Jordans Notes 9

Question 1

What are the three steps to PCR?

Answer 1

1. Denaturation
2. Annealing
3. Extension

Question 2

What are 4 important considerations in choosing
primers for PCR?

Answer 2

1. Select conserved regions
2. pairs must have similar annealing temperature (T )
3. Must select a unique sequence so that it doesn’t bind somewhere else
4. Primers should not anneal to themselves

Question 3

What two things are specific primers useful for?

Answer 3

-detection / absence in eDNA
-species confirmation

Question 4

How do universal primers allow for slight
differences in sequences between species?

Answer 4

primers contain degeneracy
different combinations of basepairs

Question 5

If no bands show in the gel electrophoresis, what
are the 5 reasons?

Answer 5

1. No DNA
2. Primers don’t match → redesign
3. Annealing temp too high → lower annealing temp
4. Old reagents → get new
5. Inhibitors present → dilute

Question 6

what is added during the binding stage of spin
column extraction?

Answer 6

Ethanol to aid in salt bridge formation between the DNA and silica

Question 7

What is fixation?

Answer 7

when an allele becomes the only one present in a population (f=1.0)

Question 8

What does high gene flow result in between
populations?

Answer 8

populations are similar with higher genetic variation

Question 9

What two things lead to homozygosity?

Answer 9

1. Genetic drift
2. Low gene flow

Question 10

What are the 5 assumptions for the hardy-weinberg
equilibrium?

Answer 10

1. No natural selection
2. No mutation
3. No gene flow (migration)
4. Population is infinitely large (preventing genetic drift)
5. Mating is random

Question 11

What are the two equations for HWE?

Answer 11

Question 12

What is the first step of calculating the Hardy-Weinberg equilibrium?

Answer 12

Question 13

What is the name of the chain termination method
of sequencing?

Answer 13

Sanger dideoxy Sequencing

Question 14

What are the chain terminators in sanger
sequencing?

Answer 14

dideoxy nucleoside triphosphates (ddNTPs)

Question 15

How many separate reactions are done for sanger
sequencing?

Answer 15

4: one for stopping each base pair A, T, G, C

Question 16

How is the Sanger product read?

Answer 16

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

Question 17

What kinds of DNA can you use Sanger
sequencing for?

Answer 17

only DNA from an individual organism

Question 18

What is a homologous genetic characteristic?

Answer 18

Aligned sequences between species
showing that the same DNA exists in both individuals

Question 19

What does a phylogenetic branch indicate?

Answer 19

time in regards to accumulated nucleotide differences

Question 20

What is a phylogenetic node

Answer 20

a hypothesised ancestor

Question 21

What is a root node?

Answer 21

The common ancestor of all organisms presented on the phylogenetic tree
At the very bottom of the tree

Question 22

What does bifurcation indicate?

Answer 22

A speciation event

Question 23

What is a new derived trait of a single lineage
called?

Answer 23

An apomorphic trait
The old derived traits are plesiomorphic

Question 24

What kind of substitution is more common?

Answer 24

Transition

Question 25

What is a neutral or synonymous mutation?

Answer 25

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

Question 26

What is the molecular clock hypothesis

Answer 26

That the rate of base pair changes is constant enough to predict times of divergence

Question 27

What was the first molecular clock?

Answer 27

The albumin protein

Question 28

What methods generate millions of parallel short
reads?

Answer 28

Second generation sequencing

Question 29

What are the two second generation sequencing
machines?

Answer 29

1. Illumina
2. Ion torrent

Question 30

Which sequencing method detects a change in
pH?

Answer 30

Ion Torrent

Question 31

What is the depth of coverage?

Answer 31

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

Question 32

What process is interested in a single nucleotide
and disregards all surrounding sequence?

Answer 32

SNPs genotyping
only the SNP is of interest

Question 33

What are the different names of each length of a
short tandem repeat unit

Answer 33

dinucleotide repeat
trinucleotide
tetranucleotide
pentanucleotide

Question 34

Where in the genome are microsatellites usually
found?

Answer 34

non-coding regions
mutations can build up without deleterious effects

Question 35

Why do short tandem repeats tend to create extra
repeat units?

Answer 35

slippage during DNA replication

Question 36

What form of genotyping looks at high
polymorphism alleles?

Answer 36

Microsatellites
SNPs tend to have very low polymorphism (only 2, rarely 3 variants)

Question 37

What makes microsatellite alleles different from
each other?

Answer 37

Different number of repeat units means different fragment length
Gel electrophoresis can read the fragment length

Question 38

How many different microsatellite loci are
necessary for accurate genotyping?

Answer 38

8 to 20 loci

Question 39

What two ways can you multiplex a microsatellite
analysis?

Answer 39

Before or After PCR
both use fluorescent primers

Question 40

Why might multiplexing microsatellites before the
PCR be an issue?

Answer 40

Shorter fragments amplify easier so longer fragments may fail (allelic dropout)