Lecture 14 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
- 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
- Make ‘adaptor’ by annealing 2 complementary oligos:
A
- Join the two cut sites together
14
Q
- 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
- 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
- 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
- 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
- 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
