Electropherograms and STR analysis

Gel electrophoresis

-separation medium: agarose or polyacrylamide gel slab

-electric-field strength: 4-10 V cm-1 to avoid excessive heating in a centimeter-thick gel

-run time: tens of minutes to hours depending on fragment size and gel length

-sample volume: microliters loaded into wells, part of the sample remains in the gel

Capillary Electrophoresis

-separation medium: Capillary filled with buffer

-electric-field strength: 300-600 V cm-1 because the thin wall dissipates heat efficiently

-run time: minutes per sample, <5min fragment sizing, 20-40 min single-base DNA sequencing

-sample volume: nanoliters injected, minimal consumption and waste

concentration in capillary electrophoresis

-varies depending on application and instrument, but common range is 0.4-3ng per 25 uL

-lower concentrations may not yield a DNA profile

-higher concentrations can cause artifacts

CE data interpretation

1.) Assess the internal size standards (ISS), allelic ladders, and controls

2.) assess each sample for the presence of extraneous peaks and determine if they may interfere with the interpretation process

3.) assess the data from each sample

Internal Size Standards (ISS)

-contains DNA fragments of known sizes that provide reference points for determining the length of a sample’s DNA fragments

-in general, the peaks or bands from an ISS are uniform in size or intensity. Lack of uniformity or miscalled peaks can indicate problems with the sample, injection, and/or run

allelic lader

-contain the more common alleles in the general population for specific chromosomal locations. Allelic ladders are used like molecular rulers to help “measure” the length of the fragments in the reference and evidentiary samples

Positive control

-included in most commercial DNA analysis kits

-amplified with each batch of samples

-shows the expected alleles

-if no peaks seen from positive control, this may indicate a problem with amplification or injection

Peak Height Percentage

-heterozygous peaks within a locus should be similar in height to each other

-heterozygous alleles have peak heights that are within 70% of each other

-peak height percentage for two heterozygous peaks is determined by dividing the peak height of the smaller peak by the peak height of the larger peak

-this result is expressed as a percentage

-degraded DNA may have peak height percentages much lower than 70%

mixtures

-the presence of more than two alleles at any one locus

-imbalance and/or unexpected peak height percentages

mixtures with known contributors

-subtract the contribution of the known donor from the mixed profile

-common approach for intimate samples: fingernails, vaginal swabs, etc.

mixtures with unknown contributors

-two people or more

-commonly use the approach of estimating percent contribution or percent peak height ratios

calculating percent contribution

-if there appears to be a major and a minor contributor

-mixture proportions are estimated by summing the peak heights of the two major alleles and dividing by the peak heights of all four alleles, this is then multiplied by 100

reporting guidelines

-inclusion/match: all loci match between a questioned and known sample

-exclusion/non-match: one locus does not match

-inconclusive/uninterpretable: a potential determination with complex mixtures or degraded samples

-no results: no discernable allelic activity observed at locus

common interpretation problems

-thresholds: established peak-height levels for “scoring” alleles

-only if fluorescence exceeds this value

-each laboratory must set its own as part of its validation procedure

common interpretation problems

-spurious peaks

-dye blobs, noise, spikes

-not reproducible

-run the sample again

common interpretation problems

-stutter: by-product of the amplification of STR loci - a minor product, typically one repeat smaller than the primary allele is generalted

-due to slipped strand mispairing during the amplification

common interpretation problems

-degradation

-can result in a partial profile

common interpretation problems

-allele drop out: a sample is typed and one or more alleles are not present

-the initial input quantity of DNA is too low, resulting in the failure to amplify one or more alleles in the sample

-a mutation in the primer binding site is present, which causes a failure in the amplification of the allele

comparing genotype from two tissue samples

-difference in genotype: exclusion

-exact same genotype: 2 possibilities

• tissue samples came from the same person (or identical twins)

  • tissue samples came from 2 different people with the same genotype

population genetics

-study of genetic variation within and between populations

-frequencies of the various alleles for STRs also show differences among populations, therefore there are considerations depending on what dataset you use

Product rule

-for heterozygous loci

• P=2qp

-P is probability; p and q are frequencies of allele in a given population

likelihood ratios

-a single number that supports a simple hypothesis (especially for DNA mixture interpretation)

info gain in hypothesis= odds (hypothesis data)/ odds (hypothesis)

genotype information gain

= probability (evidence genotype) / probability (coincidental genotype)

CODIS

-combined DNA index system

next generation sequencing advantages

-any one region of the genome can be covered by hundreds of reads, enough to provide reasonable certainty in the sequence, despite artifacts introduced during PCR and sequencing

-cover more STRs: or other variants

-uses one small sample, not multiple

-avoid allelic dropout

Forensic use of epigenetics

-modification of gene expression without altering the genetic code

DNA phenotyping

-prediction of appearance from DNA

-unknown sample donor

-typically uses SNPs (single nucleotide polymorphisms)

CentiMorgans

-measure length of SNA

-used for genealogy

forensic use of eDNA

-eDNA = environmental DNA

-plant material: geolocation, associative

-microbial: PMI, associative

-eDNA to detect the presence of invasive Asian carp