microsatellites and SNPs Flashcards
what are molecular markers
genetic markers
specific DNA sequences with a known location in the genome that show variation between individuals, populations or species
what is a locus
the fixed position where the allele is found on each chromosome
what is the genome and what is its role
DNA
store genetic material
what is the transcriptome and what is its role
RNA
message transmission
what is the proteome and what is its role
protein
enact cellular response
what is the possible result of a change in DNA sequence
change in amino acid sequence and protein structure
what were the first molecular markers
proteins
what are allozymes
variant proteins (enzymes) encoded by different alleles
what is important about the structure of allozymes
it varies
how can allozymes be separated
gel electrophoresis based on size / charge
what do different allozymes indicate
genetic variation in a population
what followed allozymes in use as a molecular marker
RFLP’s
what does RFLP mean
restriction fragment length polymorphisms
what are RFLPs and what do they do
restriction enzymes which recognise and cut short specific stretches of DNA sequence
what is the result of mutations on RFLPs
loss or addition of cut sites
how can the DNA fragments be separated in RFLP
using a gel, separate based on size
what can be used to hybridise fragments of interest
DNA probes
give an example of when RFLPs were used as molecular markers and how
detection of sickle cell disease
mutation caused loss of restriction site
what was used in 1st generation molecular markers
sequence data and microsatellites
what is used in 2nd and 3rd generation molecular technique
SNP’s, sometimes still microsatellites
what are 4 benefits of 2nd and 3rd generation molecular techniques
more sensitive,
greater genome coverage - whole genome sequencing,
high throughput,
cheaper
what does high throughput mean
lots of sequences produced at once
where does taq polymerase come from
isolated from heat tolerant bacterium
what is the benefit of using taq polymerase
can use higher temperatures = faster
what are the PCR primers (how long are they)
18-20 base pair oligonucleotides
what are the 3 steps of PCR and what temperatures do they occur
- denaturing, 96C
- annealing, 45-65C
- extension, 72C
what happens during denaturation
heat denatures DNA strands (breaks H bonds) leaving a single-stranded template
what happens during annealing
cooling allows primers to bind to complementary sequences
what happens during extension
optimal temperature for Taq polymerase t0 synthesise new strands of DNA
how many genes can be done in Sanger sequencing
1
what is the cost of Sanger sequencing
low
what type of DNA is Sanger sequencing more useful for
mitochondrial
how does Sanger sequencing work
using dye-labelled deoxyribose nucleotides (ddNTP),
picked up by machine
what is a neutral marker
infers no reproductive disadvantage or advantage
where are neutral markers usually found
mitochondrial DNA
how does DNA barcoding work
taxonomic identification based on sequence variation
reads are compared to existing databases
what is a well known primer in DNA barcoding
CO1
what are microsatellites
repetitive DNA, widespread in eukaryotic organisms
what is another word(s) for microsatellites
short tandem repeats
how many base pairs in a microsatellite
2-6
where are microsatellites usually found
non-coding regions
how does DNA fingerprinting work
sampling multiple markers simultaneously
how much of the population will share one microsatellite allele
5-20%
how many microsatellite markers are used in DNA fingerprinting in the UK
17
what people can DNA fingerprinting not distinguish between
monozygotic twins
what occurs in DNA fingerprinting after DNA has been extracted
PCR using specific set of primers to amplify microsatellite of interest
how are the fragments from PCR separated
gel electrophoresis or capillary electrophoresis
how are the results from DNA fingerprinting interpreted
unique set of DNA fragments is matched to database or family members
how does next generation sequencing work
parallel sequencing of millions of DNA templates simultaneously
what molecular marker is most often used in NGS
SNP’s
what is a single nucleotide polymorphism (SNP)
a single base change in a DNA sequence
SNP’s are biallelic, what does this mean
the base change / mutation occurs on both alleles of the chromosome
where are SNP’s usually found
non-coding / regulatory regions
what is an SNP chip and which animals have them
SNP chips hold up to 500k SNP probes allowing identification of individuals
most animals now have an SNP chip made
what happens if there is no SNP chip for your species
whole genome must be sequenced
how do we know what the faces of those that sunk on the Henry VIII flagship in 1545 look like
genotyping based on SNP’s
compare genotypes to databases of known traits e.g. height, weight, eye and hair colour, diseases
why do SNP’s outperform microsatellites
clearer population delimitation and more populations identified
what is the goal of Darwin’s tree of life programme
to sequence all described species in the UK
