FRS3055 Forensic Application of Biological Specialism - DNA Extraction Notes

Previous Lectures

• Forensic Serology
• Body fluid identification
• Presumptive and confirmatory tests
• Sample collection and preparation for DNA profiling
• Sample collection from crime evidence
• Sample preparation for DNA profiling
• Assess the obstacles to retrieving DNA from any given sample

Session Overview

• Assess the obstacles to retrieving DNA from any given sample.
• Compare and contrast different DNA extraction methods.

Aim of DNA Extraction

• To separate DNA from all other cellular material.
• To remove substances that may inhibit downstream reactions:
  • Humic acids (soils)
  • Haem compounds (blood)
  • Dyes (indigo from denim)
• To produce an aqueous solution of DNA.

Sources of DNA

• Blood
• Semen
• Epithelial cells
• Muscle
• Bone
• Hair roots (mtDNA can be obtained from hair shafts)
• Faeces
• Urine

General Principles of DNA Extraction

Three stages:

• Disruption of the cellular membranes - cell lysis.
• Protein denaturation.
• Separation of DNA from the denatured protein and other cellular components.

Cell Lysis

• Breaking the cell open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by treating the sample with lysis buffer.
• Removing membrane lipids by adding a detergent.

Protein Denaturation

• Removing proteins by adding a protease (optional but almost always done).

Separation of DNA

• Precipitating the DNA with alcohol - usually ice-cold ethanol or isopropanol.
• Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation.
• This step also removes alcohol-soluble salt.

Essential Components of a DNA Extraction Procedure

• Maximise DNA recovery
• Remove inhibitors
• Remove or inhibit nucleases
• Maximise the quality of DNA

Commonly Used DNA Extraction Procedures in Forensic Science

• Organic (phenol-chloroform) extraction.
• Chelex (ion exchange resin) extraction.
• FTA paper (collection, storage and isolation).
• Solid phase extraction (spin-column, beads)
  The method utilised may be sample dependant, technique dependant or analyst preference.

Organic Extraction

• Perhaps the most basic of all procedures in forensic molecular biology is the purification of DNA.
• The key step, the removal of proteins, can often be carried out simply by extracting aqueous solutions of nucleic acids with phenol and/or chloroform.

Organic Extraction Procedure

• Cell lysis buffer - lyse cell membrane, nuclei are intact, pellet nuclei.
• Resuspend nuclei, add Sodium Dodecly Sulfate (SDS), proteinase K. This will lyse nuclear membrane and digest protein.
• DNA released into solution is extracted with phenol- chloroform to remove proteinaceous material.
• DNA is precipitated from the aqueous later by the addition of ice cold 95% ethanol.
• Precipitated DNA is washed with 70% ethanol, dried under vacuum and resuspended in elution buffer.

Organic Extraction Reagents

• Cell lysis buffer - non-ionic detergent, salt, buffer, EDTA designed to lyse outer cell membrane of cells but will not break down nuclear membrane.
• EDTA (Ethylenediaminetetraacetic disodium salt) is a chelating agent of divalent cations such as $Mg^{2+}$. $Mg^{2+}$ is a cofactor for DNase nucleases. If the $Mg^{2+}$ is bound up by EDTA, nucleases are inactivated.
• Proteinase K - it is usual to remove most of the protein by digesting with proteolytic enzymes such as pronase or proteinase K, which are active against a broad spectrum of native proteins, before extracting with organic solvents. Proteinase K is approximately 10 fold more active on denatured protein. Proteins can be denatured by SDS or by heat.
• Phenol/chloroform - the standard way to remove proteins from nucleic acids solutions is to extract once with phenol, once with a 1:1 mixture of phenol and chloroform, and once with chloroform. This procedure takes advantages of the fact that deproteinisation is more efficient when two different organic solvents are used instead of one.
• Also, the final extraction with chloroform removes any lingering traces of phenol from the nucleic acid preparation.
• Phenol is highly corrosive and can cause severe burns.
• Phenol - often means phenol equilibrated with buffer (such as TE) and containing 0.1% hydroxyquinoline and 0.2% β-mercaptoethanol (added as antioxidants). The hydroxyquinoline also gives the phenol a yellow colour, making it easier to identify the phases (layers).
• Chloroform - often means a 24:1 (v/v) mixture of chloroform and isoamyl alcohol. The isoamyl alcohol is added to help prevent foaming.
• The phenol/chloroform/isoamyl alcohol ratio is 25:24:1.

Concentrating DNA - Alcohol Precipitation

• The most widely used method for concentrating DNA is precipitation with ethanol. The precipitate of nucleic acid, forms in the presence of moderate concentrations of monovalent cations (salt, such as $Na^+$), is recovered by centrifugation and redissolved in an appropriate buffer such as TE.
• The technique is rapid and is quantitative even with nanogram amounts of DNA.
• The four critical variables are the purity of the DNA, its molecular weight, its concentration, and the speed at which it is pelleted.
• DNA concentration as low as 20 ng/ml will form a precipitate that can be quantitatively recovered.
• Typically 2 volumes of ice cold ethanol are added to precipitate the DNA.
• Very short DNA molecules (<200 bp) are precipitated inefficiently by ethanol.
• The optimum pelleting conditions depend on the DNA concentration. Relatively vigorous microcentrifuge steps such as 15-20 minutes at or below room temperature at 13,000 rpm are designed to minimise the loss of DNA from samples with yields in the range of a few micrograms or less.
• Solutes that may be trapped in the precipitate may be removed by washing the DNA pellet with a solution of 70% ethanol. To make certain that no DNA is lost during washing, add 70% ethanol until the tube is 2/3 full. Vortex briefly, and recentrifuge. After the 70% ethanol wash, the pellet does not adhere tightly to the wall of the tube, so great care must be taken when removing the supernatant.
• Isopropanol (1 volume) may be used in place of ethanol (2 volumes) to precipitate DNA. Precipitation with isopropanol has the advantage that the volume of liquid to be centrifuged is smaller.
• Isopropanol is less volatile than ethanol and it is more difficult to remove the last traces; moreover, solutes such sodium chloride are more easily coprecipitated with DNA when isopropanol is used.

Resuspension and Storage of DNA

• TE Buffer - Tris-EDTA Buffer: 10 mM Tris-HCl pH 8.0, 1 mM EDTA, or TE-4 which is 10 mM Tris, 0.1 mM EDTA. DNA is resuspended and stored in TE buffer. DNA must be stored in a slightly basis buffer to prevent depurination, and the EDTA chelates any $Mg^{2+}$ helping to inactivate DNases.
• DNA can be stored at 4oC for extended periods, however for long term storage, -20oC is preferable.
• Avoid repetitive freeze thawing of DNA, since this can cause degradation.

Using Nucleases to Remove Unwanted DNA or RNA

• Depending on when nuclease treatment is performed, it may be necessary to repeat purification steps for protein removal.

Phenol-Chloroform Extraction (Organic Extraction)

• Advantages:
  • High recovery of DNA.
  • Also captures small DNA fragments.
• Disadvantages:
  • Longer time.
  • Toxic

Chelex Extraction

• Chelex 100, Molecular Biology Grade resin from BioRad is a highly pure, nuclease and ligase inhibitor-free chelating resin, certified not to interfere with downstream PCR.
• Specifically designed to complement the inherent requirements of PCR, this pure, pipettable, small-scale resin is ready for downstream use.
• Ensuring the complete removal of PCR inhibitors, contaminating metal ions that catalyse the digestion of DNA.
• Chelex 100 is an ion exchange resin that is added as a 5% solution (wt/vol).
• Chelex is composed of styrene divinylbenzene copolymers containing paired iminodiacetate ions that act as chelating groups in binding polyvalent metal ions such as magnesium ($Mg^{2+}$).
• By removing the $Mg^{2+}$ from the reaction, nucleases are inactivated and the DNA is protected.
• A 5% solution of Chelex is added to a blood stain or liquid blood and incubated at 56oC for 30 minutes. This step is used to lyse red cells and remove contaminants and inhibitors such as heme and other proteins.
• The sample is then heated at 100oC for 8 minutes. This causes the DNA to be denatured as well as disrupting membranes and destroying cellular proteins.
• The tube containing the Chelex is centrifuged, the resin is pelleted, the supernatant containing the DNA is removed.
• The Chelex extraction process denatures double stranded DNA and yields single stranded DNA.
• It is advantageous for PCR-based typing methods because it removes inhibitors of PCR and can be done in a single tube, which reduces the potential for laboratory- induced contamination and sample switching.
• Care should be taken not to have any residual Chelex with the DNA extract, since $Mg^{2+}$ is required for the Taq Polymerase.
• Capture divalent ions which are needed for the Dnase activation.
• Chelex inhibit downstream processes if carried over.

FTA Paper

• FTA originally stood for “Fitzco/Flinder Technology Agreement.
• FTA paper is an absorbent cellulose-based paper that contains chemical substances to protect DNA molecules from nuclease degradation and preserve the paper from bacterial growth.
• As a result, DNA on FTA paper is stable at room temperature over a period of several years.
• A paper matrix laced with a proprietary mixture of chemicals that lyse cells and stabilise nucleic acids on contact for long term storage at room temperature.
• Direct PCR amplification or DNA extraction.
• Blood spots on FTA card.
• FTA card punches (1.2 or 2mm).

What Does FTA Paper Do?

• kills blood borne pathogens on contact
• immobilises DNA within the matrix
• protects DNA from degradation
• allows for long-term storage at room temp

Solid-Phase Extraction

• Solid phase extraction such as using a spin-column based extraction method takes advantages of the fact that DNA binds to silica.
• The sample containing DNA is added to column containing silica gel or silica beads and chaotropic salts (guanidine salts).
• The chaotropic salts disrupt the hydrogen bonding between strands and facilitate binding of the DNA to silica by causing the nucleic acids to become hydrophobic.
• This exposes the phosphate residues so they are available for absorption.
• The DNA binds to the silica, while the rest of the solution is washed out using ethanol to remove chaotropic salts and other unnecessary constituents.
• The DNA can then be rehydrated with aqueous low salt solution allowing for elution of the DNA from the column/beads.
• Solid-phase extraction methods for DNA have been developed in recent years in formats that enable high- throughput DNA extraction.
• One of the most active efforts in this area is with silica- based extraction methods and products from QIAGEN.
• For many years, QIAamp spin columns have proven effective as a means of DNA isolation.

Solid-Phase Extraction - Advantages

• Pure/clean DNA extract
• Less DNA solution transfer
• Automation

Solid-Phase Extraction - Disadvantages

• Loss of small DNA fragments

Challenging Samples - Seminal Stain

• Acrosome very resistant to digestion due to di-sulphide bridges
• Addition of dithiothreitol (DTT), a reducing agent that breaks the disulphide bridges allowing the release of DNA

Differential Extraction

• Modified version of the organic extraction procedure. First described by Gill et al. 1985, and Guisti et al. 1986.
• Process to isolate the male and female DNA from a sexual assault evidentiary sample.
• From a single evidentiary sample, a female fraction containing the DNA from the victim’s epithelial cells, and a male fraction containing the DNA from the sperm of the assailant are isolated.
• The procedure involves preferentially breaking open the female epithelial cells with an incubation in a SDS/Proteinase K mixture.
• Sperm heads remain intact during this incubation.
• The sperm heads are pelleted and the supernatant containing the female fraction is collected and saved.
• The sperm pellet is washed several times to remove any residual DNA from the victim.
• The sperm are subsequently lysed by treatment with a SDS/proteinase K/ dithiothreitol (DTT) mixture. The DTT is required to breakdown (reduce) the protein disulfide bridges that make up the sperm head. The sperm are impervious to lysis without the addition of the DTT.
• Both the male fraction and the female fraction are then extracted with phenol-chloroform, and the DNA precipitated with ethanol.

Challenging Samples - Hair

Hair roots:

• Plucked hair shafts contain cellular material around the root
• Nuclear and mtDNA can be recovered
  Hair shaft:
• Consists of keratin, trace metals and pigments
• DNA released through mechanical grinding or digestion with DTT
• Often only mtDNA recovered
• Cellular material may get trapped in matrix (a possible source of nuclear DNA)

Challenging Samples - Bone

• Human identification:
  • Air crashes
  • Clandestine graves
  • Mass graves
  • Unidentified bodies
  • Inheritance issues
• Which bone should we use?

Preparing Bone Samples

1. Remove any contaminating DNA
   • Remove soft tissue / Bleach / Abrasion
2. Mechanical disruption of the bone
   • Drilling / Liquid N2

Processing Bone Samples

Breakdown of bone matrix

• In addition to mechanical breakdown:
  • Chemical
    • Ethylene glycol tetraacetic acid (EGTA). Compared to EDTA, it has a lower affinity for magnesium, making it more selective for calcium ions.
    • Proteinase K
    • Collagenase
      Removal of protein and other cellular components
• Chaotrophic reagents (Phenol/chloroform)
  Concentration of DNA
• Ethanol Precipitation
• Silica binding (columns)

DNA Extraction Methods Comparison

• Organic extraction method yields higher amount of DNA.
• But solid-phased extraction yields high-quality DNA and can be automated. Also lowers the risk of contamination making it very useful for forensic extraction of DNA. However, solid-phased extraction commercial kits are more expensive than organic and Chelex extractions.

PCR Inhibition and DNA Degradation

• When extracting biological materials for the purpose of forensic DNA typing, it is important to try to avoid further degradation of the DNA template as well as to remove inhibitors of PCR where possible.
• The presence of inhibitors or degraded DNA can lead to complete PCR amplification failure or a reduced sensitivity of detection usually for the larger PCR products.

PCR Inhibition

• Two PCR inhibitors commonly found in forensic cases are hemoglobin and indigo dyes from denim.
• Melanin found in hair samples can be a source of PCR inhibition when trying to amplify mitochondrial DNA.
• These inhibitors likely bind in the active site of the Taq DNA polymerase and prevent its proper functioning during PCR amplification.

DNA Degradation

• DNA degrades through a variety of mechanisms including both enzymatic and chemical processes.
• Once an organism dies, its DNA molecules face cellular nucleases followed by bacterial, fungal and insect onslaughts depending on the environmental conditions.
• In addition, hydrolytic cleavage and oxidative base damage can limit successful retrieval and amplification of DNA.

How Much DNA Can We Recover?

• A diploid cell contains approximately 6 pg of DNA
• Sperm contains approximately 3 pg of DNA
• The average WBC of an adult is 5-10 X $10^6$ cells per ml of blood. Therefore, the theoretical recovery of DNA per ul of blood is 10-60 ng.

How Much DNA Do We Need?

• The PCR reactions call for an average 1 ng of DNA.
• This is the equivalent of 1/20 of 1 ul of blood, or 350 sperms (3 pg/sperm).
• Many of the commercially available kits are sensitive below 1 ng of DNA (100-250 pg).

Issues During Extraction Procedures

• Contamination
• Good lab practice
• Autoclaving/UV
• Clean working surfaces
• Lab coats
• Mask
• Separation of crime scene samples and reference samples by space and time
• Calibration of equipment
• Pipettes
• Water-baths
• Fridges/freezers

Further Reading

• Butler “Advanced Topics in Forensic DNA Typing: Methodology” - Chapter 2
• Goodwin, Linacre & Hadi “Introduction to Forensic Genetics” - Chapter 4