Chapter 6: DNA Quantification

Why is there an optimal quantity of DNA for most commercial STR kits?

• All sources of DNA are extracted when biological evidence is processed

• Non-human DNA like bacterial, fungal, plant, or animal may also be present in the total DNA recovered

• Requirement of human-specific DNA quantitation so that appropriate levels of human DNA can be included in the subsequent PCR amplification

Why is quantification important?

• Estimate extraction method efficiency: methods yield different amounts and quality of DNA

• Multiplex STR typic works best with a fairly narrow range of human DNA

• Dilute sample to know concentrations for PCR: conserves DNA and reduces possibility of inhibitors

What would happen on a multiplex PCR if there was too much DNA?

• Off-scale peaks

• Split peaks (+/-A)

• Locus-to-locus imbalance

What would happen on a multiplex PCR if there was too little DNA?

• Heterozygote peak imbalance

• Allele drop-out

• Locus-to-locus imbalance

What are the methods for quantification?

• Slot Blot

• UV absorbance: Nanodrop/Spectrophotometer

• Intercalating Dye Assays: PicoGreen and Quibit

What is the Slot Blot method?

• Primate-specific probe binds to satellite sequence D17Z1

• DNA bound to nylon membrane, then probed

• Intensity of signal from probed DNA compared to standards prepped via serial dilution

• Comparison is most often subjective

What is the UV absorbance: Nanodrop/Spectrophotometer method?

• Nucleic acids absorb UV light at a maximal wavelength of 260nm

• A linear relationship exists between the absorption of light and the concentration of nucleic acid in the cuvette

What are the disadvantages of UV absorbance: Nanodrop/Spectrophotometer?

• Poor limit of detection

• Low sample volume required

• Cannot differentiate between different types of nucleic acids

• Not human DNA specific

• Contaminants give false signals

What is the Intercalating Dye Assays: PicoGreen and Quibit method?

• Certain molecules bind to dsDNA

• dsDNA molecule provides a hydrophobic environment that allows PicoGreen molecules to fluoresce differently than when they are in aqueous solution

• Molecules are excited by light and fluoresce in proportion to amount of DNA they bind, where stringer emissions indicate more DNA

Why does qPCR matter?

• PCR in the products are analyzed and monitored

• Once per thermal cycle, fluorescence is measured and recorded as a normalized reported

• Monitor the PCR during the exponential phase where the first significant increase in amount of PCR product correlates to initial amount of target template

What is qPCR analysis?

• When PCR is close to 100% efficiency

• The C_T is defined as the number of cycles required for the fluorescent signal to exceed background levels

• Inversely proportional to the amount of target nucleic acid in the sample

• Low C_T = greater amount of nucleic acid

What are the 2 types of qPCR assays?

• TaqMan - two primers and a fluorescent probe

• SYBR green - two primers

How does TaqMan work?

• Probe contains a reporter dye and quencher dye

• When probe is intact the quencher suppresses the fluorescence

• As TaqMan amplifies target sequence, it cleaves the probs causing fluorescence of the amplified product

How does SYBR Green work?

• Dye fluoresces when bound to dsDNA

• When DNA is denatured, SYBR green dye is released, and fluorescence is reduced

• PCR products are amplified

• When polymerization is complete, dye binds to the dsDNA product resulting in a net increase in fluorescence

What is the specificity of TaqMan and SYBR Green?

TaqMan

• Detects specific amplification products only

SYBR Green

• Detects all amplified dsDNA, including non-specific reaction products

What is the advantages of TaqMan and SYBR Green?

TaqMan

• Specific hybridization between probe and target reduces false positives

• Label probes with different reporter dyes allowing for the amplification of two distinct sequences in one reaction tube

SYBR-Green

• Monitor the amplification of any dsDNA sequence

• No probes are required, which reduces assay setup and costs

• Multiple dyes can bind to a single amplified molecule, increasing sensitivity

What is the disadvantages of TaqMan and SYBR Green?

TaqMan

• A different probe has to be synthesized for each unique target sequence

SYBER-Green

• Dye binds to any dsDNA, including non-specific sequences, possibly generating false positive signals

What are the advantages of qPCR?

• Sensitive to the same inhibitors faced in a traditional STR assay

• Availability of commercial qPCR kits

• Higher throughput of reduced and user intervention

• High sensitivity

• Large dynamic range

What are the disadvantages of qPCR?

• Accurate quantitation assumes that each unknown sample is amplified at the same efficiency as the calibrant samples in the dilution series

• Less sample subjects to variability/uncertainty

• Subject to inibition

• In highly degraded samples, assays that simplify short target sequences

What can results using qPCR kits aid in determining?

• If the sample contains enough human DNA and/or human male DNA to proceed with STR analysis

• The relative quantities of human male and female DNA in a sample that can assist in the selection of the applicable STR chemistry

• If PCR inhibitors are present, may require purification before STR analysis

• If the sample is degraded

What is the Quantifier Duo DNA Quantification Kit?

• Assays consist of the following single-copy targets (two primers + one TaqMan probe per locus)

  • Human

  • Human Male

  • IPC

• IPC assay includes novel synthetic DNA that is spiked into reaction

What is the PowerQuant DNA Quantification Kit?

• Assays consist of the following proprietary targets (two primers + one TaqMan probe per locus)

  • Human (Auto)

  • Human (Deg)

  • Human Male (Y)

  • IPC

• IPC assay includes novel synthetic DNA that is spiked into reaction

What are qPCR passive reference dyes?

• ROX/CXR

• Used to normalize well-to-well fluorescence signal differences

  • Variation in the optical paths between wells

  • Minor differences in volumes due to pipetting errors

How do you calculate the ratio of male DNA to female DNA in a sample based on qPCR results?

Male DNA: Female DNA = [Male DNA]: [(Human DNA) - (Male DNA)]/(Male DNA)