DNA Extraction

We aim to achieve the following with DNA extraction (3)

1. successful homogenization of tissue

2. lysis of the cell

3. intact acquisition of the DNA

Tissue homogenization may be done (3)

1. Mechanical homogenization

2. Chemical homogenization

3. Enzymes/enzymatic treatment

Chemical homogenization makes use of

amphipathic molecules such as detergents to disrupt the integrity of the cell membrane

In chemical homogenization, ___ occurs through ___ of the cell membrane

lysis, emulsification

Example of enzyme

Proteinase K

Most methods take advantage of the nucleotide's ability to ___

stay soluble in an aqueous environment

Separation is done through ___

centrifugation

Precipitation of the DNA is finally done through the ___

addition of ethanol

2 methods in doing DNA extraction

1. phenol:chloroform extraction method

2. Chelex method

DNA dissolves in the ___ and all undesired particles are left in the ___

aqueous layer, organic layer

3 main steps of PCI method

1. Tissue digestion

2. Phenol:chloroform extraction

3. DNA precipitation

4 basic steps of tissue digestion

1. Add sample to microfuge tubes

2. Add lysis buffer and homogenize tissue

3. Add Proteinase K then vortex

4. Incubate and vortex every so often until completely lysed

7 basic steps of PCI extraction

1. Vortex lysates

2. Spin down

3. Transfer supernatant while avoiding pellet

4. Add 25:24:1 phenol: chloroform: isoamyl alcohol. Mix

5. Spin down

6. Transfer aqueous layer to fresh tube

7. Repeat

9 basic steps of DNA precipitation

1. Add sodium acetate then vortex

2. Add 100% alcohol

3. Incubate overnight

4. Spin down

5. Pour out ethanol without disturbing DNA pellet

6. Add 70% ethanol

7. Spin down

8. Pour out ethanol and air dry

9. Resuspend DNA pellet in ddH₂O or TE buffer in -20˚C

Description for Chelex method

quick and dirty approach

9 basic steps to Chelex method

1. Chelex solution in microfuge tube

2. Add sample

3. Vortex

4. Spin down

5. Heat at 100˚C

6. Cool down to room temperature

7. Spin down

8. Transfer supernatant into a fresh tube

9. Store at -20˚C

DNA is found in (3)

1. Nuclear DNA

2. Mitochondrial DNA

3. Chloroplast DNA

For what samples are PCI method typically for

Plants and animal samples

For what samples are Chelex method typically for

tissue and biological fluids

Three layers in PCI solution

Aqueous phase, interphase, and organic phase

What organelles have a monolayer (3)

lysosome, vacuole, ER

What organelles have a bilayer (3)

nucleus, mitochondria, chloroplast

Describe the phospholipid bilayer

• hydrophilic/polar head

• hydrophobic/non-polar tail

Polarity of phospholipid bilayer

Amphipathic

What model does phospholipid bilayer follow

Fluid mosaic model

Why is DNA negative

Phosphate group

Complementary bases connected via

hydrogen bonds

Adjacent nucleotides are connected by

phosphodiester bonds

Kind of mechanical homogenization using shaken metal/porcelain beads inside to crush

Bead ruptor

3 factors of lysis buffer

1. pH

2. Ionic strength

3. type of surfactant/detergent

4 components of lysis buffer

1. Buffering salt

2. Ionic salt

3. Chelating agent

4. Surfactant/Detergent

Surfactant commonly used in lysis buffer

Sodium dodecyl sulfate

Buffering salts that could be used (2)

Tris-HCl and Tris base

Purpose of buffering salt

Maintains optimum pH of the solution

Ionic salts that could be used (3)

NaCl, KCl, MgCl₂

Purpose of buffering salt (3)

1. Establish ionic strength

2. Prevents DNA degradation

3. Increases concentration of solute outside the cell ⇒ hypertonic ⇒ shrinkage

Purpose of surfactant (3)

1. Anionic detergent that disrupts membrane structures

2. Forms micelles which are SDS aggregates interrupting phospholipid bilayer

Describe proteinase K

A serine protease that digests proteins and degrades nucleases

4 other types of enzymes for DNA extraction

RNAse, chitinase, cellulase, DNAse

Describe principle of PCI method

Liquid to liquid extraction of biomolecules; Separates based on solubility

Phenol purpose in PCI (2)

1. Can digest or cleave amide bonds

2. Revert structure of protein to primary structure

Chloroform purpose in PCI (3)

1. increase efficiency of phenol in denaturing proteins

2. protects DNA

3. Can digest lipids

Isoamyl alcohol purpose in PCI

reduces foaming at interphase

What is found in the aqueous phase (2)

1. DNA

2. Carbohydrates

Goal of DNA precipitation

make DNA insoluble

DNA becomes insoluble without the ___

hydration layer

Non-polar/hydrophobic resins purpose

Lipids and proteins will stay there

Chelex solution is ___ to disrupt membrane

highly alkaline

Expose to high temperature

lysis then denaturation

Conformational change of proteins to ___ due to boiling step

expose non-polar

Structure of DNA after Chelex method

single-stranded due to boiling step