Molecular ecology 1

Question 1

What is the point of molecular ecology?

Answer 1

Helps us;
*know if an organism is a hybrid
*understand the relationships between different taxa
*find the Genetic diversity
*understanding breeding programmes
*with forensics
*look at dispersal (infectious diseases)
*look at adaptations

Question 2

What is restriction fragment length polymorphism (RFLP)?

Answer 2

a technique that exploits variations in homologous DNA sequences, known as polymorphisms, populations, or species or to pinpoint the locations of genes within a sequence. Helps us identify a species.

Question 3

What are the steps of RFLP to find out a species?

Answer 3

There main steps:
1. DNA extraction
2. DNA amplification: Polymerase Chain Reaction (PCR)
3. Examination of DNA sequence/fragments

1. extract DNA from a battered fish
2. amplify a region of the MT-CYB gene using Polymerase Chain Reaction (PCR)
3. cut the PCR fragment with a restriction enzyme
4. run an agarose gel electrophoresis to separate the DNA fragments (bands)
5. analyse the pattern of fragments and compare with a known standard to identify the fish species (or sequence & analyse the PCR-amplified
   fragment)

Question 4

Why do we need to extract the DNA?

Answer 4

• break down cell walls/membranes
• remove all cellular material (e.gRNA) except DNA
• clean and resuspend DNA either in water or a low concentration buffer suitable for storage

Question 5

What are the factors that affect the type of technique that is used?

Answer 5

• the type of tissue
• what you need to do with your DNA
• what techniques other people have used for similar organisms
• time/money/resources/expertise

Question 6

Describe HotShot?

Answer 6

(quick, cheap and easy)
Boil in NAOH (highly alkaline) to release DNA from cells, then neutralize by adding buffer at pH 8

Question 7

How do you know you have successfully extracted DNA from your tissue sample?

Answer 7

*Run an agarose gel (takes an hour too long)
*spectrophotometer (measures absorbance of solution) relies on the fact that DNA absorbs around 260nm

Question 8

What is the absorbance of proteins?

Answer 8

mainly 280nm

absorbance at 260 divided by absorbance at 280 to find how pure DNA is.
phenol absorbs at 230nm

Question 9

need to quantify the DNA in the sample what calculation can we use (dilution)?

Answer 9

• C(initial) x V(initial) = C(final) x V(final)

Question 10

What is DNA replication in cells like?

Answer 10

• highly controlled
• starts from multiple origins of replications along chromosomes
• only happens in S-phase (in eukaryotic cells)
• results in a “double copy” of the genome

Question 11

What is the (simple) central dogma of molecular biology?

Answer 11

DNA -> RNA -> protein

Answer 12

5’ to 3’

Question 13

DNA replication

Answer 13

DNA replication is semi-conservative and starts with “RNA primers”

Question 14

What do you do in vitro (in tube) PCR?

Answer 14

*Denature, heat to 95 degrees C= one molecule of double stranded DNA is denatured, by breaking hydrogen bonds.

• Anneal heat to 40-60 degrees C= Annealng of the RNA/DNA primers oligonucleotides around 25 bases then attach to specific region of DNA.
  primers are designed to specific region you want to amplify, so they have the complementary sequence.

*extension, 72 Degrees C (for polymerase)= continue the cycles. Exponential amplification

forward primer=3 to 5
reverse primer=5 to 3

Question 15

What do you need to have in the tube before starting PCR?

Answer 15

1.Template DNA
2. Forward and reverse primers
3. dNTPs (deoxyribonucleotides)
4. DNA polymerase
5. Buffers and catalysts