Lecture 14

Question 1

Molecular markers and their relative popularity since 1966:

Answer 1

• Allozyme
• RFLP
• Minisatellite
• Microsatellite
• RAPD
• AFLP
• DNA sequencing
• SNP
• NGS

Question 2

Allozyme:

Answer 2

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

Question 3

RFLP:

Answer 3

• Genetic variability between restriction fragment length polymorphism

Question 4

Minisatellite

Answer 4

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

Question 5

Microsatellite:

Answer 5

• Short/simple sequence repeats (SSR)

- Popular in the 90’s in population genetics

Question 6

RAPD:

Answer 6

• Not popular anymore

- Hard to reproduce the same results more than once

Question 7

AFLP:

Answer 7

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

Question 8

DNA sequencing

Answer 8

• Popular now

Question 9

SNP:

Answer 9

• Single nucleotide polymorphism

- Streamline the genotyping assay, involving biophysics and bioengineerying

Question 10

NGS:

Answer 10

• Next Generation Sequencing

- High throughput sequencing

Question 11

Amplified fragment length polymorphism has 6 steps

Answer 11

• A detailed process with a series of enzymes

Question 12

1. Digestion of gDNA with EcoRI and MseI:

Answer 12

• ## gDNA is genomic DNA (good DNA) because it must be pure in order for the next step to work

Question 13

2. Make ‘adaptor’ by annealing 2 complementary oligos:

Answer 13

• Join the two cut sites together

Question 14

3. Adaptor ligation:

Answer 14

• Attach the adaptor, we know the sequence of (which will help us identify the sequence of the test DNA
• The adaptor structure

Question 15

4. Pre-amplification PCR using ‘Eco+1’ and ‘Mse+1’ primers to enrich templates

Answer 15

• PCR, using the same primers, which recognise the adaptor sequences we will use

Question 16

5. Label an ‘Eco+3’ primer with radioactive istotope or flourescence

Answer 16

• 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

Question 17

6. Selective amplification using labelled ‘Eco+3’ and unlabelled ‘Mse+3’ primers

Answer 17

• Labelled sequences can be scanned to show bands with the tag attached

Question 18

7. Gel electrophoresis and expose gel to X-ray film

Answer 18

• 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

Question 19

AFLP advantages:

Answer 19

• 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

Question 20

AFPL disadvantages:

Answer 20

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

Question 21

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

Answer 21

• 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

Question 22

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

Answer 22

• Smaller than that you can’t score it

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

Question 23

Dominance:

Answer 23

• 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

Question 24

How do you make it no longer anonymous

Answer 24

• 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