DNA Forensic Analysis

Harry R Erwin, PhDCIS308

Faculty of Applied SciencesUniversity of Sunderland

Topics

• Introduction• DNA structure and the genome• DNA handling procedures• PCR• The use of short tandem repeat data• DNA statistics and evidence• DNA databases• Other markers

References

• Goodwin, Linacre, and Hadi (2007) An Introduction to Forensic Genetics, Wiley.

• Butler (2005) Forensic DNA Typing, 2nd edition, Elsevier.

Lecture Plan

• DNA processing• Short tandem repeat data• DNA statistics• Discussion: Bayesian analysis and DNA

evidence

DNA Processing

• Aims of DNA extraction– Quality– Quantity

DNA Extraction Process

1. Free the chromosomes by cell lysis.2. Denature the proteins (histones, etc.) that

keep the DNA packaged in the chromosomes.3. Separate the DNA from the rest of the cell

without disrupting it irretrievably.

Extraction Techniques

• Chelex® 100 resin– Fast– Simple– Avoids mishandling– Low cost– Avoids harmful chemicals– Compatible with many forensic procedures– Relatively crude, single-stranded output

Silica-Based Extraction

• Standard research approach• Incubate with lysis buffer that contains

proteinase K and detergent• Add a chaotropic salt to disrupt the protein

structure• Add silica• Produces clean high-molecular weight DNA• Takes longer and creates a risk of mishandling

Phenol-chloroform-based extraction

• Older research procedure• Phenol is toxic• Still used with bone and soil samples• Phenol-chloroform is used instead of the chaotropic

salt in silica-based extraction• Multiple centrifuge steps.• Produces clean DNA• Significant risk of mishandling• Labour-intensive

FTA® Paper

• Cellular material lyses on contact• Stable for years• Wash off the non-DNA components from a

piece of the paper• Very simple• Avoids mishandling

Challenging Samples

• Semen is hard to lyse and is sometimes mixed with epithelial cells. DTT should be used instead of or after proteinase K

• Hair shafts are notoriously hard to analyze. There is a serious risk of contamination since DNA is at a low concentration.

• Hard tissues can preserve DNA in decomposed samples. Extraction involves cleaning and grinding and takes much longer.

Quantification of DNA

• Must know the amount and quality to get the best PCR results. (This is a mandatory step in America.)

• One approach is to visualise it on an agar gel. This allows estimation of quantity and of the size of the extracted molecules.– Quick and easy– Subjective, with no reference standards– Incompatible with Chelex® 100.

Other Approaches to Quantification

• Ultraviolet spectrophotometry—used in molecular biology labs but not in most forensics labs

• Fluorescence spectrophotometry—very sensitive, but not human-specific

• Hybridization—common approach using standard targets in human DNA. Human-specific.

• Real-time PCR—measure the output of PCR. Highly sensitive, human-specific, and easy.

• DNA IQ™—uses carefully measured amounts of silica so that the maximum concentration is known. Not human-specific.

PCR

• Procedure amplifies specific regions of DNA.• Can handle trace evidence and highly degraded

samples (sometimes)• Initially was used to amplify VNTR loci (5-10

kilobase).• Not initially suitable for degraded samples.• Now, with a large number of STRs characterised,

can now be used with degraded samples.

PCR Components

• Template DNA—the DNA to be amplified.• At least two primers—synthetic DNA that binds to

the borders of the DNA region to be amplified.• A thermostable DNA polymerase that can be used

at high temperatures (80-95° Celsius)• MgCl to stabilise the interaction• DNA bases—to be used in the reaction.• A buffer solution• In a clean room

How PCR Acts

• Split the DNA.• Bind primers• Use DNA polymerase to duplicate the DNA

between the primers.• Repeat.– Each cycle almost doubles the DNA– 32-34 cycles magnifies the DNA by a factor of 109 or

more.– Results are visualised on agar gel.

Problems with PCR

• Polymerase inhibitors can be a problem– Haem compounds from blood– Bile salts– Faeces– Humus– Urea (urine)– Ca or Mg ions in high concentration– Blue dye in denim

• Silica binding methods can overcome these problems, as can the addition of bovine serum albumin.

PCR Contamination• PCR is very sensitive to contamination by cellular material from:

– People unconnected with the incident– Victims– People discovering the incident– Police– Scene of crime officers– Laboratory staff– Other cases

• In particular, laboratory technicians must either specialise in pre-PCR/post-PCR or avoid working in pre-PCR areas when they have worked recently in post-PCR areas of the lab.

• Suspect analyses must be done separately from scene of crime analyses to avoid contamination.

• Positive and negative controls are required.• A lab that fails to do all of these is unprofessional.

Short Tandem Repeat (STR) data

• Core repeat region of 1-6 base pairs• Alleles generally less than 350 base pairs• Many STR loci have been characterized, but 20

are in common forensic use.• Currently, 67 STR loci on the Y chromosome

are used for establishing paternity.

Widely Used STR Loci

• 4-5 base pair core-repeat motif• Four categories– Simple repeat– Simple repeat with non-consensus repeats (a

second motif occurs at specific places)– Compound repeat (multiple elements)– Complex repeat (even more elements at specific

points in the marker)

Characteristics of ‘Good’ STR Loci

• Discrete and distinguishable alleles• Amplification should be robust• High power of discrimination• Not genetically linked with other loci• Low levels of artefact formation• Compatible with amplification as part of a

multiplex PCR.

Interpretation

• The end result of PCR analysis is an electropherogram with a series of peaks representing different alleles– Value– Size– Peak height– Peak area

• This allows each allele present to be classified.• The results should be repeatable.

Artefacts

• Stutter peaks—mutations during PCR. Usually +/-1.

• Split peaks—an adenine residue is usually added to the DNA sequence. If it isn’t consistently present, it produces peak splitting.

• Pull-up—small peaks at the same place as larger peaks can result from signal processing errors and over-amplification.

Artefacts (II)

• Template DNA—the template amount may be smaller or larger than optimal, producing noise in the profile.

• Overloaded profiles—too much DNA is produced so that peaks are off-scale.

• Low copy number DNA—extremely low amounts of DNA may be relevant to the investigation. It is difficult to optimise PCR to analyse this while avoiding artefacts.

Artefacts (III)• Peak balance—most STR loci are heterozygous, producing

two peaks that are frequently different-sized. If the area of the smaller is less than 60% the area of the larger, it suggests an error in the laboratory procedure. You can even get peak drop-out.

• Mixtures—more than one person present in the sample. Even two-person mixtures can be hard to interpret.

• Degraded DNA—exposed to the environment for hours, days, months, or years. Results in over-amplification of smaller loci. You should use multiplexes specially designed for degraded DNA.

DNA statistics

• DNA statistics test hypotheses about population genetics– A population is a group of people sharing common

ancestry.– This can be quite general.

• An ideal population satisfies Hardy-Weinberg equilibrium. This needs to be demonstrated before DNA statistics can be applied to a community.

Hardy-Weinberg Equilibrium

• Within a randomly mating population, the genotype frequencies at any single genetic locus remains constant.

• Discuss in the context of homosexuality.• This needs to be statistically demonstrated to

make claims about the likelihood of a combination of alleles.

• Punnett square

HW Conditions

• Infinite population—no genetic drift, requires relatively large populations

• Random mating—however, STRs don’t appear to be affected by mate choice

• No migration—an issue for human populations• No natural selection—no evidence that the loci used in

forensics are selected• No mutations—actual mutation rates

(<0.5%/generation) are sufficiently low that selective pressure is probably not important.

Testing HW Equilibrium• The population database needs to contain at least 100

individuals per locus. Several hundred individuals is good practice.

• Allele databases should be corrected to avoid inaccurate frequency estimates.– The minimum allele frequency should be at least 1% in the analysis (or

5/(2*N) for a database with N individuals if that is a smaller number).– Correct for sampling bias.– Allow for subpopulations.

• You may also set the required match probability to a small number (1 in a billion in the UK).

• Use a population frequency database that reflects what is known about the person being identified.

Discussion: Bayesian analysis and DNA evidence