Forensic Genetics - Week 12 Lecture Notes

Topic = DNA Technology Advancements

A Brief History of Forensic DNA

• Forensic applications of DNA have evolved significantly since Sir Alec Jeffreys.

Current Innovations in DNA Technology

• Key advancements in DNA technology include:

  • Next-generation sequencing (NGS)

  • Methods include:

    • Hybridization

    • Synthesis

    • Pyrosequencing

    • Ion Torrent

    • Bridge amplification

    • PacBio long-read sequencing (3rd generation)

  • Rapid DNA

  • Genetic Genealogy

  • Anticipating future developments in DNA innovations.

Next-Generation Sequencing – Sequencing by Synthesis

• The most widely used technology: Illumina technology.

• The sequencing process involves four major steps:

  1. Sample preparation

  2. Cluster generation

  3. Sequencing

  4. Data analysis

Sequencing by Synthesis – Sample Preparation

• The preparation process includes:

  • DNA extraction from the sample.

  • Purity and quantity assessment.

  • Library preparation: Conversion of whole genomic DNA into a library of fragments, categorized as:

  • Shotgun libraries: contain fragments of the entire DNA present in the sample.

  • Targeted libraries: contain fragments focused on specific regions of interest.

• Targeted libraries are commonly used in forensic applications, enabling the targeting of regions such as:

  • Short Tandem Repeats (STRs)

  • Single Nucleotide Polymorphisms (SNPs)

  • Mitochondrial DNA (mtDNA)

Sequencing by Synthesis – Adapter Ligation

• Adapters are ligated to the ends of the DNA fragments, which contain:

  1. Sequencing primer binding site

  2. Index or barcode for sample identification

  3. Flow cell binding sequence

Sequencing by Synthesis – Cluster Generation

• Involves multiple stages:

  • Flow cell setup: The flow cell is coated with two different oligonucleotide sequences.

  • Conversion: DNA fragments are converted to single strands and bound to the flow cell via adapter sequences.

  • DNA copying: The bound DNA is amplified using polymerase; the template fragment is washed away, retaining a complementary sequence attached to the flow cell.

  • Bridge amplification: Bound fragments fold over to bind to a second oligo; polymerase synthesizes a second strand, forming a double-stranded structure.

  • Continuous amplification: This bridge amplification is repeated, resulting in clusters of amplified sequences distributed across the flow cell.

  • Cutting and retention: Reverse strands are cut away, leaving only forward strands in each cluster.

Sequencing by Synthesis – Sequencing Procedure

• Steps involved in sequencing:

  • A sequencing primer is added, binding to the adapter.

  • Fluorescently labeled deoxynucleotides (dNTPs) are introduced; each has a unique color and a terminator molecule.

  • In each cycle, only one base is added.

  • Clusters are illuminated at the end of each cycle; emitted color signals are detected and classified by software.

  • Following imaging, the terminator is cleaved, and new dNTPs are introduced.

  • The procedure is repeated 300-500 times, yielding reads of 300-500 base pairs (bp).

  • Can conduct either single reads or paired-end reads by repeating in the reverse direction.

Sequencing by Synthesis – Data Analysis

• The data generated is vast and more error-prone compared to Sanger sequencing.

• Relies on high sequence coverage; a technique known as Massively Parallel Sequencing.

• Consensus sequences are derived from sequence alignments.

Advantages of Sequencing by Synthesis

• Detection of additional variation: Enables the discovery of diverse STR alleles, which may not have identical sequence compositions.

• Limitations: Cannot analyze longer STRs effectively.

Forensic Applications of Sequencing by Synthesis

• Hybridization-based target enrichment: Manages capture libraries.

  • Probes carrying biotin are utilized to target specific DNA regions, such as STRs, SNPs, and mtDNA.

  • Streptavidin-coated magnetic beads draw specific fragments from the solution, while unwanted fragments are removed, enhancing sequencing coverage for targeted regions.

• Capture Library Advantages:

  • Greatly increase target areas coverage, consequently minimizing sequencing errors.

  • A washed library can create additional capture libraries which assists in analyzing degraded DNA.

Traditional PCR vs. Capture Library Sequencing

• Traditional PCR involves no amplification, focusing solely on probe capture while sequencing libraries.

Rapid DNA Profiling

• An automation of the standard STR profiling technique rather than a new technique itself.

• Standard DNA profiling process includes several steps:

  1. Collect the sample.

  2. Lyse the sample.

  3. Purify the sample.

  4. Quantify the sample.

  5. Amplify the sample.

  6. Electrophorese the sample.

Rapid DNA Profiling Process

• Steps include:

  1. Sample is collected.

  2. Cartridge insertion: The sample (cheek swab or evidence) is inserted into a special cartridge.

  • Analysis tools may be used at the point of action, whether in the lab or field.

  1. Instrument insertion: The cartridge is inserted into an instrument.

• The system performs all analysis automatically, requiring no human intervention, with DNA results produced in 90 minutes.

• The RapidLINK Software centralizes data management and control of results.

Rapid DNA – Sample Cartridge

• The cartridge contains all necessary reagents for DNA extraction and PCR.

  • All reagents, swabs, and cartridges are monitored digitally.

Rapid DNA - Primary Cartridge Components

• Contains:

  • A gel cartridge

  • A capillary

  • Sample chamber

  • Waste chamber

  • Reagent vials

  • A PCR chamber

Rapid DNA - Results Interpretation

• Results are color-coded for ease of understanding:

  • Green: Successful run with good-quality output.

  • Yellow: Requires user verification.

  • Red: Failed run.

• The system stores information for all users, ensuring quality control of samples and user inputs.

• Results are output as regular STR traces, allowing for manual editing of results while documenting all changes.

Rapid DNA – Uses and Limitations

• The system can only process up to 1 or 8 samples at a time.

• Interpretation of results still required in most cases.

• Currently, it is not approved for fieldwork in Australia but is allowed for specific uses by the FBI.

Genetic Genealogy

• Represents a novel approach for analyzing forensic samples.

• The gold standard for identification remains STR profiling.

• Challenges Addressed: Offers solutions when there is no suspect or existing profile in the database.

Genealogy Defined

• Genealogy is essentially the study of family history, traditionally through historical records.

• Introduction of DNA testing into genealogy emerged in the 1990s, primarily focusing on Y-chromosome and mtDNA analyses.

Advancements in Genetic Genealogy

• Current technological advances allow the analysis of autosomal DNA, thus providing a more comprehensive profile.

Accessibility of Genetic Genealogy

• Numerous companies offer ancestry services with minimal requirements (typically just a saliva sample and a fee).

• The analysis process is often user-friendly, with options to upload data to wider databases for enhanced connections.

SNP Microarrays

• Focuses on Single Nucleotide Polymorphisms (SNPs) via specialized SNP chips.

• Microarrays can test millions of SNPs simultaneously.

  • Predominantly utilizes BeadChip technology where silica beads are coated with probes targeting specific SNPs across the chip surface.

SNP Microarrays – Amplification Process

• Begins by amplifying all the DNA from the sample using non-specific primers that initially amplify across the genome, incorporating 5’ caps that do not bind.

• Following the initial amplification, cap-specific primers are utilized for further amplification.

SNP Microarrays – Fragmentation and Binding

• The amplified DNA is fragmented to sizes of 300-600bp.

• The fragmented DNA is washed over the chip, wherein it sticks to complementary sequences on the silica beads while stopping one base short of the SNP for subsequent analysis.

SNP Microarrays – Base Information Capture

• Single base extension is utilized to obtain data from the base following the probe.

• Dideoxynucleotides (ddNTPs) function similarly to Sanger sequencing, where C and G bases are tagged with biotin, while A and T bases are tagged with dinitrophenyl.

• Fragmented genomic DNA is then washed from the chip to detect the added base.

SNP Microarrays – Staining Process

• Involves a two-step staining process incorporating fluorescent molecules and antibodies:

  • Step 1: Green fluorescent streptavidin attaches to biotin and red fluorescent anti-DNP binds dinitrophenyl.

• The initial signal strength is insufficient for reliable readings, necessitating more antibody steps.

SNP Microarrays – Signal Enhancement

• Step 2: Additional antibodies that are tagged for binding enhance the fluorescent signal.

  • Each step is followed by multiple washes to improve signal intensity.

Imaging the Chip

• Two imaging steps are required:

  1. Detect the green fluorescence.

  2. Detect the red fluorescence.

• Output Interpretation:

  • Green beads indicate homozygous C,C or G,G.

  • Red beads indicate homozygous A,A or T,T.

  • Yellow beads indicate heterozygous combinations.

• Outputs are clear and will always yield green and red options such as: AC, AG, TC, TG.

Genetic Genealogy – Forensic Applications

• SNP data assists in deducing information about samples, which can include:

  • Ancestry: Helps in narrowing suspect or missing persons lists.

  • Biological profiles: Infers certain physical characteristics from DNA.

  • Familial relationships: Provides investigative leads from related individuals.

• Genetic genealogy has resolved numerous cold cases across the globe.

SNP Arrays in Forensics

• Targeting single base mutations is especially useful for old and degraded DNA.

• This approach improves chances for reopening cold cases and addressing instances without STR profile matches.

• Utilizes relational databases where relatives upload their information to public ancestry sites, blending genetics and traditional detective work.

Investigative Genetic Genealogy

• While still seeking relationships, it is distinct from general public genealogy practices.

• Typically arises when investigators possess a suspect's STR profile but lack a match; the focus is on ancestry markers.

Building Family Trees

• Investigators construct trees from related individuals, allowing the elimination of candidates based on age or gender.

• Personal histories help further narrow down potential suspects to 2-3 individuals of interest.

Detailed Examination of Potential Suspects

• Sample Collection: Obtains public DNA samples or from close relatives.

• STR Analysis: Aims for a matching STR profile which would confirm a suspect.

• A drive towards collecting single-source samples to confirm STR matches and resolution of high-profile cold cases, including notable cases such as:

  • Golden State Killer: Joseph James DeAngelo.

  • Tanya Lee Glover.