Forensic Biology: DNA Extraction, Quantitation, PCR Amplification, and Detection

Comprehensive vocabulary flashcards covering DNA extraction techniques, quantitation assays, PCR principles and parameters, and direct/indirect detection systems in forensic biology.

Melting Temperature ($T_m$)

The temperature at which $50\%$ of double-stranded DNA molecules are denatured into single strands, influenced by salt concentration, GC content, pH, and fragment length.

Hot-Start PCR

A PCR technique that minimizes nonspecific primer annealing and primer dimer formation during room-temperature setup, typically utilizing an enzyme such as AmpliTaq Gold DNA polymerase which remains inactive until activated by heat (e.g., $95^\circ\text{C}$) that lowers buffer pH.

Multiplex PCR

The simultaneous amplification of more than one target region of template DNA in a single reaction vessel using multiple primer sets designed to have compatible annealing temperatures and distinct amplicon sizes.

Stochastic Effect

An unequal sampling phenomenon occurring when amplifying low copy number (LCN) DNA templates, leading to disproportionate detection of two alleles in a heterozygous locus or complete allele drop-out.

Reverse Transcriptase

An enzyme—commonly encoded by the pol gene of avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (MMLV)—that catalyzes the synthesis of single-stranded complementary DNA (cDNA) from an mRNA template.

RNase H

A retroviral endonuclease domain of reverse transcriptase that degrades the RNA template strand in RNA-DNA hybrid duplexes during cDNA synthesis.

Oligo(dT) Primers

A class of universal single-stranded primers that hybridize specifically to the $3'$ poly(A) tails of eukaryotic mRNAs, allowing cDNA synthesis exclusively from transcribed genes.

Proteinase K

A proteolytic enzyme used during cell lysis and tissue disruption to digest cellular proteins, degrade nucleases, and release DNA from nucleoprotein complexes.

Dithiothreitol (DTT)

A reducing agent used during DNA extraction that cleaves disulfide bonds in crosslinked proteins, enabling the lysis of chemically resistant sperm cell membranes.

Chelex 100

A chelating resin used in boiling extraction methods that binds polyvalent metal ions such as $Mg^{2+}$, preventing DNase activation and enzymatic degradation of DNA.

Chaotropic Salts

Substances (such as guanidinium salts) that disrupt hydrogen bonding networks and water hydration shells, causing nucleic acids to become hydrophobic and reversibly adsorb onto silica matrices.

Differential Extraction

An extraction method designed for sexual assault evidence containing sperm and epithelial cells; it lyses nonsperm cells first using SDS and proteinase K, pellets intact sperm heads, and subsequently lyses the sperm pellet using proteinase K supplemented with DTT.

Phenol-Chloroform Extraction

A solvent-based extraction using phenol:chloroform:isoamyl alcohol ($25:24:1$) that separates cellular components into an upper aqueous phase (containing DNA), an interphase (containing proteins), and a lower organic phase (containing lipids).

Centrifugal Ultrafiltration

A method employing devices such as Microcon or Centricon with molecular weight cutoff filters (typically 100) to wash, desalt, and concentrate purified DNA solutions.

Reagent Blank

A negative control tube containing all extraction reagents without biological material, carried through the entire extraction and amplification protocol to monitor for chemical or carryover contamination.

Ethidium Bromide (EtBr)

A planar, fluorescent intercalating dye that inserts between stacked base pairs of double-stranded DNA, permitting visualization under ultraviolet light at sensitivities down to approximately $10\,ng$.

Silver Staining

A polyacrylamide gel staining method where positively charged silver ions ($Ag^+$) bind to DNA and are subsequently reduced by formaldehyde to metallic silver ($Ag$), providing approximately 100-fold higher sensitivity than ethidium bromide.

Lumigen PPD

A chemiluminescent substrate dephosphorylated by the enzyme alkaline phosphatase (AP) to produce long-lasting photon emissions detectable on X-ray film.

Tetramethylbenzidine (TMB)

A colorimetric substrate oxidized by horseradish peroxidase (HRP) at acidic pH to yield an insoluble blue precipitate, commonly utilized in slot blot quantitation and reverse blot typing.

Fluorophore

The component of a fluorescent dye molecule that absorbs light energy at an excitation wavelength and emits a photon of longer wavelength as an electron relaxes to the ground state.

Jablonski Diagram

An energy diagram illustrating the absorption of an excitation photon by a molecule promoting an electron to an excited state ($S_2$), non-radiative internal conversion/relaxation to the lowest vibrational level ($S_1$), and subsequent fluorescence emission upon returning to the ground state ($S_0$).

Fluorescence Resonance Energy Transfer (FRET)

A distance-dependent physical process wherein excitation energy is non-radiatively transferred from an excited donor fluorophore (reporter dye) to a nearby acceptor molecule (quencher moiety).

DNA Advisory Board (DAB) Standard 9.3

A forensic quality assurance standard requiring human-specific DNA quantitation of evidentiary samples prior to PCR amplification to ensure optimal template input (typically $0.5$ to $2.0\,ng$).

Slot Blot Assay

A membrane-based hybridization assay that immobilizes denatured single-stranded DNA and hybridizes it to a primate-specific probe to quantify human DNA in the range of $150\,pg$ to $10\,ng$.

D17Z1 Locus

A primate-specific alpha-satellite sequence targeted by a 40-nucleotide biotinylated or alkaline phosphatase-conjugated probe in slot blot quantitation assays.

Cycle Threshold ($C_T$)

The PCR cycle number at which the accumulating fluorescent signal crosses an established baseline threshold, inversely proportional to the logarithm of the initial template quantity.

TaqMan Probe

A sequence-specific oligonucleotide probe dual-labeled with a $5'$ reporter dye and a $3'$ quencher dye that relies on the $5'$ exonuclease activity of Taq polymerase during extension to separate the dyes and generate fluorescence.

Minor Groove Binder (MGB)

A chemical modification attached to the $3'$ end of TaqMan probes that binds to the minor groove of duplex B-DNA, stabilizing hybridization and raising probe $T_m$ to permit shorter probe lengths.

Internal Positive Control (IPC)

A synthetic or exogenous target DNA sequence co-amplified in a real-time qPCR reaction to distinguish true low-template samples from those suffering from PCR inhibition (which causes an elevated IPC $C_T$).

Split Peaks ($\pm A$)

An STR typing artifact arising from overloading template DNA ($>2.0\,ng$) in which incomplete non-template adenylation by DNA polymerase produces twin peaks differing by a single adenine nucleotide.