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Molecular Genetics And Evolution > Week 2 > Flashcards

Week 2 Flashcards

1
Q

What is an extract?

A
  • a solution obtained by soaking something in it.
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2
Q

What is a supernatant?

A
  • the liquid left over from precipitation or centrifugation
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3
Q

What is the molecular genetics pathway to success?

A
  • sample collection
  • DNA extraction
    1. whole genome sequencing (NGS)
    1. PCR
  • gel electrophoresis
    1. genotyping, or 5. sanger sequencing (standard one)
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4
Q

Why would we need to extract DNA?

A
  • need purified DNA for use in things like PCR
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5
Q

Goals of purifying DNA and extracting it?

A
  • removes proteins and nucleases and other things
  • more stable and less degradation (also smaller than tissue samples)
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6
Q

Why do we need to get rid of nucleases?

A

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

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7
Q

Objectives of a DNA extraction?

A
  • maximize DNA recovery
  • concentrate DNA
  • remove or inhibit nucleases
  • remove PCR inhibitors
  • maximise DNA quality.
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8
Q

Which DNA extraction method?

A
  • depends on the type of sample and purpose of analysis
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9
Q

What are the two main parts of DNA extraction?

A
  • Lysis (solubilize DNA)
  • purification (removes contaminants (proteins, RNA, macromolecules)
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10
Q

Steps in lysis?

A
  • tissue maceration (cutting and grinding)
  • heating in the buffer - increases cell breakdown.
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11
Q

What does a lysis buffer contain?

A
  • 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.
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12
Q

What are 5 different DNA purification methods?

A
  • spin columns (silica membrane) - lab
  • magnetic beads
  • phenol/chloroform
  • ethanol precipitation
  • chelex - do also in lab - quicker and cheaper
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13
Q

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

A
  • elution - nucleic acids become hydrated and will release from the membrane.
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14
Q

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

A
  • 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.
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15
Q

Magnetic beads purification?

A
  • 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.
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16
Q

Whats good about phenol/chloroform purification?

A
  • yields pure and high weight DNA, but has bad chemicals.
17
Q

Steps to ethanol precipitation?

A
  • 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.
18
Q

Pros and cons to ethanol precipitation?

A
19
Q

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

A
  • 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.
20
Q

How does Chelex work?

A
  • 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.
21
Q

Pros and cons to Chelex?

A
  • super fast and cheap and nontoxic
  • cons: prone to degradation (so keep in freezer). Ineffective at removing pigments and inhibitors.
22
Q

Summarise the extraction methods:

A
23
Q

How do you store DNA?

A
  • 4c for short-term storage in the fridge
  • -30c for long-term storage
  • avoid repetitive thawing and freezing (degradation)
24
Q

How do you check the quality of DNA?

A
  • we will be using a nanodrop
  • but often you’ll use gel electrophoresis
25
Q

Why would you run your sample through an agarose gel?

A
  • 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).

Molecular Genetics And Evolution (20 decks)

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