FSCT 8150 Week 3 - DNA Extraction and Quantification Notes
DNA Extraction Methods
Two commonly used methods:
Organic extraction: Uses organic solvents like phenol and chloroform to separate DNA from proteins and other cellular debris. This method is effective for a variety of sample types but is more labor-intensive.
Silica-based extraction: Employs silica membranes or beads to selectively bind DNA. This method is faster and more amenable to automation.
Examples: DNA IQ, PrepFiler, EZ1 kits.
Both methods are used in North America for various DNA evidence samples (questioned/known).
Direct PCR amplification is frequently used for known samples, skipping extraction/quantification. This approach is particularly useful for reference samples that are expected to have high-quality DNA.
Direct vs. Differential extractions:
Direct: Typical extraction method applied when there is no need to separate different cell types.
Differential: Used for samples suspected of containing sperm to separate sperm and epithelial cells, which is crucial in sexual assault cases.
Goal of DNA Extraction
Isolate DNA (nuclear or mitochondrial).
Separate DNA from cellular components (proteins, lipids) and extracellular material, including PCR inhibitors.
Concentrate DNA into a final extract to increase the DNA quantity for downstream applications.
Nuclear DNA:
Located in the nucleus.
base pairs (BP) per genome.
2 meters in length, highly organized into chromosomes.
Mitochondrial DNA:
Located in the mitochondria.
Circular loop of base pairs.
Difference in copy number:
Nuclear DNA: Two copies per cell (one from each parent).
Mitochondrial DNA: Hundreds or thousands of copies per cell (due to multiple mitochondria), making it useful for degraded samples.
Mitochondrial DNA may be more sensitive due to higher copy number, but nuclear DNA offers better individualization due to its unique genetic markers.
PCR Inhibitors
Extraction must remove known and unknown PCR inhibitors to allow successful amplification. Inhibitors can compromise the accuracy and reliability of PCR results.
Examples:
Blue denim (indigo dye): Commonly found on clothing.
Heme (in blood): Can interfere with DNA polymerase activity.
Humic acid (in soil): Present in environmental samples.
PCR (Polymerase Chain Reaction) is like photocopying DNA; inhibitors prevent/limit this process, reducing the ability to amplify target DNA sequences.
DNA Extraction Steps
Get cells (containing nucleus/nuclear DNA) into a tube with lysis buffer.
Lysis buffer breaks down cell walls, releasing DNA. The composition of lysis buffer is critical for efficient cell lysis and DNA recovery.
Extraction methods should handle different biological samples, including blood, saliva, tissue, and bone.
Bones and teeth samples require a freezer mill or cryogenic grinder.
Bones are cut into small pieces and placed in a vial with a metal impactor and metal stoppers.
Liquid nitrogen is used to make the bones brittle, facilitating efficient grinding.
The grinder pulverizes the bone into a powder that can be added to lysis buffer, maximizing DNA yield.
Organic DNA Extraction Method
Process:
Cellular lysis using lysis buffer.
Incubation (e.g., overnight at 56°C; time/temperature can vary) to allow Proteinase K to digest proteins.
Vortex and centrifuge to mix and separate cellular components.
Use phase contrast gel to separate organic and aqueous phases. Phase-lock gels help to sharpen the separation.
Remove aqueous phase (containing DNA) and discard organic phase, which contains proteins and lipids.
Use Amicon microconcentrator to concentrate the DNA by spinning it down. Centrifugal filters with specific molecular weight cut-offs are used to retain DNA while removing smaller contaminants.
Wash a few times and concentrate into TE buffer; store at -20°C to preserve DNA integrity.
Extraction buffer components:
Tris HCl: Maintains pH to protect DNA from degradation.
Salt: Provides ionic strength to optimize DNA solubility.
EDTA: Chelating agent that binds metals and inhibits nucleases (enzymes that degrade DNA).
SDS: Detergent that solubilizes cell membranes, aiding in cell lysis.
Proteinase K (ProK): Enzyme that degrades proteins, releasing DNA.
Phase-lock gel:
Aqueous phase (DNA) at the top, retained for further steps.
Gel in the middle separates phases, trapping unwanted proteins.
Organic phase (phenol chloroform) at the bottom, discarded.
Microconcentrator:
Removes trace contaminants/PCR inhibitors via size exclusion.
DNA stays in the top (aqueous) phase and is washed with TE buffer.
Larger DNA molecules are retained, while smaller inhibitors filter through.
Benefits:
Reliable method with high DNA recovery.
Works for multiple sample types.
Few PCR contaminants.
Drawbacks:
Time-consuming (typically manual extraction).
Not easily amenable to high throughput robotics.
Silica Extraction Method (DNA IQ Example)
Newer method (but has existed for a long time).
DNA IQ basic kit; add-on tissue and hair extraction kit for difficult samples. These kits include optimized buffers and reagents for specific sample types.
Amenable to robotic systems, allowing for high-throughput processing.
Uses silica paramagnetic resin that binds DNA reversibly, facilitating easy separation of DNA from contaminants.
RCMP lab uses this method, demonstrating its reliability and widespread use.
Resin capacity:
Limited DNA binding capacity, preventing the need for dilution. This feature ensures consistent DNA yields.
More blood leads to more DNA up to a certain point due to resin saturation. Understanding the resin capacity is critical for optimal DNA recovery.
Sample types: Database samples, swabs, paper, FTA cards, liquid blood, stained items, swabbed items. The versatility of this method makes it suitable for various forensic samples.
Components: Resin, lysis buffer, wash buffer (2x), elution buffer. The specific contents and concentrations of these buffers are crucial for efficient DNA extraction.
Buffer contents and concentrations are often trade secrets, highlighting their importance in the extraction process.
Tissue and hair extraction kit add-on: Includes DTT to break down tissue and hair, improving DNA yield from these challenging samples.
DTT is also used in differential extraction to break open sperm cells, demonstrating its role in cell lysis.
Process:
Resin slurry added to the sample; DNA binds under acidic conditions (pH < 7.5). Maintaining the correct pH is critical for efficient DNA binding to the resin.
Paramagnetic resin retained using magnetic rack or stand. Using magnetic separation simplifies the washing steps.
Solution is discarded; wash buffer is added, vortexed, and removed to eliminate contaminants.
Elution buffer (changes pH, heats up) releases DNA from resin. Changing the pH and applying heat disrupts the DNA-silica binding, releasing pure DNA.
Pure DNA solution is removed to a new tube for downstream applications.
Benefits:
No centrifuge needed, simplifying the extraction process.
Works for a variety of samples, especially with the tissue/hair kit.
Less tedious, quick, and easy to automate.
No phenol chloroform (toxic), enhancing safety.
Few PCR inhibitors, improving the reliability of downstream PCR.
More amenable to robotics, allowing for high-throughput processing.
Drawbacks:
Poor yields for degraded samples (e.g., bones), making it less suitable for ancient DNA analysis.
More expensive than organic extraction (due to purchasing kits).
Prep Filer (Applied Biosystems/Thermo Fisher)
Silica bead method (similar to DNA IQ).
Amenable to robots, facilitating high-throughput DNA extraction.
Smaller silica beads provide a larger surface area, binding and retaining more DNA, enhancing DNA recovery.
Steps: Cell lysis, substrate removal, DNA binding, wash, DNA purification, elution. Each step is optimized for efficient DNA extraction.
QIAGEN
EZ1 kit.
Silica bead and silica spin column extraction kits available, offering flexibility in DNA extraction methods.
DNA Extraction Strategy
Direct approach (silica or organic): Non-semen containing samples, simplifying the extraction process.
Differential extraction: Samples with semen, separating sperm and epithelial cells for targeted DNA analysis.
Differential Extraction Approach
Biochemical method to physically separate sperm from epithelial cells, which is crucial in sexual assault cases.
Takes advantage of sperm's hardiness, as sperm cells are more resistant to lysis than epithelial cells.
Sequential lysis of cells using increasingly harsh conditions, final with DTT to break down sperm. DTT reduces disulfide bonds in the sperm cell membrane, facilitating lysis.
Fractions:
F1: Least harsh conditions mainly extracts non-human DNA, may contain some degraded or highly concentrated epithelial/sperm cells).
F2 (non-sperm/epithelial fraction): Mainly epithelial cells, may contain some sperm cells (if degraded or sample contains only sperm).
F3 (sperm fraction): Mainly sperm cells, may have some carryover of epithelial cells. Careful optimization is needed to minimize carryover.
Many labs skip the F1 step to streamline the process.
Example protocol (with F1):
Stained item in tube with PBS buffer and SarcoSil overnight at 4°C. This step hydrates and loosens cells from the substrate.
Centrifuge to pellet epithelial and sperm cells; remove supernatant (F1 fraction).
Add F2 lysis buffer (Pro K); vortex to resuspend pellet and incubate for 2 hours at 37°C. Proteinase K digests epithelial cells.
Centrifuge; collect supernatant (F2 fraction), which contains epithelial cells.
Add F3 lysis buffer (Pro K, DTT) to sperm pellet; vortex and incubate for 2 hours at 37°C (F3 fraction). DTT breaks down sperm cells.
Proceed with organic extraction for F2 and F3 fractions (or store F1) to purify DNA.
DNA Quantification
Goal: Determine the amount of human DNA in a DNA extract. Accurate quantification is essential for optimal PCR amplification.
Methods: Slot blot and real-time PCR. Real-time PCR is now the preferred method due to its accuracy and sensitivity.
Must be human-specific to avoid amplifying non-human DNA.
Most labs have switched to real-time PCR because it is more accurate, automatable, and provides real-time data.
Purpose of Quantification:
Avoid wasting time, effort, and money on samples with no human DNA.
PCR requires a specific range of input DNA for reliable results. Too much or too little DNA can lead to inaccurate results.
PCR kit examples: Profiler Plus, Identifiler Plus, Minifiler, Globalfiler. Each kit is designed for specific applications and DNA concentrations.
Globo philer could be pg to one ng.
Too much DNA: Off-scale peaks, pull-up, increased baseline artifacts, leading to interpretation difficulties.
Too little DNA: Subthreshold peaks, stochastic effects (e.g., allelic dropout), or no results, compromising the accuracy of DNA profiling.
Slot Blot Analysis (QuantiBLOT Kit)
Chemiluminescence detection method. DNA is hybridized with a probe, and the resulting signal is detected using chemiluminescence.
Human/primate-specific to ensure accurate quantification of human DNA.
Human DNA Quantification
Standards and unknowns are placed on the blot. Standards are used to create a calibration curve.
Band intensity indicates DNA quantity. The intensity is measured using a densitometer.
Example calculation:
Band intensity matches ng standard.
ng / 10 µL loaded = ng/µL.
If sample was diluted 100x, then ng/µL * 100 = 8 ng/µL.
Disadvantages of Slot Blot:
Not very precise compared to real-time PCR.
Requires multiple dilutions to ensure accurate quantification.
Semi-quantitative, providing only an estimate of DNA concentration.
Interpretation:
Check ladder and controls to ensure the accuracy of the assay.
Verify negative controls are negative and positive controls contain DNA.
Estimate sample concentrations based on standards.
Factor in dilutions to calculate the original DNA concentration.
Only semi-quantitative, so results should be interpreted cautiously.
Real-Time PCR
Developed by Applied Biosystems, a leader in biotechnology.
Human/primate-specific to ensure accurate quantification of human DNA.
'Real-time' means PCR process is monitored as it happens, unlike slot blot which is endpoint evaluation. This allows for more accurate and sensitive quantification.
Mechanism:
Each target sequence is amplified by two primers and a fluorescently labeled probe. Primers bind to specific DNA sequences, and the probe hybridizes to the amplified region.
Accumulation of PCR product is monitored by detecting cycle-to-cycle changes in the fluorescent signal. The fluorescent signal increases as more PCR product is generated.
Fewer cycles to reach detectable fluorescence = more DNA in the sample. This relationship allows for accurate quantification of the initial DNA concentration.
Uses TaqMan technology, a widely used and reliable method for real-time PCR.
Reagents:
PCR reaction mix: AmpliTaq Gold DNA polymerase, dNTPs, passive ROX reference dye (corrects for pipetting variations). The polymerase amplifies DNA, dNTPs are building blocks, and ROX dye normalizes fluorescence.
Primer mix: Forward and reverse primers (human/primate-specific).
Probe: Reporter dye (FAM or VIC) at 5' end, minor groove binder, and non-fluorescent quencher at 3' end. The probe hybridizes to the target DNA sequence.
Internal positive control (IPC): Synthetic, non-human DNA to monitor PCR inhibition. The IPC ensures the PCR reaction is functioning correctly.
Minor Groove Binder (MGB):
Increases the melting temperature and sequence specificity of the probe. MGB enhances the probe's ability to bind to the target DNA sequence.
TaqMan Probe
Reporter dye (R, FAM or VIC) at the 5' end and quencher (Q) at the 3' end. The reporter dye emits fluorescence when separated from the quencher.
MGB on the 3' end to enhance probe specificity.
The forward primer binds and as the Taq polymerase extends the strand, it cleaves the reporter dye from the probe, and the quencher no longer quenches the fluorescent signal. This cleavage releases the reporter dye, allowing it to fluoresce.
Fluorescence is proportional to PCR product. The more PCR product, the higher the fluorescence signal.
Real-Time PCR Instrument
Small footprint; can run many (e.g., 96) samples at a time. This allows for high-throughput quantification.
Software screenshots:
Amplification plot: Shows different colored lines representing different samples. Each line represents the amplification of a specific DNA sample.
Cycle threshold (CT): Software records the PCR cycle number when the fluorescent signal crosses a certain threshold. The CT value is inversely proportional to the initial DNA concentration.-
Threshold is manually set within the exponential phase of PCR amplification to ensure accurate quantification.
Compare CT values to standards to estimate DNA quantity. By comparing CT values to a standard curve, the DNA concentration can be accurately determined.
Internal Positive Control (IPC):
Should cross the cycle threshold at around 28 cycles. A consistent CT value for the IPC indicates no PCR inhibition.
If the IPC crosses much later (e.g., 36 cycles) or is undetected, then the sample is inhibited. This indicates the presence of PCR inhibitors in the sample.
Standard Curve:
Plot of CT values versus log concentration for samples of known concentration. The standard curve is used to quantify unknown samples.
Negative slope, indicating an inverse relationship between CT value and DNA concentration.
System automatically generates standard curve, plots samples, and reports concentrations, streamlining the quantification process.
Interpreting Real-Time PCR Results
Look at the amplification plot to assess the overall PCR performance.
Check IPC values to identify potential PCR inhibition.
Examine the standard curve to ensure accurate quantification.
Review report view for concentrations and IPC values to obtain a comprehensive overview of the results.
Real-Time PCR vs. Slot Blot
Feature | Slot Blot | Real-Time PCR |
|---|---|---|
Bench space | More | Less |
Time | More | Less |
Samples analyzed | More | Less |
Sensitivity | Less | More |
Internal positive control | No | Yes |
Pipetting error protection | No | Yes (ROX passive dye) |
Dark room, cleaning | Required | Not required |
Newer Real-Time PCR Kits
Applied Biosystems Quantifiler Duo Kit:
Detects both male and human DNA to determine the male/female DNA ratio (useful for sexual assault cases). This allows for targeted DNA profiling based on the sex of the DNA source.
Decision-making based on DNA ratio:
High male, low female: Proceed with autosomal STRs to generate a comprehensive DNA profile.
Low male, high female: Proceed to Y-STR kit to focus on male-specific DNA markers.
PROMEGA Kit:
Quantifies total human and total male DNA, providing additional information for sexual assault cases.
Detection limit: picograms (down to one cell since one cell contains approx. 6 pg of DNA). This high sensitivity allows for the analysis of trace DNA samples.
Automatable with robotics, streamlining the DNA quantification process.
Difference: Plexor has the amplification curve starts at the top and moves downwards whereas Quantifiler amplification curve starts at the bottom and moves up. This difference is due to the detection method used by each kit.
QuantiFiler HP (Human Plus) and QuantiFiler Trio (Applied Biosystems):
Latest versions of the QuantiFiler kits, incorporating advanced features for improved DNA quantification.
Same IPC as Quantifiler, ensuring consistent PCR performance monitoring.
Trio Kit has degradation index (DI) to assess the quality of the DNA sample.
The TrioKit degradation index is an index that assesses allele dropout due to degradation based on fragment size. This is important for interpreting results from degraded DNA samples.
QIAGEN:
Investigator Quantiplex Pro and Investigator High Res kits, offering alternative options for DNA quantification.
Manual vs. Automated Sample Handling (Robotics)
Labs are increasingly adopting robotics to meet demand and maintain quality. Automation improves efficiency and reduces human error.
Robotics reduce repetitive tasks and allow analysts to focus on data interpretation and evidence screening, improving overall lab productivity.
Robotic platforms: TEACAN, QIAGEN, Promega, Applied Biosystems, Beckman, Hamilton. These platforms vary in their capabilities and throughput.
Some robots handle multiple steps (extraction, quantification, PCR setup), while others are limited to DNA extraction only. The choice of robotic platform depends on the specific needs of the lab.