Understanding STR Allele Values and Genotyping Techniques

Size standard

An external standard run with each sample to correlate sizes.

Allelic ladder

A standard reference for comparison that is run as an individual sample.

Fluorescent signal intensity

The initial plotting of peaks as a function of time passing the detector.

Internal Lane Standard (ILS)

Used to determine the size of the peak by comparison.

Detection thresholds

Used to separate signal from noise.

Typical varying detection thresholds

Range from 50 RFU to 200 RFU (relative fluorescence units).

Analytical threshold

Detection threshold impacted by instrument baseline noise.

Stochastic threshold

Detection threshold important in mixture interpretation.

Genotype determination

Done by comparing allelic ladder to sample results.

Accepted range for allele bins

+/- 0.5 with the genotyping software.

Independent calls for genotyping

Most labs require 2 independent calls.

PQV

Process quality values used for assessing sample quality.

Biology related artifacts

stutter peaks, non-template addition, microvariant alleles, null alleles, mutations.

Technology related artifacts

Includes spike, pull-up, dye blobs, noisy baseline.

Allelic drop outs

one of the two alleles present in a sample fails to amplify during PCR

Can occur due to degradation or inhibition.

Stutter

A biological artifact resulting from the PCR process, usually small peaks one repeat smaller than the main parent peak.

Frequency of stutters

Occurs 5-15% of the time in tetranucleotide repeats STR loci.

Non-template addition

DNA polymerases adding an extra nucleotide to the 3' end of the PCR product.

Result of non-template addition

A PCR product that is one bp longer than the actual target sequence.

Impact of DNA amount on PCR

Too much DNA causes off scale peaks; too little DNA causes heterozygote peak imbalance and allele drop out.

Null allele

An allele present in the DNA sample that fails to amplify due to a nucleotide change in a primer binding site.

Mutations

An inheritable change in DNA sequence.

Spikes

narrow, sharp peaks

Appear in all lanes.

Pull-up

observed when high signal in one base appears to "pull up" the peaks of other bases

Occurs due to excess fluorescence.

Peak height imbalance calculation

(Peak A RFU (lower RFU peak)/ Peak B RFU (higher RFU peak)) * 100.

SWGDAM

Scientific working group on DNA Analysis methods.

Only public labs

Statistics for evidence examination

Required when there is a 'match'/inclusion.

RMP

Random match probability, the chance of a specific profile occurring in a specific population based on observed allele frequencies.

RMP calculations usage

Used for single source profiles.

Likelihood ratio (LR)

Compares the probabilities of the evidence under two alternative theories or hypothesis.

Probability of exclusion (PE)

The percentage of the population that can be EXCLUDED as potential contributors to a DNA mixture profile.

Probability of inclusion (PI)

The percentage of the population that can be included as potential contributors to a DNA mixture profile.

Combined probability of exclusion (CPE)

CPE= 1-CPI or CPI= 1- CPE.

CODIS

Combined DNA Index System.

Use of CODIS

Linking serial crimes and unsolved cases with repeat offenders; requires 20 core STR markers.

Database vs. databank

Database: collection of computer files containing entries of DNA profiles; databank: collection of the actual samples.

NDIS

National DNA Index System.

Profiles in CODIS

Convicted offender, arrestees, forensic unknown, biological relatives of missing persons, missing persons, unidentified human remains (UHR).

Expungement

Removing a profile from a database and destroying the sample.

STR

Single tandem repeat.

miniSTR

Smaller versions of STR loci that can work well on degraded DNA; helps improve the success rate and recovery of compromised DNA fragments.

SNPs

Potential for identifying ethnicity of evidence sample; Single nucleotide polymorphism; any polymorphic variable at a single nucleotide.

mtDNA

Passed from mother to offspring without change; DNA is outside nucleus; used most often when only hair, bones, or teeth are left behind or highly degraded specimen

Y-STRs

Permits examination of male only DNA.

Y chromosome

Passed from father to son without change; tested using Y-STR and Y-SNPs.

Hair phases

Anagen phase: growth phase BEST FOR NUCLEAR DNA; Catagen phase: transition phase; Telogen phase: resting phase.

Quality assurance (QA)

Programs designed to ensure that a lab's results are scientifically valid.

Quality control (QC)

Day to day operational techniques and activities used to fulfill quality requirements.

Proficiency test

Used for QA; stimulated forensic cases with known results; done externally and internally.

TWGDAM

Technical working group on DNA analysis methods.

NRC

National Research Council.

Validation

A process by which a procedure is evaluated to determine its efficacy and reliability in forensic casework analysis.

Validation 3 R's

Reproduceable, robust, reliable.

What are non-human DNA testing for?

Cat DNA, dog DNA, plant DNA, marijuana DNA