DNA Extraction: Comprehensive Forensic Biology Notes

Learning Objectives for DNA Extraction

• MLO5: Explain the range of techniques used for the extraction of biological trace materials.

• Session Objectives:

  • Describe the three fundamental steps involved in DNA extraction.

  • Compare various methods of DNA extraction used in forensic contexts.

DNA Processing Overview

• DNA Processing is a multi-step workflow within forensic science. The sequence typically follows:

  1. DNA Recap: Initial review of biological principles.

  2. DNA Extraction: The focus of this session.

  3. DNA Quantification: Determining the amount of DNA recovered.

  4. PCR & qPCR: Amplification of DNA samples.

  5. STR Separation I: Short Tandem Repeat analysis.

  6. STR Separation II: Advanced separation techniques.

• Associated Learning Components: The curriculum includes three associated labs (Online, PCR, and Electrophoresis) and two workshops.

The Rationale for DNA Extraction

• DNA must be extracted because it is contained within the complex environment of the cell.

• Extracellular Environment: Outside the cell, there are various substances such as gases, steroids, glucose, and molecules like $H_2O$ and ATP.

• Intracellular Environment: Inside the cell, DNA is protected by layers but also exposed to various ions.

• Extraction is required to isolate the genetic material from these cellular and extracellular components to prepare it for analysis.

Nucleases and DNA Degradation

• Nucleases: These are enzymes that degrade nucleic acids by facilitating the hydrolysis of phosphodiester bonds (often abbreviated as s-p-b).

• Metaphor: Frequently referred to as "molecular scissors."

• High-Molecular Weight (MW) DNA: This type of DNA degrades more quickly because it contains a greater number of sites available for hydrolysis.

• Role in Extraction: Nucleases must be either removed or inhibited during the DNA extraction process to minimize degradation.

• Temporal Factor: The longer a biological sample remains unextracted, the higher the degree of degradation.

Quality Goals for Extracted DNA

• The ultimate goal of extraction is to produce a stable solution of high-quality DNA.

• Stability: High-quality DNA will not easily degrade over time.

• Forensic Utility: Stable DNA extracts can be "revisited" years later, which is essential for investigating cold cases.

Identification of DNA Sources

• Before extraction can begin, potential DNA sources must be identified by Forensic Examiners and Search and Recovery teams (such as those in examination labs).

• Common Biological Sources & Presumptive Testing:

  • Blood: Requires a presumptive test to confirm the presence of hemoglobin or related components.

  • Semen: Requires a presumptive test to identify acid phosphatase or prostate-specific antigen.

  • Saliva: Requires a presumptive test to identify amylase.

Categorization of Extraction Samples

• Simple Samples: These contain easily accessible DNA. Examples include liquid blood, dried blood, and saliva.

• Specialist Samples: DNA cannot be easily removed from the specific cell type or tissue. An example includes bone fragments.

• Differential Samples: Requires the separation of two distinct sample types within the same matrix. A common example is a mixture of semen and epithelial cells (often found in sexual assault evidence).

The Three Fundamental Steps of DNA Extraction

1. Lysis:

   • This step releases DNA into the solution by breaking down the phospholipid bilayer (PL-BL).

   • This is typically achieved using a combination of detergents and enzymes.

2. Precipitation:

   • Ethanol is added to the mixture.

   • Because DNA is insoluble in ethanol, it precipitates out of the solution, allowing it to be separated from unwanted cellular debris such as proteins and nucleases.

3. Purification:

   • This step involves the final removal of cellular debris and any other unwanted materials to leave only the DNA in solution.

Specific Reagents Used in Extraction

• Reagents for Lysis:

  • Sodium dodecylsulfate (SDS): A detergent that breaks the phospholipid bilayer (PL-BL), often used in conjunction with heating.

  • Proteinase K (Pro K): An enzyme that breaks proteins down into their constituent individual amino acids; it also helps inactivate nucleases.

  • Dithiothreitol (DTT): Utilized for difficult samples, specifically seminal samples. It reduces disulfide bonds to dithiols, which is critical for releasing DNA from the tightly packed structure of sperm heads.

• Reagents for Precipitation and Washing:

  • Ethanol: DNA is insoluble in ethanol, causing it to precipitate. Ethanol is also used to "wash" away residual debris and contaminants from the DNA.

Simple Extraction Methods

Phenol-Chloroform Method

• This is an older method that presents many safety and efficiency issues.

• Lysis: Uses SDS and Pro K combined with heating to break the plasma membrane and release DNA. Proteins are broken down, and enzymatic activity is halted.

• Precipitation: Phenol and chloroform are added, followed by vortexing.

  • Phenol (P): Denatures proteins.

  • Chloroform (C): Increases the density of the organic phase, improving separation.

  • DNA remains in the aqueous portion as it is more soluble there.

• Purification: The sample is centrifuged to separate the cellular debris into a distinct layer.

Chelex Method

• Uses Chelex 100, which consists of "styrene divinylbenzene copolymers containing paired iminodiacetic ions."

• Mechanism: The Chelex beads act as a chelating group that binds to metal ions.

• Inhibition: Magnesium ions ($Mg^{2+}+$) are strongly and irreversibly complexed by the beads. Since nucleases require $Mg^{2+}+$ as a cofactor, removing the ions inactivates the enzymes.

• Lysis: A $5\%$ Chelex suspension is added to the sample and boiled.

• Precipitation: After boiling, the sample is centrifuged, which pulls the Chelex beads to the bottom of the tube.

• Purification: The aqueous layer (supernatant) containing the DNA is removed for further use.

Solid-Phase Spin Columns

• This method involves DNA selectively binding to a substrate, typically a silica spin column.

• Lysis: Utilizes SDS detergent, Pro K, and a specialized buffer (often called "AL") that binds and removes lipids.

• Precipitation: $100\%$ ethanol is added to precipitate the DNA. This causes the DNA to become more attracted to the silica material within the spin column.

• Purification: Wash buffers (containing ethanol to maintain DNA binding) are passed through the column to wash away contaminants.

• Elution: The DNA is unbound from the silica using an elution buffer (such as water or Tris-EDTA) for final storage.

Specialist Methods: Differential Extraction

• Context: Common in sexual assault cases, including those involving pre-ejaculation. The goal is often to identify the seminal origin specifically.

• The Challenge: Epithelial cells from the victim often "swamp" the male spermatozoa. If not separated, the resulting DNA profile may show the male contributor as absent, at a low level, or uninterpretable.

Step 1: Pre-Extraction (Examination)

• Examiners perform an initial separation using a spin-basket.

• This allows some epithelial cells to be washed (eluted) away.

• This results in a "cleaned" pellet of cells with a higher percentage of seminal cells.

Step 2: Sperm Elution (Water Elution Method)

• Procedure:

  1. The sample is placed in a spin basket with water.

  2. The sample is vortexed and centrifuged to produce a pellet.

  3. The supernatant is retained, and the pellet is processed further.

  4. Lysis reagent is added, followed by incubation.

  5. The sample is returned to a spin basket and centrifuged again to produce a new pellet.

  6. The pellet is retained, and the supernatant is sent for further processing.

• Success Rates: Research indicates that initial recovery of spermatozoa in a cell pellet via standard water extraction is often less than $20\%$. However, the "Sperm Elute" method has shown recovery rates of $71\%$, compared to only $25\%$ for standard water-eluted samples.

Step 3: Differential Extraction Workflow

• Initial Lysis: Add SDS and Proteinase K to the mixture.

• Incubation/Centrifugation: This breaks down the epithelial cells but leaves sperm heads intact.

• Separation: The supernatant (containing the Epithelial Lysate Fraction) is transferred and retained.

• Resuspension: The remaining pellet (the Spermatozoa Pellet) is resuspended.

• Secondary Lysis: SDS, Proteinase K, and Dithiothreitol (DTT) are added to the pellet to break down the resilient sperm heads.

• Final Result: This produces the Spermatozoa Lysate Fraction.

• Purification: Both fractions are then isolated/purified using organic (Phenol-Chloroform) or solid-phase extraction.

Specialist Methods: Bone and Faeces

DNA Extraction from Bone and Teeth

• Source Quality: Bone is a good source of DNA because the hydroxyapatite mineral (essential for bone strength) stabilizes the DNA and limits enzymatic degradation.

• Process:

  1. The bone/tooth is cleaned to remove surface contaminants.

  2. The sample is ground into a fine powder.

  3. Decalcification: EDTA and SDS are used to remove the mineral matrix.

  4. Extraction: This is followed by a standard method such as solid-phase extraction.

DNA Extraction from Faeces

• Complexity: Faecal DNA extracts contain very little human DNA relative to other sources.

• Challenges: High concentrations of inhibitors, nucleases, and microbial communities.

• Methodology: The process is completed on cold tube racks to prevent degradation. Specific buffers are used to remove nucleases and inhibitors, followed by solid-phase extraction.

Cleaning and Concentrating DNA

• DNA often requires additional cleaning to remove impurities that can interfere with downstream processes (like PCR).

• Target Impurities: Haem (from blood), nicotine (from cigarettes), and dyes (from clothing).

• Filtration Method:

  • Uses centrifugal filters that function as a molecular sieve.

  • Process: During centrifugation, DNA remains on top of the filter while smaller impurities flow through to be discarded.

  • Recovery: The DNA is recovered from the sieve by turning the filter upside down and centrifuging slowly to collect the concentrated, cleaned DNA.