Forensic Biology Final

What is genetics?

The study of inheritance

Who determined the rules of inherence in the late 1800s

Gregor Mendel

How does DNA regulate cell activity?

By telling the cell what proteins to synthesize and how

What did forensic biologists use to individualize biological evidence before DNA

Proteins

What are genetic differences that enable people to be distinguished

Genetic polymorphisms

What are the five classical or conventional genetic markers

1. Blood groups
2. Isoenzymes
3. Plasma (serum proteins)
4. Hemoglobin variants
5. HLA

Explain what a gene is

Region of DNA that codes for a particular protein. there are 2 genes of everything that are paired

What is a gene locus?

An area on the chromosome where a particular trait is located

What is an allele?

The genes making up the pair at a locus. They can be homogeneous or heterogeneous

What is DNA

a double-stranded helical structure. It is a polymer consisting of monomer units known as nucleotides. Nucleotides consist of a nitrogenous base, five-carbon sugar, and a phosphate. Bases are ATCG

Explain a genome

The entire complement of DNA in one cell. There are about 3.5 billion base pairs. Coding portions are known as the genetic code. The non-coding portion is of forensic importance because it contains tandemly repeated portions that differ within the population

Explain nuclear DNA

Found in nucleus. Every cell except mature RBC and germ cells contain the entire genome

Explain mitochondrial DNA

Only inherited through the maternal line. Not individualizing. HV1 and HV2 are the variable region within the DNA that make it of any forensic value

What are the steps of the DNA profiling process

Extraction, quantitation, amplification, separation, detection and interpretation

What is the preferred outcome of DNA profiling?

Yields sufficient quantity, good quality and high purity. Most common method involves isolating total cellular DNA

What are the steps in nucleic acid extraction?

1. Cell and tissue disruption
2. Lysis of cellular membrane
3. Removal of proteins and cytoplasmic constituents

How are cells and tissues disrupted?

-Enzymatic digestions like proteinase K
- Boiling
-Alkali treatment (tissues)
-Mechanical methods like grinding or using decalcification with EDTA for bones/teeth

How is the lysis of cellular and organelle membranes done?

They release DNA from the nuclei and the mitochondria
The lysis buffer consists of:
1. Detergent like SDS that destroy membranes
2 Buffer like tris that maintain pH and avoid enzyme degradation by Dnase
3. High salt conetrations (guadinimum salts) which dissociate histones from DNA
4. Reducing agents like DDT that inhibit oxidation that can damage DNA
5. Chelating agents that capture divalent metal ions that are Dnase cofactors

How is the removal of protein and cytoplasmic constituents completed?

Cytoplasmic components and proteins interfere with DNA isolation and they need to be removed. They can be dissolved by phenol-chloroform-isoamyl mixtures, the DNA binds to a solid matrix and the contaminants are washed away

What are the four methods of extraction?

1. Organic extraction (phenol-chloroform)
2. Chelex Extraction
3. Silica-based extraction
4. Differential extraction

What is proteinase K

proteolytic enzyme that breaks down proteins and releases the contents of the cells

How is organic extraction performed?

Proteins removed using phenol-chloroform-isoamyl mixtures which forms 2 layers, the organic (lower) and the aqueous (upper) (DNA in aqueous). Lipids in organic and proteins in middle. Then DNA is precipitated out using ethanol and salts. Then ultrafiltration is used

What are the advantages and disadvantages to organic extraction

Good: Large size, double stranded, can be used in PCR and RFLP
Bad: Time consuming, hazardous reagents, requires sample transfers between tubes

How is Chelex extraction done?

First washing is used that removes contaminants and inhibitors. Then the sample is pre-boiled at 56 for 20min which softens membranes and separates cellular clumps. The sample is boiled where the cells are lysed and the chelex resin binds polyvalent molecules like Mg which is a cofactor of Dnas. It is then centrifuged

What are the advantages and disadvantages of chelex extraction?

Good: it is simple and fast and uses one tube
Bad: heating can denature DNA, DNA is single stranded and is not suitable for RFLP

What is the principle of Silica based extraction

DNA is reversibly absorbed by silica in the presence of chaotropic salts. The chaotropic salts disrupt the H bonds which cause nucleic acids to become hydrophobic and then the phosphate groups interact with the silica

How is Silica based extraction performed?

1. Cell Lysis & Protein Digestion
• Done using Proteinase K
• Cells membranes break open, DNA is released
2. DNA Adsorption onto Silica
• Silica is the stationary phase to which DNA binds • Occurs in the presence of chaotropic agents
• Double-strandedDNAisadsorbed
• Cellular contaminants do not bind
3. Washing
• Removes chaotropic salts & contaminants
• Certain solvents (EtOH) will not affect DNA's interaction with silica
4. Elution of DNA
• DNA is eluted by rehydration with low salt solutions

What is the principle of differential extraction?

Useful for extracting DNA in sexual assault cases
• Usually mixtures of spermatozoa from males and epithelial cells
from females
• Mixed DNA profiles complicate data interpretation
• Sperm and nonsperm cells are lysed at separate times
• Based on the properties of the different cell membranes • Sperm and nonsperm cell fractions can be isolated

How is differential extraction completed?

Lyse non-sperm cells with proteinase K
• Sperm cells are resistant
• Membranes contain proteins crosslinked by disulfide bonds
Centrifugetube
• Supernatant contains nonsperm cell fraction
• Clumpofcellsattubebottomcontainssperm cells
• Lyse sperm cells with Proteinase K and DTT
• DTT is a reducing agent
• The DTT breaks down the protein di-sulphide bridges that make up the sperm nuclear membrane.
• Sperm nuclei are impervious to digestion without DTT
One sample generates two fractions of DNA
• SpermandnonspermcellDNAmay not be completely separated
• Poor storage conditions can cause sperm cells to lyse
• Overabundanceofnon-sperm DNA may cause carry over into sperm fraction

How should extracted DNA be stored?

Usually stored in TE buffer
• Short term storage at 4°C or -20°C • Long term storage at -80°C
• Avoid frequent freeze-thaw cycles
• Temperature fluctuations can break the DNA
• Increased stability with increased purity

How does contamination occur?

Introduction of exogenous DNA
• Can occur between
• Person and sample
• Sample to sample
• Samples and amplified DNA
• Material from other organisms

How is contamination prevented?

Process evidence and reference samples separately in both space and time
• Process evidence first
• References contain more DNA
• Process evidence samples away from amplified DNA
Make sure disposables and reagents are DNA-free • Use aerosol-resistant pipette tips
• Create reagent blanks for each extraction • Only reagents, no sample
• Monitors for contamination in
• Reagents
• Handling procedures

How is RFLP done?

Genetic variation in the distance between restriction enzyme sites
• Template DNA digested by enzymes, electrophoresed, detected via Southern blotting
• Power of discrimination in the range of 106-108 for a six probe analysis
-analysis of VNTR loci.
It involved cutting DNA with enzymes known as restriction endonucleases, separating the fragments by electro-
phoresis, transferring the
fragments to a nylon membrane, and detecting alleles with DNA probes

What was the first case DNA was profiled?

Collin Pitchfork case
Two young women raped and murdered in Narborough, England
5,000 local men are asked to provide blood/saliva samples
1st exoneration (Richard Buckland) and conviction (Colin Pitchfork) on forensic DNA evidence

What are the disadvantages of RFLP

RFLP testing requires a relatively large amount of HMW DNA (~50ng = thousands of cells)
• Not ideal for forensic evidence, in which small, degraded samples are common

How does PCR work

Polymerase Chain Reaction = molecular Xeroxing
• Three temperature phases, carried out in a Thermal Cycler, replicate or "amplify" the desired DNA fragment(s)

What are the advantages of PCR?

First forensic application is the DQα locus, later multi-plexed with PolymarkerTM loci using dot- blot detection method.
• Works with lower quantity (1-2ng), lower quality samples.
• Power of discrimination goes from 102-106

What are the standards of DNA quantitation

All sources of DNA are extracted when biological evidence from a crime scene is processed to isolate the DNA present.
• Thus, non-human DNA such as bacterial, fungal, plant, or animal material may also be present in the total DNA recovered from the sample along with the relevant human DNA of interest.
• For this reason, the DNA Advisory Board (DAB) Standard 9.3 requires human-specific DNA quantitation so that appropriate levels of human DNA can be included in the subsequent PCR amplification.
• Multiplex STR typing works best with a fairly narrow range of human DNA - typically 0.5 to 2.0 ng of input DNA works best with commercial STR kits.

What is the point of quantifying DNA?

If we can confidently determine the amount of DNA in an extract we can then ask questions:
• Will mitochondrial sequencing be required (skip STR analysis)?
• Should we use low copy number LCN methods for STRs?
• Re-extract the sample?
• If problems occur in the STR typing process we can have confidence that the DNA template is not the source (CE, cycler, kit).

What is the principle of DNA quantitation?

Quantitation determines the amount of human DNA present in an extract.
• A narrow concentration range is required to "seed" the Identifiler PCR reaction for Amplification
• Too much or too little DNA gives rise to artifacts (false positive or false negative alleles)
• Must use human-specific DNA quantitation • Bacterial DNA may be present (e.g. saliva)
• Test should measure quality as well as quantity of DNA

What are the three most common quantitation methods

Three common methods
1. Slot Blot Assay
2. Interchelating Dye
3. Quantitative PCR
• Method of choice in most modern crime labs • We'll use this method in lab

How does the slot blot method work?

• Detects primate DNA
• Genomic DNA is denatured (made single-stranded) and small volume is spotted onto a nitrocellulose membrane
• Single stranded denatured DNA links to the nitrocellulose membrane
• DNA immobilized on a nylon membrane
• Hybridization with labeled 40-nucleotide probe
• Complementary to primate-specific α-satellite sequence at the D17Z1 locus
Radioisotope detection was replaced by: • Alkaline phosphatase-labeled probes
• Chemiluminescent detection
• Biotinylated probes
• Chemiluminescent detection • Colorimetric detection

How was the intercalated dye method performed

Fluorescent dye used
• Quant-iT PicoGreen dsDNA reagent
• Not specific to human DNA
• Useful with known reference blood samples
• Not useful for questioned samples or buccal swab samples
• Fluorescence measure by spectrofluorometer
Measurement of unknown compared to a standard curve of known amounts of DNA
Detection limit is approximately 250 pg

How is Quantitative PCR (qPCR or "real time PCR") performed

• 1990s
• More sensitive
• Large range of detection
• Amount of PCR product amplified during exponential phase of PCR correlates with the initial concentration
• Real-time PCR most common method in forensic lab
• Analyzes the amplification of a target sequence at each cycle of PCR

Why is DNA quantitation important and what is some of the equipment used

DNA quantitation is important to determine how much human DNA (as opposed to bacterial DNA) is present in a sample
• A commonly used DNA quantitation kit is called Quantifiler (sold by Applied Biosystems)
ABI 7500: an instrument used to perform "real-time quantitative PCR"

What are the phases of qPCR

Exponential phase
• 100% efficiency (plenty of primers
and dNTPs)
• High degree of precision in accumulation of PCR products with time: doubling per cycle
• Linear phase
• One or more components fall below critical concentration; amplification efficiency drops
• Precision in accumulation of PCR products drops
• Plateau ("end point")
• Reaction slows to a halt; components consumed

What are the advantages to qPCR

The availability of commercial qPCR kits (labs have almost entirely switched to this method for DNA quantitation)
• Higher throughput and reduced user intervention • Automated set up
• Simple data analysis
• Experimental data rapidly analyzed in software; interpolating into the
calibration curve
• qPCR will be sensitive to the same inhibitors as faced in a traditional STR test (both PCR based)
No post PCR manipulation (reduced contamination issues) - amenable to automation
• High sensitivity (down to a single copy number ?)
• Large dynamic range: ~30 pg to ~30 ng
• More accurate measurements of small quantities of DNA
• Assays are target specific (autosomal, mito, Y) and can be multiplexed - to a degree...

What are the disadvantages to qPCR

qPCR is subject to inhibition
• internal PCR controls (IPC) can help
• qPCR quantitation precision suffers at low copy numbers (below 30 pg by a factor of 2)
• When working below 100 pg qPCR is still subject to variability and uncertainty
qPCR quantitates specific target sequences, it does not quantify "DNA"
• In highly degraded samples, assays that amplify short target sequences will detect and measure more DNA than assays that amplify long target sequences (relevant to STR typing)
• Accurate qPCR quantitation assumes that each unknown sample is amplified at the same efficiency as the Calibrant sample in the dilution series
• Results are relative to the Calibrant (these can vary)

Current methods of DNA Typing

Autosomal Short Tandem Repeats • Non-coding, tetranucleotide sequences which vary greatly from person to
person in the number of repeating units.
• Requires<1ngofDNAtotype13-15STRloci
• Power of discrimination ranges from 1014-1023. World population is 109 so bring on the database!

What about the loci of STR analysis

13 STR loci were initially chosen for analysis as they provided a high level of
individualization.
• The choice of 13 STR loci
was also based on the
desire to have all forensic laboratories contribute profiles of the same genetic markers to the DNA database

How does PCR work?

Polymerase chain reaction (PCR) is a technique for replicating small quantities of DNA or broken pieces of DNA found at a crime scene, outside a living cell.
• Polymerase chain reaction is the outgrowth of knowledge gained from an understanding of how DNA strands naturally replicate within a cell.
• For the forensic scientist, PCR offers a distinct advantage in that it can amplify minute quantities of DNA many millions of times.
Using multiplex PCR, specific regions of the human genome are simultaneously targeted with sequence-specific oligonucleotide PCR primers and copied with a DNA polymerase and deoxynucleotide triphosphate building blocks.
• Close to a billion copies of each specific region of the genome can be generated in a matter of a few hours by subjecting the DNA sample to typically 28-32 cycles of heating and cooling that permit the replication process to occur.
• ThePCRamplificationprocessincorporatesfluorescently-labeledprimersinto the PCR products that enable multi-color fluorescence detection.
• In order to protect from contamination by post-PCR products, pre- and post- PCR steps are typically segregated in different laboratory space.

What are the advantages to PCR

Advantages of PCR amplification with
forensic specimens;
- Very small amounts of template required 1ng
- Effective with degraded dna
- Specific DNA sequences can be amplified simultaneously - multiplexing
- Human specific primers used - no contaminant DNA
- Commercial kits available

PCR Amplification principle

PCR = polymerase chain reaction; first described by Kary Mullis in 1985 (Nobel Prize in Chemistry in 1993).
• PCR is an enzymatic process in which a specific region of DNA is replicated over and over again to yield many copies of a particular sequence - 'molecular Xeroxing'
• Heating and cooling samples in a precise thermal cycling pattern over ~30 cycles
• During each cycle, a copy of the target DNA sequence is generated for every molecule containing the target sequence
Process that copies a particular region of DNA using two "primers" (short pieces of DNA)
• Each strand of DNA is used as a template to create a replicate that permits a doubling of the number of target molecules with each cycle of heating and cooling

Components of PCR reaction

Most important components of a PCR reaction are the two primers, which are short DNA sequences that precede or 'flank' the region to be copied.
• A primer acts to identify or 'target' the portion of the DNA template to be copied.
• It is a chemically synthesized oligonucleotide that is added in a high conc relative to the DNA template to drive the PCR reaction.
• Other components; template DNA, dNTP building blocks (supply 4 nucleotides) and a DNA polymerase (adds the building blocks in proper order based on template DNA sequence).
• Thermal stable Taq polymerase- ensures does not fall apart during denaturation; comes from bacterium Thermus aquaticus found in hot springs.

Multiplex PCR

PCR permits more than one region to be copied simultaneously by simply adding more than one primer set to the reaction mixture. The simultaneous amplification of 2 or more regions of DNA is multiplexing.

PCR Reaction Setup

DNA sample is added (about 1 ng based on DNA quantitation performed) - 10 μL
• PCR primers and other reaction chemicals from an STR typing kit are added - 15 μL
• Always include a negative no template control reaction to ensure reagent purity.
The polymerase chain reaction (PCR) copies sections of DNA through heating and cooling the sample
• Each DNA strand is copied with each temperature cycle
• A thermal cycler heats and cools DNA samples (usually 28 cycles)

Steps in the thermal cycling

1. First, the DNA is heated to separate it (denaturation).
2. Second, primers (short strands of DNA used to target specific regions of DNA for replication) are added, which hybridize with the strands.
3. Third, DNA polymerase and free nucleotides (A, T, G, and C) are added to rebuild each of the separated strands.
4. Now, this cycle is repeated 25 to 30 times.
• Within a few hours a short strand of DNA can be multiplied a billionfold.

PCR/qPCR What is the Difference?

In the PCR the products are analyzed after the cycling is completed (static)
• gel, CE, UV, fluorimeter • End point assay
• qPCR the products are monitored as the PCR is occurring (dynamic)
• Once per thermal cycle
• Fluorescence is measured • Kinetics of the system

Factors affecting PCR

Template quality
• Inhibitors
• Contamination

Template Quality

• Prevent degradation during collection and processing of DNA evidence
• Degradation causes DNA to break into fragments
• Breaks occurring at the target region can results in failure of
amplification
• Low copy number (LCN) template is often occurs in forensic samples
• Can result in stochastic effect - two alleles in a heterozygous sample are unequally detected
• Cycle number may be increased for LCN samples

Inhibitors

Inhibitors interact with DNA template or polymerase, causing amplification to fail
• Can be detected using an internal positive control
• Common inhibitors in forensic samples: • Heme from blood
• Indigodyefromfabric
• Melanin in hair samples
• Inhibitors must be removed during extraction

Contamination

PCR is very sensitive - avoid risk of contamination
• Pre- and post-PCR sample should be processed in separate areas or
at different times
• Reagents for pre- and post-PCR should be separated
• Protective gear such as gloves, lab coats, facial masks, and hair caps
• Aerosol-resistant pipet tips
• DNA-free solutions and test tubes
• Controls must be used to detect contamination
• Extraction reagent blanks monitor contamination during extraction
process, extraction reagents
• Amplification negative controls monitor contamination of amplification and PCR reagents
• DNA profiles of lab members should be available for comparison

General facts about Capillary Electrophoresis

The most commonly used CE systems are the single capillary ABI Prism 310 Genetic Analyzer and the multi-capillary ABI 3100 or 3130xl.
• These CE instruments electrokinetically inject the negatively charged DNA molecules from a formamide-diluted sample of the PCR products mixed with an internal size standard.
• The size standard is labeled with a separate fluorescent dye to enable calibration of each analysis so that comparisons can be made between samples run at different times on the same instrument.
• A polymer solution inside the capillary permits resolution of DNA fragments differing by as little as a single basepair (bp) over a size range of approximately 100 to 400 bp.

Methods of labeling DNA with a fluorescent dye:

1. Using a fluorescent intercalating dye to bind to double-stranded DNA post-PCR
2. Incorporating fluorescently labeled deoxynucleotides (dNTPs) during PCR
3. Incorporating a fluorescent dye into the amplicon during PCR through a 5'end labeled oligonucleotide primer
• Fluorescent dyes are present on one strand of each PCR product due to incorporation of a PCR primer during multiplex PCR amplification.
• These dyes are excited by laser as they pass a detection point in the CE instrument.
• Since the four or five fluorescent dyes used in STR analysis have different chemical properties, they emit light at slightly different wavelengths enabling detection in different color channels.
• Because there is overlap with the emitted light from the different dyes, mathematical algorithms are used to perform a "matrix correction" or "spectral calibration" so that individual DNA peaks in an electropherogram appear to be labeled with a single color.

Separating and "Seeing" STR's

• Electrophoresis
Separates amplification products based on size
• Fluorescent detection
• Amplification products have a fluorescent "label" attached to the primer
• Label is seen through excitation via a laser and corresponding emission captured with a camera

Components of a CE instrument:

• Fused-silica capillaries
• Twobuffervials
• Two electrodes connected to a high-voltage power supply
• Laser excitation source
• A fluorescence detector
• Auto sampler to hold the sample tubes
• Computer to control the sample injection and detection

Components of ABI 3130xl

Chemistry
• STR kits, fluorescent dyes, matrix samples, capillaries, buffers,
polymer, formamide • Hardware
• CCD camera, laser, electrodes, pump block, hot plate for temperature control, autosampler
• Software
• Data collection, color separation, peak sizing & calling, genotyping, stutter removal

Advantages of CE

• The injection, separation and detection steps can be fully automated
• Multiple samples can be run unattended by CE
• Tiny quantities of sample are consumed with an injection
• Samples can easily be retested if needed without more preparation

Disadvantages of CE

• Single capillary instruments cannot process high numbers of samples.
• A high start-up cost
• Maintenance and supplies for the instrumentation can be expensive

Size Standards and Allelic Ladders

A sizing standard is used in all samples and allelic ladders.
The known standard is used to determine the size of the allelic ladders and the unknown samples.
When genotyping, it is critical to use an allelic ladder run under the same conditions as the samples because:
• Size values obtained for the same sample can differ between instrument • platforms because of different polymer matrices and electrophoretic
conditions.
• Variation in laboratory temperature can affect migration speed
The red internal lane standard establishes the allele size. This size is compared to an allelic ladder run earlier.

Autosomal Short Tandem Repeat Profiling

• Short tandem repeat (STR): region of genomic DNA containing an array of short repeating sequences
• Arrays range from several to 100 repeat units
• Number of repeat unit varies among individuals • Most commonly used STR are 100-500 bp long

Core Repeat and Flanking Regions

• Core repeat region = tandemly repeated sequences • Flanking regions = surrounding core repeat
• PCR primers complementary to flanking regions are used to amplify core repeat regions
Core repeat and flanking regions of CSF1PO STR locus. It consists of eight repeating units of tetrameric nucleotides (TAGA); thus, it is designated as allele 8.

Repeat Unit Sequences

• Simple repeats = identical repeat unit sequences
• Compound repeats = more than one type of simple repeat
• Complex repeats = Several clusters of different tandem repeats with intervening sequences
• Nonconsensus alleles, or microvariants, differ from common alleles by one or more nucleotides

STR Loci Used for Forensic DNA Profiling

Desired characteristics of forensic STR loci:
• Highlyvariableamongindividuals
• Loci should not be linked or inherited together
• Forensic STR loci are usually on different chromosomes to be sure they are not linked. However, loci that are far apart on the same chromosome may be used
• Have few amplification artifacts, such as stutter
• Short amplicons are best for multiplex PCR and analysis of degraded DNA

Value of STR Kits

Advantages
• Quality control of materials is in the hands of the manufacturer
• Improves consistency in results across laboratories - same allelic ladders used
• Common loci and PCR conditions used - aids DNA databasing efforts
• Simpler for the user to obtain results
Disadvantages
• Contents may not be completely known to the user (e.g., primer sequences)
• Higher cost to obtain results

Requirements for Accurate STR Typing

1. High precision (to permit comparison of allelic ladders to sequentially processed STR samples)
2. Color separation of different dye sets used (to avoid bleed through between different colors)
3. Resolution of at least 1 bp to >300 bp (to detect microvariants)
4. Reliable sizing over 75-450 bp region

Current Forensic STR Multiplexes

All amplified fragments are less than 500 bp in length which can be beneficial in analyzing degraded DNA samples. The red color represents the internal lane standard which is added to each reaction. Since the sizes of the red fragments are known, they can be used to determine the size of the amplified fragments. An allelic ladder, containing all commonly observed repeats is also run and sized. Comparison of the unknowns to the ladder fragments allow the identification of the genotype.

Allele Calls

Comparison of the unknown fragments to the migration pattern of the allelic ladder
allows determination of the genotype. Software programs use macros to automatically
make the comparison.

Human Y Chromosome Genome

The Y chromosome is patrilineal
• Inherited from father and passed on to male offspring
• Encodes genes for sex determination and spermatogenesis
• 59 million base pairs
• 50-60genes
• Pseudoautosomal region and male-specific Y region

Pseudoautosomal Region on Y Chromosome

• Pseudoautosomal regions (PARs) = homologous nucleotide sequences on X and Y chromosomes
• 2 PARs on each X and Y: PAR1 and PAR2
• Y chromosome PAR1
• Terminal region of short arm • 2.6Mb,24genes
• Y chromosome PAR2
• Tip of long arm
• 320 kb, 4 genes
• PARs are part of proper segregation of X and Y chromosomes during meiosis
• Allows X and Y to pair
• Crossing over and recombination can occur

Polymorphic Sequences

• Y chromosome contains many polymorphic markers • Y-STRs are used for Y chromosome DNA testing • High-through put and good discrimination power
• DYF155S1 first characterized Y chromosome minisatellite • Highly polymorphic
• Potential marker for forensic casework
• However, analysis method is labor intensive

Y-STR

• Investigation of sexual assault
• Mixture of high levels of female DNA and low levels of male DNA
• Y chromosome-specific loci can be examined without interference from female DNA
• Determining number of unrelated male perpetrators
• Forensic Y-STRs on nonrecombining section of Y so patrilineage can be
established
• Paternity and missing persons
• Lower discriminating power than autosomal loci

Core Y-STR Loci

• Development of multiplex systems with additional Y-STR loci
• Improve discriminating power
• Commercial kits with additional loci have been validated for forensic use
• Some additional Y-STR are highly discriminating
• High mutation rates - discrimination of related individuals

Mitochondrial DNA Profiling

• Referred to as mtDNA
• Helpful in identifying missing persons & mass fatalities
• Maternally inherited
• Your mtDNA profile is identical to your maternal relatives
• Useful tool for human identification when nuclear DNA is insufficient or not available
• Number of copies of mtDNA genome per cell is higher than nuclear DNA
• Hair shaft, bones, decomposed samples are good candidates • Circular & more stable

Human Mitochondrial Genome

• Mitochondria are subcellular organelles which are energy-generating
• Each cell contains 100's of mitochondria
• Each mitochondria contains numerous copies of the mtDNA genome • Estimated that 100's of copies exist in most cells
• Sequenced in 1981 by Fred Sanger
• Known as the Cambridge Reference Sequence
• 10 substitution errors detected in the original mtDNA sample
• Modified in 1999 & is now called the Revised Cambridge Reference
Sequence (rCRS)
• Used as the standard for sequence comparisons

Maternal Inheritance of mtDNA

• Non-Mendelianinheritance
• Sperm mitochondria reside in the midpiece
• Only head enters the egg for fertilization • Eggcontainsmitochondria
• Individuals from the same maternal lineage will have the same mitotype
• Occasionally paternal mtDNA may enter the egg cell
• If it does, it is tagged with ubiquitin (Ub) and destroyed
• mtDNA type is called the mitotype
• Homologous DNA recombination has not been observed • It is a haplotype
• Treated as a single locus
• 10x higher mutation rate than nuclear DNA • Increases variability

mtDNA Polymorphic Regions

Most polymorphic region located in D-Loop
Hypervariable regions: HV1 (342 bp)*
HV2 (268 bp)*
HV3 (137 bp)
*Most commonly analyzed for forensic purposes

General Considerations of Forensic mtDNA Testing

• Often used on decomposed or skeletal remains
• Clean surface to remove debris & contaminants • Pulverize bone & teeth to facilitate extraction
• Extract DNA using similar method to nuclear
• Can get idea of mtDNA amount based on nuclear DNA amount • Can also do special quant for mtDNA

Forensic STR Analysis-Data transfer

Following data collection, the data (.fsa) files are typically transferred from the lab computer to one in an office where data analysis is performed
• A USB thumb drive permits rapid and easy transfer of data files

Data Analysis

The analyst carefully reviews the DNA data (electropherogram) and checks software genotype calls and edits out artifacts
• Software designates sample genotypes via comparison to an allelic ladder (mixture of common allele possibilities)
• STR loci are amplified with fluorescent dye-labeled primers • Multiple dyes label each amplicon in a mutliplex STR system
• Different fluorescent dyes are resolved by the detector
• Signals corresponding to each fragment are identified with
specialized software
• An electropherogram is generated: a profile of peaks corresponding to each DNA fragment
• DNA fragments are sized by comparison to an internal standard

STR Results

• Individuals will differ from one another in terms of their STR profile
• STR genotype can then be put into an alpha numeric form for search on a DNA database

Data is Tabulated

The number of repeats observed for each locus is tabulated
This data format is stored in databases and used for comparisons/matches
Finally a case report is written based on tabulated STR genotype calls
Electropherogram of GeneScanTM 500 size standard (Applied Biosystems). RFU represents relative fluorescence unit.

Detection Thresholds

• Thresholds are set to separate signal from noise - in other words, are we confident that a peak is real?
• Signal peak height is measured in relative fluorescence units (RFUs) that are related to the amount of DNA present in the sample loaded onto the analysis instrument

Thresholds for Measuring DNA
Data

These thresholds for reliable data are determined through
validation studies
Peak is called (deemed "reliable")
50 RFUs
Peak is NOT called (deemed "unreliable")
• Detection (analytical) threshold
• Dependent on instrument sensitivity
~50 RFU (relative fluorescence units)
• Impacted by instrument baseline noise
• Dropout (stochastic) threshold
• Dependent on biological sensitivity ~150-200 RFU
• Importantinmixtureinterpretation

CE and STR

Capillary electrophoresis separation of amplified STR products. Fluorescent dye- labeled amplification products are separated and subsequently detected. Various fluorescent dye colors are resolved by the detector. The peaks corresponding to each DNA fragment are identified.

Determining the Genotypes of STR Fragments

• Genotype for an STR locus is the number of repeat units of the allele
• Genotype is determined by using an allelic ladder
• Ladder = collection of synthetic fragments corresponding to
common alleles for a given set of STR loci
• Electropherogram of sample is compared to ladder to determine genotype
• Off-ladder allele: a rare allele that fails to match alleles in the ladder

Three Types of "Off-Ladder" Alleles

Above, below or variant

Interpretation of STR Profiling Results

• Inclusion (Match)
• Peaks of compared STR loci show identical genotypes
• Exclusion
• Genotypes differ; profiles originated from different sources
• Inconclusive Result
• Insufficient information to reach a conclusion

Amplification Artifacts
Stuttering

• Peaks that show up primarily one repeat less than the true
allele as a result of strand slippage during DNA synthesis
• Short repeat unit loci are more likely to stutter
• Loci with complex repeat sequences have less stutter
• Caused by slippage of polymerase during amplification
• Stutter peaks make mixture analysis more difficult
Proposed mechanism for stutter products. During the DNA synthesis step of PCR amplification, a DNA polymerase slips, and a region of the primer- template complex becomes unpaired, causing the template strand to form a loop. The consequence of this one-repeat loop is a shortened PCR product smaller than the template by a single repeat unit.

Amplification Artifacts
Nontemplate adenylation

• Taq polymerase will often add an extra nucleotide to the end of
a PCR product; most often an "A" (termed "adenylation") • DNA adds adenosine, to the 3'-end of an amplicon
• Amplicon 1 bp longer than parental allele, +A peak
• Multiplex STR kits use conditions that favor adenylation, so all
peaks have the extra adenosine
• Too much template is used in PCRmixture of -A and +A peaks
• Excess amounts of DNA template in the PCR reaction can result in incomplete adenylation (not enough polymerase to go around)