FORENSIC BIOLOGY EXAM 1

Primer

short DNA, single stranded DNA oligonucleotide (around 20-35 bases) that binds (anneals) to a complementary sequence on the template DNA during PCR. It provides a free 3’-OH group for Taq polymerase to start adding nucleotides.

Probe

A labeled oligonucleotide ( with a fluorescent reporter and quencher) that hybridizes to a specific DNA sequence between primers during qPCR. Fluorescence is released when Taq polymerase cleaves the probe, allowing real-time detection.

Allele

A specific variant of a DNA sequence at a given locus. In STR typing, alleles differ by the number of repeat units (example: TH01 9 vs TH01 9.3 microvariant). Individuals inherit one allele from each parent.

Microvariant

An allele that differs by a partial repeat or base change (example: allele 9.3 at TH01 has 9 full repeats + 3 extra bases). Reported with decimals.

Plateau Effect

In later PCR cycles, product amplification slows/stops because reagents (dNTPs, primers) are depleted and product molecules re-anneal to each other instead of primers. Results in no further exponential increase.

Electrophoresis

A technique to separate charged molecules by size. DNA (negatively charged due to phosphate backbone) migrates towards the positive electrode in a gel or capillary matrix.

Capillary Electrophoresis (CE)

High-resolution separation of DNA fragments inside thin silica capillaries filled with polymer. Fluorescently labeled fragments are detected as they migrate past a laser. Faster and more precise than gels.

Electrokinetic Injection

DNA sample is loaded into the capillary by applying an electric field, which drives negatively charged DNA fragments into the polymer.

Fluorophore

A dye covalently attached to primers/probes that absorbs light (excitation) at one wavelength and emits light (emission) at another. Each dye is spectrally distinct (blue, green, yellow, red).

Fluorescence detection

A laser excites the fluorophore, emission light passes through a spectral grating, and a CCD camera records signal intensity.

RFU

Relative Fluorescence Units - The quantitative measure of fluorescence intensity in electropherograms; reflects the peak height of an allele signal.

Allelic Ladder

A mix of known STR allele fragments used as a reference “ruler” to a size and label sample peaks at each locus. Ensures consistency across labs.

Internal Lane Standard (ILS)

A set of fragments of known size run alongside each sample in CE.

Stutter

A minor peak one repeat shorter (N-1) than the true allele, caused by DNA polymerase slippage during PCR. Less commonly N-2 or N+1.

Pull-up

Artificial peaks in one dye channel caused by spectral overlap (bleed-through) of a strong allele signal in another channel.

Spike

Sharp, non-reproducible peaks caused by electrical or instrument noise (usually very narrow).

Dye Blob

Diffuse peaks caused by unincorporated fluorescent dye molecules.

Analytical Threshold

Minimum RFU value above which peaks can be considered potential alleles (commonly 50-150 RFU depending on lab).

Stochastic Threshold

RFU level above which a peak can be reliably considered a true homozygote; below this, allele dropout of the sister allele is possible (200-900 RFU depending on instrument/protocol)

Stochastic effect

random variation that happens when the DNA template amount is very low. This could be allele dropout (one allele in a heterozygote fails to amplify making it look falsely homozygous) and peak imbalance ( one allele much taller than the other).

What is Organic Extraction (phenol-chloroform)

A chemical method where Proteinase K digests proteins, then phenol-chloroform separates phases. DNA stays in the aqueous phase, proteins in the organic phase. DNA is then concentrated by ethanol precipitation. Produces high-molecular-weight dsDNA (good for RFLP/STR). Drawback: slow, hazardous chemicals.

What is Chelex Extraction?

Uses Chelex resin beads to bind metal ions (Mg2+), which protects DNA from nucleases. Sample is boiled to lyse cells, DNA remains in supernatant. Produces ssDNA (single stranded DNA), suitable for PCR only. Pros: fast, simple, one tube. Cons: can denature DNA, not for RFLP.

What is Silica-based Extraction?

DNA binds to silica in the presence of chaotropic salts. Contaminants are washed away, then DNA is eluted with low-salt buffer. Used in Qiagen kits, DNA IQ (magnetic silica beads), FTA paper. Produces clean DNA, scalable, widely used in forensic labs.

What is Differential Extraction?

Used for sexual assualt samples. Epithelial cells lyse with proteinase K + detergent; sperm cells remain intact (due to strong disulfide bonds). Adding DTT later breaks sperm heads open, releasing male DNA separately.

What is RFLP (restriction fragment length polymorphism)

An old method of DNA profiling that uses restriction enzymes to cut DNA at specific recognition sites. Variation in the number of repeats (VNTRs = Variable Number Tandem Repeats) produce fragments of different lengths.

What is STR (short tandem repeat)

A modern DNA profiling method that analyzes short sequences of DNA (2-6 bp) repeated in tandem at specific loci.

How much DNA is in a diploid cell?

~6 picograms (pg)

Which body fluids are the richest in DNA?

Blood (20-40 ng/uL), semen (150-300 ng/uL), saliva (1-10 ng/uL), buccal swabs (100-1500 ng/swab).

What are some common inhibitors of DNA extraction?

Hematin (from blood), humic acid (soil), dyes, collagen, tannins. These can block PCR by binding DNA or Mg2+.

What are nucleases and why are they a problem?

Enzymes (exonucleases, endo nucleases) that degrade DNA. Exonucleases trim ends; endonucleases cut internally. Must be inactivated/removed during extraction.

How is contamination prevented in extraction?

Process evidence before reference samples, use DNA-free reagents, run reagent blanks, use aerosol-resistant pipette tips, separate pre- and post- PCR areas.