Week 2

Question 1

What is an extract?

Answer 1

• a solution obtained by soaking something in it.

Question 2

What is a supernatant?

Answer 2

• the liquid left over from precipitation or centrifugation

Question 3

What is the molecular genetics pathway to success?

Answer 3

• sample collection
• DNA extraction
• 3. whole genome sequencing (NGS)
• 3. PCR
• gel electrophoresis
• 5. genotyping, or 5. sanger sequencing (standard one)

Question 4

Why would we need to extract DNA?

Answer 4

• need purified DNA for use in things like PCR

Question 5

Goals of purifying DNA and extracting it?

Answer 5

• removes proteins and nucleases and other things
• more stable and less degradation (also smaller than tissue samples)

Question 6

Why do we need to get rid of nucleases?

Answer 6

-nucleases break down DNA ???? so need to remove them in purification

Question 7

Objectives of a DNA extraction?

Answer 7

• maximize DNA recovery
• concentrate DNA
• remove or inhibit nucleases
• remove PCR inhibitors
• maximise DNA quality.

Question 8

Which DNA extraction method?

Answer 8

• depends on the type of sample and purpose of analysis

Question 9

What are the two main parts of DNA extraction?

Answer 9

• Lysis (solubilize DNA)
• purification (removes contaminants (proteins, RNA, macromolecules)

Question 10

Steps in lysis?

Answer 10

• tissue maceration (cutting and grinding)
• heating in the buffer - increases cell breakdown.

Question 11

What does a lysis buffer contain?

Answer 11

• detergent - breaks apart membranes by attaching to lipids
• proteinase enzymes - breaks down proteins and nucleases which can break down dna
• chaotropic salts - destabilises hydrogen bonds in proteins and creates a hydrophobic environment in which the silica membrane (next step) is the most suitable binding partner for DNA.

Question 12

What are 5 different DNA purification methods?

Answer 12

• spin columns (silica membrane) - lab
• magnetic beads
• phenol/chloroform
• ethanol precipitation
• chelex - do also in lab - quicker and cheaper

Question 13

What do you do after cell lysis and DNA purification in spin column purification?

Answer 13

• elution - nucleic acids become hydrated and will release from the membrane.

Question 14

What are the purification steps in spin column purification? (separates purified DNA from cell material and any PCR inhibitors)

Answer 14

• chaotropic salts and ethanol are added to enhanced the binding of DNA to silica
• load sample to spin column
• centrifuge
• outcome; DNA binds to silica membrane and flow-through lysate discarded.
• there is a washing step afterwards which removes any residual impurities.
• all ethanol is removed so that DNA can be removed successfully from silica m,membrane
• centrifuge until completely dry.

Question 15

Magnetic beads purification?

Answer 15

• similar to spin column, but instead of binding to the silica membrane, DNA binds to the beads, then you use a magnet to keep it there while you wash everything away.

Question 16

Whats good about phenol/chloroform purification?

Answer 16

• yields pure and high weight DNA, but has bad chemicals.

Question 17

Steps to ethanol precipitation?

Answer 17

• lyse cells and add denaturation buffer to denature proteins and incubate
• add isopropanol to precipitate the DNA. Then centrifuge which forms a dna pallet at the side.
• isolated so then wash it with ethanol and centrifuge to keep the pallet there. remove all the ethanol again by airdrying it.
• Then elution buffer and suspend DNA in that.

Question 18

Pros and cons to ethanol precipitation?

Answer 18

Question 19

What do we do to Chelex at the start? What does it do?

Answer 19

• turn the beads into a 5% solution.
• it contains iminodiacetate ions that act as chelating groups to bind metal ions such as mg2+ to the chelex resin.
• nucleases need these to be activated. So Chelex stops anything from breaking down your DNA.
• doesn’t do anything else.

Question 20

How does Chelex work?

Answer 20

• boil sample with %5 chelex in it. This denatures your proteins but also get single-stranded DNA (more prone to degradation)
• centrifuge it to get a pallet. Extract supernatant that has DNA.

Question 21

Pros and cons to Chelex?

Answer 21

• super fast and cheap and nontoxic
• cons: prone to degradation (so keep in freezer). Ineffective at removing pigments and inhibitors.

Question 22

Summarise the extraction methods:

Answer 22

Question 23

How do you store DNA?

Answer 23

• 4c for short-term storage in the fridge
• -30c for long-term storage
• avoid repetitive thawing and freezing (degradation)

Question 24

How do you check the quality of DNA?

Answer 24

• we will be using a nanodrop
• but often you’ll use gel electrophoresis

Question 25

Why would you run your sample through an agarose gel?

Answer 25

- to examine the extent of DNA degradation. Good-quality DNA should migrate as a band with little or no evidence of smearing. Smearing is caused by degradation from shorter fragments (no high-quality DNA).