FRSC-3000: Lecture 3

Why is quantification important?

• Estimate extraction method efficiency

• Multiplex STR typing work best with a fairly narrow range of human DNA

• It is important to dilute samples to known concentration for use in PCR

What interpretation challenges arise from too much DNA in PCR?

• Off-scale peaks

• Split peaks

• Locus-to-locus imbalance

What interpretation challenges arise from too little DNA in PCR?

• Heterozygote peak imbalance

• Allele drop-out

• Locus-to-locus imbalance

What is normalization?

The process of obtaining a DNA concentration suitable for STR analysis.

At what range of DNA do STR kits work best?

0.5-2.0 ng of DNA.

What are the general steps of the slot blot method?

• Primate specific probe binds to satellite sequence

• DNA binds nylon membrane and is probed

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

Which quantitation method has a primate specific probe?

Slot Blot Method

What are the general steps of Nanodrop Spectrophotometer?

• Nucleic acids such as DNA and RNA absorb UV light at a specific wavelength

• A linear relationship exists between the absoprtion of light and the concentration of nucleic acid

• This relationship is used to determine the concentration of DNA in a sample using a spectrophotometer

What are some limitations of Nanodrop spectrophotometer?

• It cannot differentiate between different types of nucleic acids

• It is not a human specific procedure

• Contaminants give false signals

What are the general steps of the PicoGreen assay?

• PicoGreen molecules bind dsDNA

• The inside of dsDNA molecules provides a hydrophobic environment and causes PicoGreen molecules to fluoresce differently in aqueous solution

• The molecules are excited by light and the strength of the emissions indicates the amount of DNA present

What are the limitations of the PicoGreen assay?

• It is not human specific

• It is only dsDNA specific

What is the major difference between PCR and qPCR?

In PCR, the products are analyzed after the cycling is completed and in qPCR the products are monitored in real time.

During which phase of PCR does qPCR monitor? What does this tell us?

The exponential phase is monitored. It tells us the initial amount of target template present.

What is the Ct?

The cycle threshold is the defined number of cycles required for the fluorescent signal to exceed the baseline noise in qPCR.

What is the relationship between Ct and the amount of target DNA?

They are inversely proportional. A low Ct indicates a greater amount of DNA in the sample.

What are the two main types of qPCR assay?

• TaqMan assay with two primers and a fluorescent probe

• SYBR green with two primers

Between TaqMan and SYBR Green, which assay has superior specificity?

TaqMan

What is the major disadvantage of TaqMan?

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

What is the major disadvantage of SYBR Green?

It binds any dsDNA, including nonspecific sequences, generating false positive signals.

What are the advantages of qPCR?

• High throughput and automation

• High sensitivity

• Sensitive to the same inhibitors faced in traditional STR assay

• Lare dynamic range

What are the limitations of qPCR?

• Subject to inhibition

• <100 pg subject to variability and uncertainty

• More amplification of short target sequences in degraded samples

• Assumes that each unknown sample is amplified at the same efficiency as the calibrant samples

What is the purpose of passive reference dyes?

ROX are used to normalize well-to-well fluorescence signal differences in qPCR.

Positive amplification occurs when the Ct value for the reporter is:

<40

What is the ratio of male and female DNA in a mixture?

Male/Male:(Human - Male)/Male

What type of STR unit is most commonly used in forensics?

tetranucleotides (4 bases)

What is a microvariant STR?

An allele with a partial repeat.

Why are STRs ideal for forensic applications?

• Small product sizes compatible with degraded DNA and PCR

• Multiplex amplification with fluorescent detection = higher power discrimination

• Commercial kits

• Uniform set of core STR loci (standardization) allows for sharing of databases

STR kits vary based on:

• Loci amplified

• Fluorescent dye combinations

• DNA-strand labelled

• Allelic ladder

• Primer sequence for PCR amplification

How are commercial STR kits “16plex”?

• 13 core loci

• 2 additional loci

• AMEL sex-typing

What are the STR kit components?

• Primer mix

• PCR buffer

• DNA polymerase

• Allelic ladder

• Positive controlAlle

Allelic ladders mix common alleles to create a reference for scoring and are generating using:

The same primers used for sample amplification in the kit.

What are the solutions for allelic overlap in multiplexing?

• Use more dyes when possible

• Add non-nucleotide linkers to change mobility of PCR products

• Redesign primers

How is amelogenin used for sex determination?

It encodes a gene for tooth enamel on both the X and Y chromosomes in slightly different sizes. The gene encoded by the X chromosome has an additional intron.

What are two rare complications with amelogenin amplification?

• Rare deletion of gene on Y-chromosome (Y allele dropout)

• Rare mutations in primer binding sites in males (X allele dropout)