Lecture H2 DNA quantitation

DNA Quantitation Overview

  • Importance of DNA quantitation in forensic analysis.

Purpose of DNA Quantitation

  • Isolating DNA from Biological Evidence

    • All sources of DNA included: human, non-human (bacteria, fungi, plants, and animals).

  • Ensuring Human DNA Recovery

    • Human-specific DNA quantitation is essential.

    • FBI Quality Assurance Standards (Standard 9.4, QAS 2020): require quantification of human DNA in forensic samples before nuclear DNA amplification.

Importance in PCR-based Assays

  • DNA Amount Determination

    • Essential for most PCR assays.

    • Optimal range for multiplex short tandem repeat (STR) typing: 0.5 – 1 ng of human DNA input.

    • Standard practice to use ~1 ng of input DNA.

Impact of DNA Input Amount in PCR

  • Consequences of Excess DNA

    • Leads to off-scale peaks, split peaks (+/- A), and locus-to-locus imbalances.

  • Consequences of Insufficient DNA

    • Causes heterozygote peak imbalances, allele drop-out, and locus-to-locus imbalances.

  • Recommended Input Levels

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

Adjusting DNA Amount for PCR

  • When DNA is Outside the Target Range

    • Adjustment of DNA concentration is necessary.

    • Too much DNA: Dilute the sample.

    • Too little DNA: Employ concentration methods (remove excess liquid to concentrate DNA).

    • Low template approaches may require more amplification cycles.

DNA Quantitation Methods

  • Various Methods Used

    • UV absorbance, yield gels, slot blot, and real-time PCR.

    • Early assays were typically laboratory-prepared (“home-brew”); now, commercially available kits.

UV Absorbance Method

  • Operation and Limitations

    • Nucleic acids absorb UV light at specific wavelengths (260 nm).

    • Higher absorption indicates higher nucleic acid concentration.

    • Not highly sensitive or specific; may yield false high signals due to RNA or contaminating proteins.

Yield Gels-Fluorescence Method

  • Process

    • Extracted DNA is run on agarose gel and stained with dyes (EtBr, DAPI, Sybr-Gold).

    • Quality assessed by intactness (degraded vs. intact).

    • Subjective quantity determination; quality not human-specific.

Slot Blot Method

  • Historical Context

    • Most common forensic method in late 1990s and early 2000s; discontinued by 2006.

    • Utilized a-satellite DNA probe D17Z1; biotin-labeled for detection.

Slot Blot Detection Process

  • Nylon Membrane Capture

    • Genomic DNA captured on a positively charged nylon membrane.

    • Negatively charged DNA binds after denaturation.

  • Hybridization and Detection

    • D17Z1 probe hybridizes only with human DNA.

    • Detection via colorimetric and chemiluminescent methods.

    • Chemiluminescence allows detection down to 10-20 pg of DNA.

Real-Time Quantitative PCR (qPCR) Method

  • Purpose and Functionality

    • Determines amount of “amplifiable” DNA in real-time.

    • PCR failures may arise from inhibitors or degraded DNA.

Real-Time Data Collection in qPCR

  • Instrumentation and Assays

    • Monitors PCR process, reports cycle-to-cycle changes in fluorescence.

    • Common assays include 5' nuclease assay (TaqMan) and intercalating dyes (e.g., SYBRGreen).

    • Instruments: ABI series and Roche light cycler.

5’ Nuclease Assay (TaqMan)

  • Mechanism

    • Suppresses reporter fluorescence via energy transfer between dyes.

PCR Output Interpretation

  • Cycle Number Analysis

    • Assesses amplification growth phases: exponential growth, linear growth, plateau.

  • Threshold Cycle (CT)

    • The cycle at which the fluorescence surpasses background noise, indicating sufficient DNA for detection.

Summary of qPCR Assays

  • Various assays available for quantitating human DNA, including:

    • Quantifiler TaqMan, Quantifiler Y, Quantifiler Duo, and more, targeting specific genes or regions.

    • Assays provide standard curves for quantitation and range from 46 pg to 100 ng DNA.

Next Generation Quantitation: Quantifiler Trio

  • Features

    • Targets human Y and autosomal DNA, providing a male/female ratio and a degradation index.

    • Quick results within 1 hour, beneficial for assessing degraded samples.