FRSC-3000 Lecture Summary Notes

Alec Jeffreys

Developed DNA profiling along with Peter Gill and Dave Werrett of the Forensic Science Service (FSS)

Variable Number of Tandem Repeats (VNTRs)

Certain regions of DNA that contain repetitive sequences that are variable in number between individuals

The Pitchfork Murder Case

A case in which DNA identification exonerated a kitchen porter and led to the conviction of Colin Pitchfork for murder based on a profile match with the semen from both murders

Basic Principles of DNA Testing

The uniqueness of DNA profiles, the consistency of the genome in every cell, and the inheritance of DNA from both parents

Applications for DNA Testing

• Crime solving

• Paternity testing

• Missing persons investigations

• Immigration testing

• Disaster victim identification

• Identifying soldiers in war

• Developing convicted felons' databases

DNA Testing as a Reference

DNA analysis for identity requires a reference sample for comparison, such as crime scene evidence compared to suspects or victim's remains compared to biological evidence

Three Possible Outcomes of Evidence Examination

• Exclusion (no match)

• Non-exclusion (match with statistical evaluation)

• Inconclusive result (insufficient information to support a conclusion)

Genotype

The characterization of alleles at a locus

DNA Structure and Composition

DNA molecule includes sugar backbone, phosphate groups, and four nucleotide bases; forms a double helix through H-bonds and phosphodiester bonds

Restriction Enzymes

Enzymes that recognize specific DNA sequences and produce double-stranded cuts, used in DNA copy number variation analysis

Human Chromosome Nomenclature

Variation in chromosome size and G-banding results in a karyotype; loci described based on chromosome number and position

Explain the naming of allele D16S539

• D = DNA

• 16 = Chromosome 16

• S = Single copy sequence

• 539 = 539th locus described on chromosome 16

Characteristics of DNA for Forensic Applications

• The same in every cell

• Remains the same throughout life

• Inherited from both parents

• Unique to each individual

Types of DNA Polymorphisms

• Sequence polymorphism

• Length polymorphism

Genetic Variability

Multiple markers are examined in DNA typing to increase the chance of distinguishing between unrelated individuals

How to calculate DNA profile frequency

Multiply all genotype frequencies together

Non-DNA Based Typing Methods

• Blood group testing

• Forensic protein profiling

DNA Based Typing methods

• RFLP multi-locus markers and single-locus markers

• PCR sequence based Reverse-dot blot

• PCR length based AFLPs, silver-stained STRS, fluorescently detected STRs

• Mitochondrial DNA sequencing

Blood Group Typing

Rapid and simple tests, limited power of discrimination

Basics of RFLP-Based DNA Testing

Detection of VNTRs, cutting DNA with restriction enzymes, gel electrophoresis to separate fragments by length, radioactive probe detection

Advantages of RFLP-Based DNA Testing

• Excellent powers of discrimination

• Large number of alleles at each locus which facilitates mixed sample analysis

Limitations of RFLP-Based DNA Testing

• Limited sensitivity

• Time-consuming

• Cannot be automated

• Not suitable for degraded DNA samples

• Binning introduces complication for interpretation

• Limited number of validated loci (4 to 6 loci commonly used)

Invention of PCR

Kary Mullis invented PCR, which amplifies specific DNA loci from small amounts of starting material

Amplified Fragment Length Polymorphisms (AFLPs)

PCR products separated on a gel and detected with silver staining, improved sensitivity compared to RFLP

Advantages of AFLPs

• Improved sensitivity compared to RFLP, uses PCR

• Many alleles facilitate mixed-sample analysis

• Discrete allele calling using allelic ladder

Limitations of AFLPs

• Large allelic range is difficult to multiplex with other loci

• Poor power of discrimination as a single locus

• Allele dropout in highly degraded DNA

• Not automatable due to gel separation and silver-stain

• No high throughput sample processing

DQA1 Reverse Dot Blot Tests

Allele-specific probes used to find sequence polymorphisms, commonly used for HLA-DQA1 locus

Advantages of Reverse Dot Blot Tests

• Fast and simple compared to RFLP

• Small or degraded samples can be analyzed, PCR used

• No instrumentation needed after PCR

Limitations of Reverse Dot Blot Tests

• Poor power of discrimination

• Mixture interpretation difficult due to limited number of alleles per locus

Short Tandem Repeat Markers

Replication slippage causes microsatellite polymorphism, analyzed through silver-stained or fluorescently detected STRs

Silver-Stained STRs

PCR products separated on a gel and detected with silver staining, sensitive and works well with degraded DNA samples

Advantages of Silver-Stained STRs

• Sensitive due to PCR

• Rapid process (1-2 days)

• Works well with degraded DNA samples

• Lower start-up cost compared to fluorescent STRs

Limitations of Silver-Stained STRs

• Multiplex amplification and detection is limited to 3-4 loci (one colour channel)

• Both strands of DNA are detected, some loci complicate interpretation

Fluorescent STRs

PCR products labeled with fluorophores by sequence-specific primers, PCR product size generated

Advantages of Fluorescent STRs

• Sensitive due to PCR

• Rapid process ( a few hours - 2 days)

• Works well with degraded samples

• Multicolour fluorophore labeling enables multiplex PCR

• Commerical STR Kits available

• Automated detection and high throughput

Limitations of Fluorescent STRs

• Less discrimination power per locus compared to VNTRs

• Contamination from stray DNA in PCR

• Expensive equipment

• Stutter products and unbalanced peak heights

• Data interpretation complicated by artifacts

Replication Slippage

Occur at repetitive sequences when the new stand mis-pairs with the template strand (or visa versa)

Grim Sleeper

A serial killer who murdered 11 women in California over a span of 15 years, convicted based on a DNA partial match to his son in the California DNA Database

Familial DNA testing

A method used to identify suspects by comparing their DNA profiles to those of their relatives.

Presumptive tests

Simple, inexpensive, and non-destructive tests used to indicate the presence of biological fluids on evidence.

RNA testing

A method used to identify body fluids by analyzing the unique pattern of gene expression in different cell types.

DNA extraction

The process of isolating DNA from cellular material for further analysis.

PCR inhibitors

Substances that can interfere with the polymerase chain reaction (PCR) process.

DNA quantitation

The measurement of DNA concentration in a sample to ensure accurate PCR amplification.

Slot Blot Method

A method of DNA quantitation that uses a primate-specific probe to compare the intensity of the signal from the sample to standards.

PicoGreen Intercalating Dye Assays

A method of DNA quantitation that uses a fluorescent dye to measure the amount of double-stranded DNA present in a sample.

qPCR

Quantitative polymerase chain reaction, a method used to monitor the amplification of DNA during PCR and quantify the amount of target nucleic acid in a sample.

Proximity-based Suppression

When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by Forster-type energy transfer.

AmpliTaq Gold DNA Polymerase

AmpliTaq Gold DNA polymerase cleaves only probes that are hybridized to the target.

Cleavage and Fluorescent Signal

Cleavage separates the reporter dye from the quencher dye, which increases the fluorescent signal.

SYBR Green

SYBR Green dye fluoresces when bound to dsDNA.

Denaturation and SYBR Green

When DNA is denatured, SYBR Green dye is released, and fluorescence is reduced.

Polymerization and SYBR Green

PCR products are amplified, and the dye binds to the dsDNA product resulting in a net increase in fluorescence.

Quantitative PCR Advantages

The ability of commercial qPCR kits, higher throughput and reduced user intervention, automated set up and analysis using the standard curve, high sensitivity, large dynamic range ~30 pg to ~30 ng.

Quantitative PCR Limitations

Subject to inhibition,

qPCR for Human Quantitation

Designed to simultaneously quantify the total amount of amplifiable human DNA and human male DNA in a sample, determine the relative quantities of human male and female DNA in a sample, detect PCR inhibitors in a sample.

Polymerization and Strand Displacement

Polymerization and strand displacement occur during the PCR process.

Probe Cleavage

Probe cleavage refers to the release of the reporter dye.

When does fluorescence occur?

When the reporter dye and quencher dye are no longer in close proximity.

Passive Reference Dyes

Passive reference dyes (ROX) are used to normalize well-to-well fluorescence signal differences caused by variation in the optical paths between wells and minor differences in volumes due to pipetting errors.

Microsatellites (STRs)

Microsatellites, also known as short tandem repeats (STRs), are repetitive DNA sequences that vary in the number of repetitive motifs between individuals.

STR Criteria for Forensic Applications

• High discriminating power, a high percentage of heterozygotes

• Separate chromosomal locations

• A narrow allele range

• Work well in combination with other microsatellites

• Have a low mutation rate and low level of biological artifacts.

STR Allele Nomenclature

STR alleles are named based on the motifs using the first repeat on the 5' strand, and microvariants are named by the number of complete repeats and the number of nucleotides in the partial repeat.

STRs in Forensic Applications

STRs are commonly used in forensic applications due to their compatibility with degraded DNA, high power of discrimination, availability in easy-to-use kit formats, and capability for national and international sharing of criminal DNA profiles.

The CODIS STR Loci

The CODIS STR loci are the 13 core STRs selected to form the basis of the National DNA Database in the United States known as CODIS.

Stochastic sampling

Unequal sampling of two alleles in a heterozygous individual due to low template amounts.

Thermostable DNA Polymerase

Taq DNA polymerase isolated from Thermus aquaticus, used for its thermostability in PCR reactions.

Oligonucleotide Primers

Short DNA sequences that are specific to target regions, possess similar annealing temperatures, and do not interact significantly with themselves.

Magnesium Ions

Crucial factor affecting the performance of Taq DNA polymerase and the stringency of primer annealing by neutralizing repulsion between negatively charged DNA strands.

Bovine Serum Albumin (BSA)

Used as an additive to bind potential PCR inhibitors and minimize inhibition.

Denaturing Time and Temperature

Initial denaturing step at 94 degrees Celsius to completely dissociate template strands, followed by 30-second denaturing steps within PCR cycles.

Annealing Time and Temperature

Short annealing time to allow primers to bind in the correct position while limiting chances of mis-binding, often kept stable at 1 minute through PCR cycles.

Extension Time and Temperature

Taq DNA polymerase extends DNA at 72 degrees Celsius, synthesizing 1000bp per minute. Extension runs for 1 minute per cycle.

Cycle Number

Generally around 30 cycles in PCR amplification, with a tradeoff between amplifying desired targets and minimizing amplification of undesired regions.

Positive Control

DNA sample with a known profile used to ensure the reaction mix is functioning properly.

Negative Control

Reaction mix without DNA sample, used to monitor for contamination or non-specific product formation.

Primer Control

DNA control used to determine if a problem arose due to the primers used in the PCR reaction.

Multiplex PCR

Amplifying multiple loci in one reaction, requiring adjustment of primer and magnesium ion concentrations to avoid primer-dimer formation.

Gel Electrophoresis

Separation of DNA fragments based on size using an electric field applied to a gel matrix.

Agarose Gel

Gel matrix with larger pore size used to resolve large DNA molecules.

Polyacrylamide Gel (PAGE)

Gel matrix with smaller pore size used to resolve smaller DNA molecules, better for PCR-amplified STR alleles.

Native vs Denaturing Conditions

DNA can run through the gel as double-stranded DNA in native conditions or single-stranded DNA under denaturing conditions for better resolution.

Capillary Electrophoresis (CE)

Separation of DNA in a capillary tube using an electric field, providing higher resolution and automation compared to gel electrophoresis.

Fluorescent Detection

Detection of DNA fragments labeled with fluorescent dyes using filters and a charge-coupled device (CCD).

Spectral Calibration

Removal of spectral overlap between different fluorescent dyes by creating a calibration file using samples labeled with a single dye.

DNA Dilution

The process of diluting PCR products or allelic ladder in deionized formamide and internal lane standard before analysis.

Electrokinetic Injection

The process of injecting DNA into the capillary of a genetic analyzer using an electric field.

STR Genotype

The allele or alleles present at a specific locus in an individual's DNA.

Electrophoresis

The process of separating DNA fragments based on their size using an electric field.

Peak Detection Threshold

The minimum signal height required to distinguish a peak as a real allele.

Microvariant Alleles

Alleles that have sequence variations compared to commonly observed alleles and do not size the same as common alleles.

Non-Allelic Peaks

Peaks in the electropherogram that do not represent alleles from the sample, including PCR artifacts, analytical artifacts, instrumental limitations, or introduced artifacts.

Stutter Products

Peaks that show up one repeat unit less than the true allele due to strand slippage during DNA synthesis.

Tri-Allelic Patterns

The presence of three alleles at a locus in a single-source DNA sample, which can be a result of extra chromosome fragments.

Null Alleles

Alleles that are present in the DNA sample but fail to be amplified due to a nucleotide change in a primer binding site.