DNA Extraction Methods

Inhibitors

• Chemicals, compounds or other molecules

• Compete with the target DNA preventing full recovery of sample during extraction and/or reduce efficiency of PCR

• Can occur at any stage of processing

• Bind directly to the double or singled stranded DNA (affects PCR)

• Interfere with cell lysis (affects extraction)

• Interfere with enzyme activity (affect extraction or PCR)

• May bind in the active site of Taq polymerase (affects PCR)

• Sequester essential cofactors such as Mg2+ (affects PCR)

Sources of Inhibition

• Bacteria

• Soil compounds

  • Humic acid

  • Fulvic acid

• Chemical

  • Phenol

  • Chelex Resin

  • SDS

  • EDTA

• Dyes

  • Indigo (Denim)

• Leather

  • Tannic Acid

• Blood

  • Heme

  • Anticoagulants

• Tissue and hair

  • Melanin

• Feces

  • Bile salt

  • Sugars

• Urine

  • Urea

• Bone

  • Ca2+

Degradation

• Breakdown of DNA molecules

  • Fragments

  • Individual base modification

• Can occur in fresh and ancient remains

  • Environmental exposure (e.g. Heat, Humidity, UV, Water)

  • Nucleases

  • Bacteria, Fungi, Insects

  • Chemical (Ex. Bleach)

Individual Base Modification

Cytosine Deamination

• Hydrolytic deamination of Cytosine to Uracil

• Uracil binds with Adenine instead of Guanine

• Adenine will then bind to Thymine

• End up with Adenine and Thymine in the data where there should have been Cytosine and Guanine

Fragmentation

• DNA fragments will be too small for PCR

• Can result in allelic drop out in the data

• Often seen as a ski-slope effect because larger fragments are missing or reduced

Extraction Reagents

Proteinase K (Pro-K)

• A serine protease (enzyme)

• Cleaves peptide bonds between amino acids

Sodium Dodecyl Sodium

• Lyses cells

• Lyses nucleus to release DNA

Dithiothreitol (DTT)

• Strong protein denaturant

• Release the DNA from its protective proteins (histones)

Organic Extraction

Sample Types:

• Bone, Teeth, Hair, Nail, Tissue, and Biological Fluids

PRO: Provides a high yield of double-stranded DNA

• Great for difficult samples like ancient remains

CONS: Labor-intensive and utilizes harmful chemicals

• DNA and impurities are separated into layers or phases

• Aqueous Layer: DNA (polar loves water) and Buffer

• Interface: Proteins and Lipids = Fatty Layer

• Organic Layer: Proteins, Cellular Debris, and Inhibitors

PCIA Extraction

Isolate and purify DNA using organic chemicals

• Phenol (Harmful)

• Chloroform

• Isoamyl Alcohol

Procedure - After initial incubation

1. Sample in a tube

2. Add in PCIA chemicals to sample tube

3. Mix the tube and allow it time to incubate – the separate layers will form

4. Carefully pipette out the aqueous layer (top layer containing DNA)

5. Transfer aqueous layer to a clean new tube and discard tube containing leftover waste

Non-Organic Extraction

Sample Types:

• Bone, Teeth, Hair, Nail, Tissue, and Biological Fluids

PRO: Can be automated with the use of robots and does not utilize harmful chemicals

CONS: Not great for difficult samples like ancient remains

Qiagen Non-Organic Extraction

• DNA binds to the negatively charged Silica membrane of the filter column

• DNA also is a negatively charged molecule and will not bind unless the appropriate chemicals are present to facilitate the binding

  • Because typically, negatives will repel

Procedure - After initial incubation

1. Add sample and Buffer PB to Qiagen column and allow time for binding

2. Wash through Buffer PE to remove impurities

3. Transfer column only to a new clean tube

4. Wash through Buffer EB to elute purified DNA, discard column

Chelex Non-Organic Extraction

Most widely used Non-Organic method

• Fast, simple, and inexpensive

• Non-toxic

• Minimal tube transfers

• Qiagen Non-Organic Extraction has a lot of tube transfers = more opportunity for error or introduction of contamination

• Known as QUICK AND DIRTY

  • Not great at removing inhibitors

• Results in single stranded DNA because of a boiling step in the procedure

  • Single stranded DNA is not suitable for some quantitation methods, which would be the next step in the DNA process

Chelating resin

• Metal ions (ex. Mg2+, Ca2+, Fe2+) are present in the sample, they activate Nucleases that break down DNA

• Chelators bind to metal ions; thus, they are unable to activate nucleases, and the DNA is protected

• Does not remove cellular debris, no wash steps like with the Qiagen Non-Organic Extraction

• Chelex is inhibitory, will inhibit downstream processing

Phenol

• Strong protein denaturant

• Inhibitory, will inhibit downstream processing if not removed

Chloroform

• Protein denaturant

• Removes lipids

• Specific gravity of 1.47

  • increases density of mixture (HEAVY)

  • Ensures good separation between the organic and aqueous layers

Isoamyl Alcohol

• De-foaming agent

• Removes any bubbles that may have been created when in the chemicals to the sample

• Allows for better visualization of the layers