DNA Extraction Methods

• Extraction involves isolating and purifying proteins and other cellular materials from DNA molecules.
• The extraction process aims to remove inhibitors that can affect downstream processing.
• It is important to produce a stable solution containing high-quality DNA.
• The general steps for extraction include lysing the substrate to release DNA, isolating the DNA, and purifying it.

Types of Extractions

• Several extraction methods are available for different types of samples and applications.

Direct Amp (FTA Paper)

• Direct amp uses FTA (Flinders Technology Associates) paper, which is cellulose-based and contains chemicals to protect DNA.
• A sample is spotted on the FTA paper, chemically treated to lyse cells, and immobilize DNA.
• Direct amp paper is washed to remove inhibitors and can be added to PCR directly.

Pros of Direct Amp

• Saves time and money.
• Ideal for reference samples where a consistent amount of DNA is expected.
• Fewer purification steps lead to increased DNA retention.

Cons of Direct Amp

• Less purification may allow PCR inhibitors to be present.
• Not generally accepted for evidence samples because DNA concentration is unknown.

Organic Extraction (Phenol-Chloroform)

• Organic extraction has been traditionally used for RFLP analysis.
• It involves serial addition of chemicals to separate proteins from DNA.

Reaction Components in Organic Extraction

• Extraction buffer penetrates cells, disrupting lipopolysaccharides and containing EDTA to maintain pH.
• Sodium dodecyl sulfate (SDS) lyses the cell membrane.
• Proteinase K (Pro K) cleaves peptide bonds, denatures proteins, and hydrolyzes histone proteins to decompress DNA.
• These all separate the proteins from the DNA
• Phenol provides a lower organic layer to separate proteins and organic debris.
• Chloroform provides a sharp interphase.
• Isoamyl alcohol reduces foam at the interphase.

Pros of Organic Extraction

• Yields high-quality, high molecular weight, clean DNA, which is necessary for RFLP.

Cons of Organic Extraction

• Phenol is toxic and needs to be used in a chemical hood.
• The process is time-consuming and involves multiple transfers.

Chelex Extraction

• The Chelex extraction method is standard for STR analysis.
• Typical samples include bloodstains, dried secretions, sweat, and vaginal swabs.
• Chelex beads act as a chelating agent, binding to metal ions such as Mg2+ and inactivating DNases/nucleases.
• Incubation at 56°C lyses cell membranes, and incubation at 100°C destroys protein and denatures DNA.
• Final centrifugation separates beads and debris, leaving single-stranded DNA.
• DNases require Mg 2+ to function, Chelix binds to this, deactivating the DNases and protecting the DNA

Pros of Chelex Extraction

• Relatively fast and inexpensive procedure.
• Yields single-stranded DNA suitable for PCR-based analysis.
• The reaction takes place in one tube.

Cons of Chelex Extraction

• DNA quality is not as high as with organic extraction.
• Considered a "dirty" extraction as the purified DNA may still be mixed with unwanted proteins and PCR inhibitors.

Differential Extraction

• Differential extraction separates male and female components in sex assault samples (vaginal epithelial/sperm cell mixtures).
• It is a modified version of organic extraction.

Key Components in Differential Extraction

• PBS disengages and separates cells from the substrate and maintains an isotonic solution.
• ProK breaks epithelial cell membranes, digests unwanted proteins, and inactivates nucleases.
• Chelex sequesters polyvalent metal ions.
• Digest buffer breaks down contaminants in the solution.
• DTT breaks disulfide bonds between cysteine residues, releasing male DNA.
• Sterile water creates a hypotonic solution to rupture cells and wash away residual debris and digest buffer.

Pros of Differential Extraction

• Simplifies the DNA profile.

Cons of Differential Extraction

• Male profile may not be detectable in azoospermic males.
• Results may be compromised with a degraded sample or degraded male DNA eluted with female DNA.

Silica Column-Based Extraction

• Silica column-based extraction yields high-quality, double-stranded DNA, good for PCR and RFLP.
• Examples include QIAGEN and Magnetic Beads.

Solid Phase Extraction

• DNA is adsorbed to silica in the presence of high salts and low pH and released in the presence of low salts and high pH.
• Cells are lysed and the DNA extract is run through a silica column using a wash-bind-elute procedure.
• DNA binds to silica particles while impurities and cell debris are washed away.
• DNA is eluted using buffers to raise the pH.

Pros of Solid Phase Extraction

• Clean DNA with a technique amenable to automation.

Cons of Solid Phase Extraction

• Requires expensive equipment, time-consuming, and may cause sample loss.

pH in Solid Phase Extraction

• Optimized buffers lyse cells before DNA binds to the silica-coated particle in a high salt environment.
• Low pH favors adsorption, while high pH favors elution.

Chaotropic Salts

• Chaotropic salts may include Guanidine hydrochloride, Guanidinium isothiocyanate, Sodium iodide, and Sodium perchlorate.
• These salts break the cell membrane by interfering with intermolecular forces that hold the membrane together.
• Salts disrupt the hydration shell surrounding DNA, making it more available for binding silica-coated particles.
• They provide the ions needed to form a bridge by which the DNA is attached to silica.
• Magnets can improve the effectiveness of the extraction.
• A magnet is applied to the bottom of the tube, pulling the particles with attached DNA towards it.

ZyGEM Extraction

• ZyGEM extraction uses the forensic GEM Sperm Kit from MicroGEM.
• It produces high-quality single-stranded DNA.
• It is a one-step heat-activated sperm lysis method for sexual assault stains or swabs.
• The process rapidly degrades sperm heads and digests the remaining tissue and nucleases in the sample.

Reagents used in ZyGEM Extraction

• Acrosolv is a proprietary enzyme cocktail.
• Orange+ Buffer contains potassium chloride.
• forensicGEM enzyme, or EA1 Enzyme, contains Proteinase + Glycerol and Ultrapure water.

Pros of ZyGEM Extraction

• Suitable for male DNA screening and is fast and efficient.

Cons of ZyGEM Extraction

• Breaks open both vaginal epithelial and sperm cells in one tube, so fractions are not separated.