From the Crime Scene

to the Laboratory

Dr. Hossein Fallahi

Dep. of Biology,

School of Sceinces,

Razi University

Kermanshah

Iran

Lecture 2:

DNA Profiling

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PCR and STR profiling

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Interpreting a DNA profile

• Interpretation distinguishes alleles from artifacts and evaluates whether the data are suitable for comparison.
• Analysts consider peak morphology, analytical thresholds, stochastic effects, degradation, inhibition, and possible mixtures.
• Single-source profiles are generally simpler than profiles containing DNA from multiple contributors.
• Interpretation guidelines and validated laboratory procedures define how observations are translated into reportable conclusions.
• The scientific question is not simply whether two profiles look similar but how strongly the data support competing propositions.

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Statistics and likelihood ratios

• Population genetics provides allele-frequency information used to evaluate how common or uncommon a profile is in a reference population.
• A random match probability expresses the expected frequency of a profile under a specified population model.
• A likelihood ratio compares the probability of the observed evidence under two competing propositions.
• Large likelihood ratios can indicate that the evidence is much more probable under one proposition than another, but they do not directly equal the probability of guilt.
• Statistical conclusions must be communicated with the assumptions, population data, and limitations that produced them.

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STR markers

• Short tandem repeats consist of repeated DNA motifs located at defined genomic loci.
• Different repeat counts create alleles that can be distinguished by fragment length or sequence.
• Multiplex STR kits amplify many loci simultaneously to generate a multi-locus profile.
• Autosomal STRs inherit one allele from each biological parent in typical diploid cells.
• Because alleles occur at different frequencies in populations, the combined profile can have a very low expected frequency.

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PCR amplification

• PCR uses cycles of denaturation, primer binding, and extension to increase selected DNA targets.
• Primers determine which loci are amplified and therefore define the analytical targets of the assay.
• Multiplex PCR allows many forensic markers to be amplified in one reaction.
• PCR is extremely sensitive, which is valuable for trace evidence but increases the consequences of contamination.
• Validated reaction conditions are required to balance sensitivity, specificity, reproducibility, and artifact control.

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Capillary electrophoresis

• After amplification, fluorescently labeled PCR products are separated according to size by capillary electrophoresis.
• A detector records fluorescent signals and software converts them into peaks on an electropherogram.
• Allele calls are assigned by comparing fragment sizes with an allelic ladder and validated analytical rules.
• Peak height and morphology provide information that can affect interpretation, especially in mixtures.
• Instrument calibration, quality controls, and allele-calling validation are essential for reliable results.

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Y-STR analysis

• Y-chromosome STRs are transmitted through the paternal lineage and are inherited together as a haplotype.
• Y-STRs can be useful when male DNA is present with a large excess of female DNA.
• Because paternal relatives may share Y-STR haplotypes, Y-STRs generally have different discrimination properties from autosomal STRs.
• Y-STR evidence can be particularly informative in some sexual-assault and lineage investigations.
• Interpretation requires appropriate databases and statistical methods for haplotype evidence.

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SNP-based forensic analysis

• Single nucleotide polymorphisms are single-base genetic variants distributed throughout the genome.
• SNP panels can support identification when STR analysis is limited by degradation or other constraints.
• SNP data can also support ancestry-related or phenotype-related inference, but these applications raise additional scientific and ethical questions.
• Massively parallel sequencing can measure many SNPs simultaneously when validated workflows are available.
• Students should distinguish identification markers from inferential markers because their evidential questions are different.

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Forensic DNA databases

• DNA databases store profiles under legal and technical rules that vary by jurisdiction.
• Database searching can generate investigative leads by identifying profile associations or potential matches.
• Database hits are investigative information and require appropriate confirmation and case interpretation.
• Quality assurance is necessary because database errors, contamination, or incomplete records can propagate incorrect associations.
• Privacy, retention, access, and governance are important scientific and legal dimensions of forensic DNA databases.

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Cold cases and reanalysis

• Archived biological evidence can sometimes be reexamined as analytical sensitivity and statistical methods improve.
• Reanalysis may reveal additional loci, improve interpretation of mixtures, or apply validated methods unavailable during the original investigation.
• Old evidence may also contain contamination, degradation, or documentation limitations that must be assessed before reanalysis.
• Independent review of original data can help distinguish genuine new information from reinterpretation caused by changed assumptions.
• Cold-case DNA work illustrates why evidence preservation has scientific value beyond the original testing episode.

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Limitations of DNA evidence

• DNA can be absent even when a person was involved and present even when contact occurred for an innocent reason.
• Trace DNA may be transferred indirectly and can persist for different periods on different surfaces.
• Mixtures, degradation, low template, contamination, and allele sharing can reduce interpretive certainty.
• Database searches may generate investigative leads that require independent confirmation.
• Strong statistical evidence should still be interpreted within the biological and factual context of the case.

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