Week 2 Flashcards
What is an extract?
- a solution obtained by soaking something in it.
What is a supernatant?
- the liquid left over from precipitation or centrifugation
What is the molecular genetics pathway to success?
- sample collection
- DNA extraction
- whole genome sequencing (NGS)
- PCR
- gel electrophoresis
- genotyping, or 5. sanger sequencing (standard one)
Why would we need to extract DNA?
- need purified DNA for use in things like PCR
Goals of purifying DNA and extracting it?
- removes proteins and nucleases and other things
- more stable and less degradation (also smaller than tissue samples)
Why do we need to get rid of nucleases?
-nucleases break down DNA ???? so need to remove them in purification
Objectives of a DNA extraction?
- maximize DNA recovery
- concentrate DNA
- remove or inhibit nucleases
- remove PCR inhibitors
- maximise DNA quality.
Which DNA extraction method?
- depends on the type of sample and purpose of analysis
What are the two main parts of DNA extraction?
- Lysis (solubilize DNA)
- purification (removes contaminants (proteins, RNA, macromolecules)
Steps in lysis?
- tissue maceration (cutting and grinding)
- heating in the buffer - increases cell breakdown.
What does a lysis buffer contain?
- 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.
What are 5 different DNA purification methods?
- spin columns (silica membrane) - lab
- magnetic beads
- phenol/chloroform
- ethanol precipitation
- chelex - do also in lab - quicker and cheaper
What do you do after cell lysis and DNA purification in spin column purification?
- elution - nucleic acids become hydrated and will release from the membrane.
What are the purification steps in spin column purification? (separates purified DNA from cell material and any PCR inhibitors)
- 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.
Magnetic beads purification?
- 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.
Whats good about phenol/chloroform purification?
- yields pure and high weight DNA, but has bad chemicals.
Steps to ethanol precipitation?
- 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.
Pros and cons to ethanol precipitation?
What do we do to Chelex at the start? What does it do?
- 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.
How does Chelex work?
- 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.
Pros and cons to Chelex?
- super fast and cheap and nontoxic
- cons: prone to degradation (so keep in freezer). Ineffective at removing pigments and inhibitors.
Summarise the extraction methods:
How do you store DNA?
- 4c for short-term storage in the fridge
- -30c for long-term storage
- avoid repetitive thawing and freezing (degradation)
How do you check the quality of DNA?
- we will be using a nanodrop
- but often you’ll use gel electrophoresis
Why would you run your sample through an agarose gel?
- 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).
