Purpose of Human-Specific DNA Quantitation

• All sources of DNA are extracted when biological evidence from a crime scene is processed to isolate the DNA present.

• Non-human DNA such as bacterial, fungal, plant, or animal material may also be present in the total DNA recovered from the sample along with the relevant human DNA of interest.

Importance of Human-Specific DNA Quantitation

• The FBI Quality Assurance Standards for Forensic DNA Testing Laboratories document Standard 9.4 requires human-specific DNA quantitation for several reasons:

  • To ensure appropriate levels of human DNA are included in subsequent PCR (Polymerase Chain Reaction) amplification.

  • To evaluate the quality and quantity of the DNA before conducting PCR.

• Multiplex STR (Short Tandem Repeat) typing works best with a fairly narrow range of human DNA, typically 0.5 to 2.0 ng of input DNA, matching commercial STR kits.

• Higher quality data obtained saves time and money in forensic analysis.

Impact of DNA Quantity on PCR

• Too much DNA can cause:

  • Off-scale peaks

  • Split peaks (+/-A)

  • Locus-to-locus imbalance

• Too little DNA can result in:

  • Heterozygote peak imbalance

  • Allele drop-out

  • Locus-to-locus imbalance

• Illustration of DNA sizes and resulting fluorescence:

  • D3S1358 10 ng template (overloaded) vs. 2 ng template (suggested level)

  • Stochastic effects when amplifying low levels of DNA produce allele dropout, emphasizing the importance of DNA quantitation prior to multiplex amplification.

Considerations for DNA Quantitation

• Accurate determination of DNA concentration allows for informed decisions:

  • Whether mitochondrial sequencing is needed (potentially skipping STR analysis).

  • If a miniSTR assay should be used.

  • The necessity of employing low copy number (LCN) methods for STRs.

  • The need to re-extract the sample.

  • If issues arise during STR typing, confidence that the DNA template is not the source of problems (like CE, cycler, or kit issues).

Types of DNA Quantitation Techniques

UV Absorbance

• DNA shows maximum absorbance at 260 nm.

• Single-stranded nucleic acids and other extraction components also absorb at this wavelength and at 280 nm.

• DNA purity can be calculated using absorbance ratio measurements:

  • The ratio of A260/A280 assesses purity.

  • Pure DNA is considered if this ratio is above 1.7; however, it is not human-specific and lacks sensitivity for forensic analysis.

Yield Gel Electrophoresis

• Involves electrophoresis using an agarose gel where DNA is stained with ethidium bromide.

• Samples are compared to a set of calibrators to estimate DNA concentration.

• Distinct bands show high molecular weight DNA; streaked bands indicate degraded DNA.

• Yield gel assesses both quantity and quality but is not specific to human DNA.

Slot Blot (QuantiBlot)

• DNA is bound to a nylon membrane using a human/higher primate specific probe.

• A series of calibrators is run on the blot; detection can include:

  • Colorimetric methods.

  • Radioactive probes.

  • Chemiluminescent probes.

• Sensitive enough to detect concentrations as low as 0.03125 ng/µl.

Fluorescent Intercalating Dye Assay

• Involves the binding of fluorophores to DNA, RNA, or proteins; thus it is not specific to human DNA.

• Fluorescence is measured with a fluorometer.

• The Picogreen microtiter plate assay detects as little as 250 pg of double-stranded DNA in a 96-well format, allowing for automation.

• The Qubit system by Invitrogen is less expensive, measuring one sample at a time.

ALUQuant

• Utilizes the ALU 300 bp sequence, repeated throughout the human genome as a probe.

• Hybridization initiates reactions that result in luciferin oxidation producing measurable light.

• Human-specific and amenable to automation; detects DNA in concentrations from 0.1 to 50 ng.

End Point PCR

• A single locus is amplified using DNA samples of known concentrations (calibrators) with SYBR Green fluorescent intercalating dye for detection.

• Samples are compared against a calibration curve to adjust DNA levels for amplification based on results.

• Primarily used to monitor PCR inhibitors.

Real-time PCR (qPCR)

• Analyzes cycle-to-cycle changes in fluorescent signals during amplification, allowing closed tube detection.

• Performed in specialized thermal cyclers; quantifies amplifiable human DNA present in a sample.

• Types include:

  • Fluorogenic 5’ nuclease assay (TaqMan).

  • Intercalating dye (SYBR green).

Fluorescence Resonance Energy Transfer (FRET)

• Employs a pair of fluorescent probes in proximity, where one (donor) transfers energy to another (acceptor).

• The donor's excitation spectrum significantly overlaps with the acceptor's emission spectrum.

TaqMan (5’ Nuclease) Assay

• Utilizes a TaqMan probe labeled with reporter and quencher dyes; proximity of R and Q suppresses fluorescence.

• Relies on Taq polymerase's exonuclease activity to cleave the probe during amplification, leading to increased fluorescence which can be detected.

• Internal PCR controls (IPC) utilize a non-template synthetic DNA for quality assurance.

• Different fluorescent reporter dyes ensure spectral separation, enhancing assay accuracy.

Quantifiler Kits by Applied Biosystems

• Quantifiler Duo assesses total human DNA and male-specific DNA; Quantifiler Trio evaluates total male, small autosomal, and large autosomal DNA.

Plexor Systems by Promega

• Specially designed systems that incorporate a fluorescent label modified nucleotide paired with iso-dGTP linked to a quencher dye.

• Tailored for total human DNA and distinguishing between male and female ratios in mixtures.

PCR Amplification Phases

• Involves four phases:

  • Lag (doubling but not detected).

  • Exponential (active doubling).

  • Linear (less than doubling).

  • Plateau (minimal change).

• qPCR measurements are typically taken during the exponential phase.

Cycle Threshold (CT)

• Refers to the number of PCR cycles needed to reach amplification threshold.

• Important implications:

  • More cycles indicate less DNA; fewer cycles indicate more DNA.

  • If the IPC’s CT is high, it may signify the presence of inhibitors.

Molecular Beacons

• Single-stranded, hairpin-shaped oligonucleotide probes that fluoresce upon binding target sequences.

• The structure allows for detection only in the presence of the target, allowing specificity.

• Composed of a loop (complementary to the target) and a stem (complementary to itself).

• The 5' end features a fluorophore, while the 3' end has a quencher, preventing fluorescence in the closed loop state.

Differences Between PCR and qPCR

• PCR analyzes products after cycling (static) using techniques like gel, CE, UV, or fluorometry; an end-point assay.

• qPCR continually monitors products during PCR cycles (dynamic), measuring fluorescence at each cycle to track kinetics.

Advantages of Real-Time qPCR

• Higher throughput and automated data analysis, allowing rapid evaluations.

• Commercial kits facilitate widespread adoption for DNA quantitation.

• High sensitivity and large dynamic range (~30 pg to ~30 ng) with the ability to multiplex assays (to some degree).

Challenges of Real-Time qPCR

• Susceptibility to inhibitors; utilizing IPCs helps with this.

• Quantitation precision wanes at low copy numbers (below 30 pg), with variability introduced below 100 pg.

• Quantitation pertains to specific target sequences, not general “DNA”, and accuracy relies on the efficiency of unknown samples compared to calibrants.