Chapter 5: DNA Extraction

What are the steps of DNA extraction?

1. Lyse cells and release DNA

2. Separate DNA from cellular material

3. Isolate DNA so it can be used for STR typing

4. Store DNA at -20°C or -80°C to prevent nuclease activity

What are PCR inhibitors?

Contaminants that interfered with Taq polymerase’s ability to function during PCR

What are the DNA extraction methods?

• Organic (phenol-chloroform)

• Solid-phase extraction methods

  • Qiagen

  • DNA IQ and PrepFiler

• Chelex

• FTA paper

• Differential extraction

What are the 4 components of lysing cells and releasing DNA and what is the purpose?

• Urea - disrupts H and hydrophobic bonds holding cellular membrane together

• SDS - ionic detergents that bind proteins and unfold them to disrupt the DNA-protein complexes

• EDTA - chelating agents that bind multivalent ions (Mg²⁺ and Fe²⁺)

• Proteinase-K - stimulated in the presence of detergents to digest proteins by breaking peptide bonds to produce smaller peptides

How is DNA separated from cellular material?

• Organic solvents - removes proteins from nucleic acids

• Solid support to bind to DNA (e.g. Qiagen spin-column)

• Magnetic beads to bind DN (e.g. DNA IQ system)

How is DNA extracted using Phenol:Chloroform (Organic) extractions?

1. Cell lysed with buffer-saturated phenol: chloroform

2. DNA remains in the aqueous phase while cellular proteins are denatured and precipitated into the organic phase

3. A phase is extracted a second time with chloroform, to ensure the removal of phenol and proteins

4. A phase treats with isopropanol or ethanol is used to precipitate the DNA

5. Precipitated DNA is washed with ethanol and rehydrated in water or TE Buffer

How is DNA extracted using solid-phase DNA extraction?

1. Lyse cells

2. Releases DNA

3. DNA selectively binds to a substrate (silica resin or silica-coated magnetic bead)

4. DNA is retained while proteins and cellular debris are washed away

5. DNA is eluted

6. Clean DNA

How is DNA extracted using silica-based extraction (Qiagen)?

1. Lyse cells

2. Nucleic acids bind to a porous silicon-oxide-coated membrane in the presence of chaotropic salts and ethanol under slightly acidic conditions

3. Washed in the presence of chaotropic salt and ethanol keeps proteins/cellular debris in solution so they can flow through whole DNA is bound to the column

4. Under low salt conditions the DNA is eluted from the support

How is DNA extracted using silica-based extraction (Promega)?

• Uses magnetic beads to bind DNA

• Adding resin to the lysate allows extraction to be performed in one tube

• Various solutions are used to wash the magnetic beads and a magnet hold the DNA in place

• The amount of resin used is the limiting factor to amount of DNA captured

• Sample is incubated in an elution buffer which releases the DNA from the beads into solution

How is DNA extracted using Chelex extraction?

• 5% chelating resin is added directly to a sample of blood or semen

• Mg ions are drawn to and bound by resin

• Cells are lysed by boiling for several minutes

• Centrifuge to pellet cellular debris and the chelex resin, remove ssDNA from the supernatant

How is DNA extracted using FTA paper extraction?

Sample Collection

• Blood added to FTA paper

• Paper lyses cells, binds white blood cells to paper’s matrix, protect DNA from nuclease activity, and deters bacterial growth

DNA Extraction

• Wash punch of paper to remove heme and other PCR inhibiotrs

• Punch is added to PCR without quantification

• Possible to perform Chelex or solid-support extractions on punches

What is the goal of differential extraction?

To separate epithelial cells from sperm cells in sexual assault cases to facilitate STR interpretation

What is the process of differential extraction?

1. Remove a portion of the mixed stain

2. Add SDS, EDTA, and proteinase K to lyse cells

3. Centrifuge and incubate at 37°C

4. Remove supernatant = Non-sperm fraction

5. Add SDS, EDTA, proteinase K, and DTT to sperm pellet = Sperm fraction