Lecture 14 Flashcards Preview

Evolutionary Genetics and Genomics > Lecture 14 > Flashcards

Flashcards in Lecture 14 Deck (24)
Loading flashcards...
1
Q

Molecular markers and their relative popularity since 1966:

A
  • Allozyme
  • RFLP
  • Minisatellite
  • Microsatellite
  • RAPD
  • AFLP
  • DNA sequencing
  • SNP
  • NGS
2
Q

Allozyme:

A
  • 60’s and 70’s
  • Polymorphisms at the molecular level
  • The protein level (enzymes)
3
Q

RFLP:

A
  • Genetic variability between restriction fragment length polymorphism
4
Q

Minisatellite

A
  • Based on the use of restriction enzymes and reliant on the hybridisation phase of the probe
  • Slow
5
Q

Microsatellite:

A
  • Short/simple sequence repeats (SSR)

- Popular in the 90’s in population genetics

6
Q

RAPD:

A
  • Not popular anymore

- Hard to reproduce the same results more than once

7
Q

AFLP:

A
  • Amplified Fragment Length Polymorphism
  • Use of Restriction enzyme and PCR
  • Anonymous genome-wide markers
8
Q

DNA sequencing

A
  • Popular now
9
Q

SNP:

A
  • Single nucleotide polymorphism

- Streamline the genotyping assay, involving biophysics and bioengineerying

10
Q

NGS:

A
  • Next Generation Sequencing

- High throughput sequencing

11
Q

Amplified fragment length polymorphism has 6 steps

A
  • A detailed process with a series of enzymes
12
Q
  1. Digestion of gDNA with EcoRI and MseI:
A
  • ## gDNA is genomic DNA (good DNA) because it must be pure in order for the next step to work
13
Q
  1. Make ‘adaptor’ by annealing 2 complementary oligos:
A
  • Join the two cut sites together
14
Q
  1. Adaptor ligation:
A
  • Attach the adaptor, we know the sequence of (which will help us identify the sequence of the test DNA
  • The adaptor structure
15
Q
  1. Pre-amplification PCR using ‘Eco+1’ and ‘Mse+1’ primers to enrich templates
A
  • PCR, using the same primers, which recognise the adaptor sequences we will use
16
Q
  1. Label an ‘Eco+3’ primer with radioactive istotope or flourescence
A
  • The primer must be labelled so that we can see the product at the 5’ end
  • To select more specific sequences, so adding an extra A and G
17
Q
  1. Selective amplification using labelled ‘Eco+3’ and unlabelled ‘Mse+3’ primers
A
  • Labelled sequences can be scanned to show bands with the tag attached
18
Q
  1. Gel electrophoresis and expose gel to X-ray film
A
  • On polyacrylemide gel to separate the products based on their sizes
  • The result will either be positive or negative, and you score for the presence or absence of bands
19
Q

AFLP advantages:

A
  • There is no prior sequence required so it is suitable for any species
  • The same process is used for all species
  • Need only a small amount of DNA
  • It facilitates mapping and marker integration
  • It allows you to build a map quickly
20
Q

AFPL disadvantages:

A
  • Dominant
  • Anonymous, so it is difficult to compare across species
  • Radioactive isotopes require permits
21
Q

There are three types of products after the digests, these get used in the PCR:

A
  • A smear of sizes, but different specific ends
  • A fragment with an E end, which produce larger ends,
  • A fragment with an E, and an MSE on the ends
  • A fragment with an M end
22
Q

The range of bands you can score are about 50 - 500 base length bands (a physical limitation):

A
  • Smaller than that you can’t score it

- Larger than that they are too big and so are un-reliable

23
Q

Dominance:

A
  • As long as one allele is present you will get a band in the heterozygote
  • In the homozygote you will see the same thing,
  • You cannot tell the difference between heterozygotes and homozygotes due to dominance
24
Q

How do you make it no longer anonymous

A
  • Cut out the anonymous marker band
  • Re-amplify it using the same amplifiers to give a 200bp product
  • Sequence the marker
  • No longer anonymous
  • Convert the PCR product into a hybridisation probe, and it will be a high value genetic marker