Jordans Notes 9 Flashcards
What are the three steps to PCR?
- Denaturation
- Annealing
- Extension
What are 4 important considerations in choosing
primers for PCR?
- Select conserved regions
- pairs must have similar annealing temperature (T )
- Must select a unique sequence so that it doesn’t bind somewhere else
- Primers should not anneal to themselves
What two things are specific primers useful for?
-detection / absence in eDNA
-species confirmation
How do universal primers allow for slight
differences in sequences between species?
primers contain degeneracy
different combinations of basepairs
If no bands show in the gel electrophoresis, what
are the 5 reasons?
- No DNA
- Primers don’t match → redesign
- Annealing temp too high → lower annealing temp
- Old reagents → get new
- Inhibitors present → dilute
what is added during the binding stage of spin
column extraction?
Ethanol to aid in salt bridge formation between the DNA and silica
What is fixation?
when an allele becomes the only one present in a population (f=1.0)
What does high gene flow result in between
populations?
populations are similar with higher genetic variation
What two things lead to homozygosity?
- Genetic drift
- Low gene flow
What are the 5 assumptions for the hardy-weinberg
equilibrium?
- No natural selection
- No mutation
- No gene flow (migration)
- Population is infinitely large (preventing genetic drift)
- Mating is random
What are the two equations for HWE?
What is the first step of calculating the Hardy-Weinberg equilibrium?
What is the name of the chain termination method
of sequencing?
Sanger dideoxy Sequencing
What are the chain terminators in sanger
sequencing?
dideoxy nucleoside triphosphates (ddNTPs)
How many separate reactions are done for sanger
sequencing?
4: one for stopping each base pair A, T, G, C
How is the Sanger product read?
Put through capillary gel electrophoresis
ddNTPs fluoresce a specific color when the laser shines on them
fluorescence order is recorded in sequence from smallest fragment size to
largest
What kinds of DNA can you use Sanger
sequencing for?
only DNA from an individual organism
What is a homologous genetic characteristic?
Aligned sequences between species
showing that the same DNA exists in both individuals
What does a phylogenetic branch indicate?
time in regards to accumulated nucleotide differences
What is a phylogenetic node
a hypothesised ancestor
What is a root node?
The common ancestor of all organisms presented on the phylogenetic tree
At the very bottom of the tree
What does bifurcation indicate?
A speciation event
What is a new derived trait of a single lineage
called?
An apomorphic trait
The old derived traits are plesiomorphic
What kind of substitution is more common?
Transition
What is a neutral or synonymous mutation?
A mutation that occurs in a non-coding region or doesn’t change the resulting protein
no change in natural fitness
No change in phenotype
What is the molecular clock hypothesis
That the rate of base pair changes is constant enough to predict times of divergence
What was the first molecular clock?
The albumin protein
What methods generate millions of parallel short
reads?
Second generation sequencing
What are the two second generation sequencing
machines?
- Illumina
- Ion torrent
Which sequencing method detects a change in
pH?
Ion Torrent
What is the depth of coverage?
The number of times a nucleotide is read during sequencing
PCR increases the depth of coverage (many copies of the same strand)
i.e. high depth of coverage = more reliable reads
What process is interested in a single nucleotide
and disregards all surrounding sequence?
SNPs genotyping
only the SNP is of interest
What are the different names of each length of a
short tandem repeat unit
dinucleotide repeat
trinucleotide
tetranucleotide
pentanucleotide
Where in the genome are microsatellites usually
found?
non-coding regions
mutations can build up without deleterious effects
Why do short tandem repeats tend to create extra
repeat units?
slippage during DNA replication
What form of genotyping looks at high
polymorphism alleles?
Microsatellites
SNPs tend to have very low polymorphism (only 2, rarely 3 variants)
What makes microsatellite alleles different from
each other?
Different number of repeat units means different fragment length
Gel electrophoresis can read the fragment length
How many different microsatellite loci are
necessary for accurate genotyping?
8 to 20 loci
What two ways can you multiplex a microsatellite
analysis?
Before or After PCR
both use fluorescent primers
Why might multiplexing microsatellites before the
PCR be an issue?
Shorter fragments amplify easier so longer fragments may fail (allelic dropout)
