophoresis
- Match the unique set of DNA fragments to a database or family members
what are the Workflow (bioformatics) steps
- DNA extraction
- PCR -> amplify specific sequences (Each cycle increases the copy number exponentially)
- Gel electrophoresis -> visualise DNA fragments
- Sanger sequencing OR NGS -> Identify/confirm specific sequences
what enzyme is used in PCR amplification
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
what are PCR primers
- 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
what is the PCR process
- Denaturation (96 °C): Heat denatures DNA strands > single stranded template (separated both strands)
- Annealing (45-65 °C): cooling allows primers to bind to complementary sequences
- Extension (72 °C): Optimal temperature for Taq polymerase to synthesise new strands of DNA
what is Sanger sequencing and what are its pros and cons
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
how does Sanger sequencing work
- Dye -labelled dideoxy nucleotides (ddNTP)
- PCR Denaturing, annealing and extension
- dNTPs are added until a ddNTP is added
what is sequence data
- ‘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
