Clinical Genetics - Introduction to Forensic DNA Notes
Clinical Genetics: Introduction to Forensic DNA
Overview
This lecture provides an introduction to forensic DNA analysis, covering topics from the nature and sources of forensic DNA to extraction methods, the distinction between genomic and mitochondrial DNA, and the variability of DNA.
Nature of Forensic DNA
Challenges
Forensic DNA is often degraded, with fragments smaller than 500 bp. Contamination from external sources and mixed samples are common issues. Strong inhibitors of biochemical assays, such as humic acids, can be present, and the quantity of DNA is often low, ranging from 0.1 ng to 10 μg.
Factors Affecting DNA Integrity
Temperature, humidity, pH, microbiota, plant extracts, the presence or absence of metals and salts, time of exposure, location, and the type of tissue all affect DNA integrity.
Sources of Forensic DNA
Human Cells
Semen: Spermatozoa found in fresh and dried samples from sexual assault cases.
Blood: White blood cells found in fresh and dried samples from violent crimes.
Swabs: Epithelial cells and mixed cells found in fresh and frozen reference samples.
Bone/Teeth: Osteocyte cells ranging from fresh to 40,000 years old, found in mass graves or single skeletons.
Hair: Root tip cells found in fresh and aged samples.
Touch DNA: Epithelial cells found in fresh and aged samples.
Diagenesis and DNA Degradation
DNA is rapidly degraded by endonucleases present in the dead body. DNA repair mechanisms cease, leading to rapid damage accumulation. Microorganisms feed on organic molecules, including DNA. Certain conditions can inhibit these processes, allowing DNA to be preserved.
Cheek Cells & Touch DNA
Cheek cells are fresh epithelial cells with more DNA, while fingerprints contain debris and fewer epithelial cells, resulting in less DNA.
The Paradox of Forensic DNA From Bone
DNA is protected by the bone and teeth structure, low temperature (permafrost samples), moisture, and pH levels between 6.5 and 8.5. This protection can preserve DNA for 3,000 to 40,000 years.
Environmental Conditions
Degradation of DNA is accelerated or slowed by burial conditions (taphonomy). Lipids and proteins are more likely to be preserved than DNA. Hot, wet, exposed, and changeable conditions (e.g., open sites, deserts, floodplains, low pH) make preservation less likely. Cool, dry, pressure, and constant conditions (e.g., caves, permafrost, high altitude) favor preservation.
Sample Collection
Samples are collected by SOCOs (Scene of Crime Officers). Background samples must be collected. Dry samples are stored at room temperature, while wet samples are stored at -20 °C. High copy number samples contain more than 1 ng of DNA, while low copy number samples contain less than 1 ng. High copy number sources include blood, semen, hair roots, vaginal fluid, and secretions. Intermediate sources include preserved tissue, skin, urine, and feces. Low copy number sources include bone, teeth, and aged material.
DNA Extraction Methods
Methods Overview
Four main methods are highlighted: organic extraction, Chelex extraction, and spin column extraction.
Organic Extraction
This method involves aqueous/organic phase separation and sample precipitation. It is excellent for blood samples but medium to poor for others. Phenol can degrade small amounts of DNA.
Chelex Extraction
Chelex is a cationic resin that traps inhibitors such as heme and divalent ions, protecting DNA by inactivating nucleases. Cells are opened to release DNA, but this method leaves cellular debris present. It is considered a "quick and dirty" method.
Chelex Extraction Protocol
Swab source, swirl in solution.
Remove swab and centrifuge.
Add 5% Chelex and resuspend.
Incubate at 56 °C.
Incubate at 100 °C.
Centrifuge (DNA is in the supernatant).
Silica Gel Membrane (Spin Column)
This method uses a silicon oxide-based gel membrane. Low flows through, while high allows DNA binding.
Silica Spin Column Purification of DNA
Prepare lysate using digestion buffer.
Apply lysate to the column.
Apply wash buffer 1 to the column and spin.
Apply wash buffer 2 to the column and spin.
Elute DNA with a low salt buffer.
Spin Column vs. Chelex
Chelex is cheap and involves minimal manipulation but is not automated, and samples can be inhibited by the resin. It is good for samples > 5 ng DNA.
Spin Columns have some cost, involve multiple steps, and can be automated. There is no inhibition by components, and it is sensitive down to 0.05 ng DNA.
Equipment Required for Manual DNA Extraction
The basic equipment includes pipettes, a microcentrifuge, a water bath, and a vortex.
Automated DNA Extraction System
Automated systems are also available for DNA extraction.
Genomic vs. Mitochondrial DNA (mtDNA)
Total DNA Composition
Genomic DNA
Genomic DNA comprises the total chromosome set (i.e., karyotype) found in the nucleus.
mtDNA
mtDNA is circular mitochondrial DNA with a few genes located in the cytoplasm.
Use of DNA in Forensics
Applications
DNA is used for sex determination (Amelogenin) kinship relationship/identification, ancient and modern populations comparisons, and diachronical study of one geographic region (migrations, genetic continuity/discontinuity).
Genetic Markers
Useful genetic markers include mtDNA, Y chromosome, X Chromosome and autosomal chromosomes.
The Human Karyotype
Each chromosome contains one continuous strand of DNA. Chromosome 1 is approximately 250,000,000 bp long, while chromosome 22 is approximately 50,000,000 bp long. Chromosomes range in length from 73 mm to 14 mm. During cell division (metaphase), chromosomes are highly condensed and associated with histone proteins, which help in packaging and organizing the DNA.
Genome Composition
The human genome comprises approximately 3.2 Gb (Gigabases), while mtDNA is 16.5 kb.
Coding and regulatory regions: 2%
Genic and related sequences: 23%
Extragenic DNA: 75% (Unique/low copy: 21%, Repetitive DNA: 54%)
Repetitive DNA includes:
Tandem repeats: 9% (Microsatellite: 5%, Minisatellite: 1%, Satellite DNA: 3%)
Interspersed repeats: 45% (SINE: 13%, LINE: 21%, LTR: 8%, DNA transposon: 3%)
How Unique is Individual DNA?
Individuals share about 99% of their DNA. Genomes have diverged by only around 5% from a common ancestor with chimpanzees around 6 million years ago. Population genetics studies show more variability within populations than between geographical groupings, with allele frequencies changing gradually across regions (clines).
The Genome & Forensic Genetics
Key Properties for Forensic DNA Loci
Highly polymorphic (varying widely between individuals).
Easy and cheap to characterize.
Profiles that are simple to interpret and easy to compare between laboratories.
Not under any selective pressure.
Low mutation rate.
Forensic DNA Targets
Minisatellites, microsatellites, single nucleotide polymorphisms, mitochondrial DNA can be used.
Minisatellites or Variable Number of Tandem Repeats (VNTRS)
Minisatellites consist of tandem repeats, with alleles varying in length. For example, alleles at the D1S7 (or MS1) locus can contain over 2000 repeats. Alleles are composed of different variants of the 9 bp core repeat.
Two Important MB Concepts
DNA Denaturation
DNA denaturation is achieved using NaOH or heat.
DNA Annealing
DNA annealing involves the use of a radioactive probe.
Southern Blotting
Southern blotting involves extracting DNA from human cells, cutting it into fragments using restriction enzymes, separating the fragments by gel electrophoresis, transferring the DNA bands to a nitrocellulose filter, and hybridizing with a radioactively labeled probe to identify the gene of interest. The fragment containing the gene of interest is identified by a band on developed X-ray film.
Principle of Radioactive VNTR Analysis
VNTR analysis uses restriction sites and probes to analyze tandem sequence repeats.
Principle of Restriction Fragment Length Polymorphism (RFLP)
RFLP involves using restriction enzymes (e.g., EcoRI, SmaI) to cut DNA at specific recognition sites. The resulting fragments are then separated by gel electrophoresis to identify polymorphisms.
Multilocus vs. Single Locus VNTRS
VNTRs can be analyzed using multilocus or single locus probes.
Steps in DNA Fingerprinting Technique (Alec Jeffreys, 1984: VNTR DNA Fingerprinting)
Blood sample.
DNA is extracted from blood cells.
DNA is cut into fragments by a restriction enzyme.
The DNA fragments are separated into bands during electrophoresis in an agarose gel.
The DNA band pattern in the gel is transferred to a nylon membrane by Southern blotting.
The radioactive DNA probe is prepared.
The DNA probe binds to specific DNA sequences on the membrane.
Excess DNA probe is washed off.
The radioactivity probe is bound to the DNA pattern on the membrane.
X-ray film is placed next to the membrane to detect the radioactive pattern.
The X-ray film is developed to make visible the pattern of bands, known as a DNA fingerprint.
The Colin Pitchfork Case
In the time period 1983-1986 two young women were found dead with indications of sexual assault in Leicestershire. A young man was accused but “DNA fingerprinting” excluded the possibility. 5000 men were screened and no match was found. Colin Pitchfork paid a man to replace him in the sampling. When authorities finally sampled him…there was a match..!!
Minisatellites or Short Tandem Repeats (STR)
The structure of a short tandem repeat. This example shows the structure of two alleles from the locus D8S1179.1 The DNA either side of the core repeats is called flanking DNA. The alleles are named according to the number of repeats that they contain – hence alleles 8 and 10
Single Nucleotide Polymorphisms (SNPs)
A single nucleotide polymorphism (SNP). Two alleles are shown which differ at one position indicated by the star: the fourth position in allele G is a guanine while in allele A it is an adenine. In most cases, the mutation event at the specific locus that creates a SNP is a unique event and only two different alleles (biallelic) are normally found
Properties of Mitochondrial DNA
Characteristics
Circular
Small (only 16.5 Kb)
Contains genes that code for mitochondrial enzymes and t-RNAs
Contains variable regions = certain nucleotides differ between individuals, e.g., D-loop
Many copies = many mitochondria per cell
Robust = preserves well over long periods.
Maternal Inheritance
mtDNA is inherited maternally.
Important Technologies for mt-DNA Analysis
PCR (Polymerase Chain Reaction) and Sanger Sequencing are used.
PCR Components
DNA Sample
Primers
Nucleotides
Taq Polymerase
Mix Buffer
PCR Process (One Cycle)
Denaturing: 95°C-Strands Separate
Annealing: 55°C-Primers Bind Template
Extension: 72°C-Synthesise New Strand
Cycle Sequencing via Capillary Electrophoresis
This involves PCR with fluorescent, chain-terminating ddNTPs, size separation by capillary gel electrophoresis, and laser excitation & detection by a sequencing machine.
The D-Loop Region
Characteristics
The D-loop contains HV1 & HV2 regions (HV = hypervariable). These regions are amplified via PCR and sequenced to determine the nucleotides involved.
Differences from Reference Sequence
mtDNA sequences from tested samples are aligned with the reference rCRS sequence (e.g., positions 16071-16140). Differences are reported by the position and the nucleotide change compared to the rCRS.
Communal Grave Body Identification with the Use of mt-DNA
mtDNA sequence data from a communal grave compared with a living relative can use to identify individuals.