DNA Extraction, Quantitation, and Amplification
Extraction chemistry
Cell lysis releases DNA; proteinase K digests protein, DTT breaks disulfide bonds in sperm heads, and detergents solubilize membranes. Silica and magnetic bead methods bind DNA under chaotropic conditions; organic extraction uses phenol-chloroform; Chelex is rapid but crude. Differential extraction separates sperm (male) from epithelial (female) fractions by preferential lysis. Inhibitors such as heme, humic acid, indigo, collagen, EDTA, and SDS carry through and suppress amplification. Ethanol carryover also inhibits. Traps: more extract is not better; dirty extracts can inhibit.
Quantitation
Real-time PCR quantifies human DNA and often male DNA, with an internal positive control to flag inhibition and degradation. The degradation index compares large to small amplicons; a value near 1 suggests intact DNA, while high values suggest degradation. Quantitation guides input normalization, usually 0.5 to 1.0 ng, but the result is not a purity guarantee. Traps: a good quant with inhibition can still fail; male quant may be absent when the male contribution is low; overloading causes pull-up, split peaks, and stochastic effects.
Amplification
Multiplex STR amplification uses fluorescent primers, thermal cycling, and a hot-start polymerase. Controls include positive, negative, and reagent blanks. Inhibitor-resistant polymerases help but do not fix severe inhibition. Low-template samples risk allele drop-out and drop-in; increasing cycles raises sensitivity and artifacts.
Worked rule
If the degradation index is 8 and the quant is 0.2 ng per microliter, do not concentrate blindly; consider re-extraction or a reduced-volume amplification, and expect dropout at larger loci. Target 0.5 to 1 ng when possible, but never exceed the validated range without documenting a deviation.