ABC Forensic DNA cheat sheet

Presumptive tests: Kastle-Meyer and luminol for blood; acid phosphatase for semen; amylase for saliva. Presumptive positives are screening results, not identification.

Confirmatory tests: Takayama or Teichmann for blood; PSA/p30 immunoassay or sperm microscopy for semen. Confirmatory tests identify a body fluid but not its donor.

Alternate light source screening uses specific wavelengths and filter goggles to locate dried stains; fluorescence is only a screening indication and requires follow-up testing.

Differential extraction is selected for sexual assault samples to separate sperm and epithelial fractions; the sperm fraction requires DTT to break protamine disulfide bonds.

Sample selection prioritizes stains with visible biological material, high DNA content, and minimal substrate inhibition; a nearby substrate control is collected when possible.

Touch DNA samples are low template; collection area, pressure, and substrate affect yield, and over-sampling can increase inhibitor and background DNA.

Body fluid identification can use mRNA, miRNA, DNA methylation, or immunoassay; these methods can be more specific than catalytic presumptive tests.

When multiple stains are present, each is sampled separately and documented by location; mixing stains before extraction can create uninterpretable mixtures.

Phenol-chloroform-isoamyl alcohol extraction denatures proteins and partitions DNA into the aqueous phase; residual phenol or chloroform inhibits PCR and must be removed.

Silica-column extraction binds DNA in chaotropic salt, washes away inhibitors, and elutes in low-salt buffer; bind, wash, and elution volumes affect yield and concentration.

Chelex extraction binds polyvalent metal ions and uses boiling to lyse cells; the product is single-stranded DNA suitable for PCR but not ideal for long-term storage.

Lysis uses proteinase K to digest proteins; sperm heads require DTT to reduce protamines, and tough tissues may need longer digestion.

Common PCR inhibitors include hematin, humic acid, melanin, indigo, collagen, EDTA, and SDS; qPCR internal controls detect inhibition, and dilution or cleanup can relieve it.

qPCR quantitation is human-specific and can estimate male DNA, degradation, and inhibition; fluorometry measures total DNA and cannot distinguish human from non-human DNA.

Degradation index compares long and short qPCR targets; a high index indicates fragmentation and flags risk of allelic drop-out during STR amplification.

STR multiplex amplification uses dye-labeled primers, dNTPs, MgCl2, and thermostable polymerase; input DNA is normalized to a target amount when sufficient extract is available.

Forensic STR loci are short tandem repeats, commonly tetranucleotide repeats; core kits type the 20 CODIS core loci plus Amelogenin for sex.

Capillary electrophoresis separates dye-labeled fragments by size; injection time, voltage, polymer, and capillary length affect resolution and peak height.

An allelic ladder defines allele bins for each locus; an internal size standard converts migration time to base-pair size for sample alleles.

A spectral calibration matrix separates fluorescence dyes; poor calibration causes pull-up, bleed-through, and incorrect color separation.

Stutter peaks are usually one repeat unit shorter than the true allele and are filtered with locus-specific thresholds; stutter ratio increases at lower template.

The analytical threshold separates signal from noise; the stochastic threshold flags peak heights at which allelic drop-out is likely.

Heterozygous peak height ratios below the validated cutoff, often about 60-70%, indicate possible drop-out, suppression, or mixture.

Amelogenin X and Y fragments indicate sex, but Y deletions or mutations can cause a female-like result; Y-STR or Y-specific quantitation can confirm male DNA.

Genotype frequency is p^2 for a homozygote and 2pq for a heterozygote; the product rule multiplies locus frequencies when loci are independent.

Random match probability is the product of genotype frequencies across typed loci; Y-STR and mtDNA use haplotype frequencies and cannot use the product rule across loci.

Likelihood ratio equals the probability of the evidence under the prosecution hypothesis divided by the probability under the defense hypothesis.

For a diploid mixture, combined probability of inclusion is the product across loci of the squared sum of allele frequencies in the mixture.

The number of contributors is estimated from the maximum number of alleles at any locus; allele sharing can hide contributors and make this a minimum estimate.

Allelic drop-out removes a true allele, drop-in adds a spurious allele, and both increase at low template; replicate amplifications and probabilistic models address these effects.

Probabilistic genotyping uses peak heights and models of drop-out and drop-in; it reports a likelihood ratio and can evaluate mixtures that binary methods cannot.

Theta correction uses Fst to adjust for population substructure; it accounts for relatedness within populations and generally makes match probabilities less extreme.

FBI Quality Assurance Standards and SWGDAM guidelines define validation, interpretation, and reporting expectations; ISO/IEC 17025 accreditation demonstrates laboratory competence.

Each extraction and amplification batch includes a reagent blank, negative amplification control, positive control, allelic ladder, and internal size standard.

A failed negative control indicates contamination and triggers investigation before results are reported; a failed positive control indicates reagent or instrument failure.

Internal validation demonstrates sensitivity, specificity, reproducibility, concordance, mixture interpretation, and stochastic thresholds under local conditions before casework.

Proficiency testing includes open, blind, and external samples; unacceptable results require root-cause analysis and corrective action.

Chain of custody records each transfer, storage condition, and handler; breaks in documentation can undermine evidence integrity and admissibility.

Reports state the STR results, statistical interpretation, limitations, and conclusions; technical and administrative reviews are documented before release.

CODIS NDIS eligibility follows federal and state law for convicted persons, arrestees, missing persons, and unidentified remains; expungement procedures apply when qualifying records are removed.