Lecture 14 Flashcards Preview

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Flashcards in Lecture 14 Deck (24):
1

Molecular markers and their relative popularity since 1966:

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

2

Allozyme:

- 60's and 70's
- Polymorphisms at the molecular level
- The protein level (enzymes)

3

RFLP:

- Genetic variability between restriction fragment length polymorphism

4

Minisatellite

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

5

Microsatellite:

- Short/simple sequence repeats (SSR)
- Popular in the 90's in population genetics

6

RAPD:

- Not popular anymore
- Hard to reproduce the same results more than once

7

AFLP:

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

8

DNA sequencing

- Popular now

9

SNP:

- Single nucleotide polymorphism
- Streamline the genotyping assay, involving biophysics and bioengineerying

10

NGS:

- Next Generation Sequencing
- High throughput sequencing

11

Amplified fragment length polymorphism has 6 steps

- A detailed process with a series of enzymes

12

1. Digestion of gDNA with EcoRI and MseI:

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

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2. Make 'adaptor' by annealing 2 complementary oligos:

- Join the two cut sites together

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3. Adaptor ligation:

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

15

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

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

16

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

- 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

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

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

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

- 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

AFLP advantages:

- 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

AFPL disadvantages:

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

21

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

- 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

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

- Smaller than that you can't score it
- Larger than that they are too big and so are un-reliable

23

Dominance:

- 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

How do you make it no longer anonymous

- 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