FRSC-3000 Lecture Summary Notes

Lecture 1: Overview and History of DNA Typing

OVERVIEW AND HISTORY OF DNA TYPING

Brief History of DNA Typing:

• 1985: Alec Jeffreys, a genetic researcher at the University of Leicester, developed DNA profiling along with Peter Gill and Dave Werrett of the Forensic Science Service (FSS)

  • Developed a method to retrieve DNA from dried blood stains and a preferential extraction method to separate sperm from vaginal cells

  • Found certain regions of DNA contain repetitive sequences that are variable in number between individuals: DNA fingerprinting or DNA typing

• Variable Number of Tandem Repeats (VNTRs):

  • Minisatellites (8-100bp repeats) often found at the ends of chromosomes

  • Microsatellites (2-7bp repeats) dispersed throughout the genome

The Pitchfork Murder Case:

• A kitchen porter was arrested and confessed to murder

• Jeffreys conducted DNA identification on evidence, and the kitchen porter did not “match” (exonerated from crime)

• Investigation of ~5k local males eventually found only one “match”: Colin Pitchfork

  • His DNA profile matched with the semen from both murders

  • Convicted and sentenced to life in prison in 1988

Basic Principles of DNA Testing:

• The DNA profile of every person is unique

• The genome is the same in every cell

• The DNA profile remains the same throughout life

• Your DNA is inherited ½ from mother and ½ from father

• DNA typing must be performed efficiently and reproducibly (must hold up in court)

• Current standard DNA tests do not look at genes (little to no information about geographical ancestry, predisposal to disease, or phenotypical information is obtained

• We probe subsets of genetic variation called short tandem repeats in order to differentiate between individuals

Applications for DNA Testing:

• Involves generation of DNA profiles usually with the same core STR markers and then matching to reference sample

• Crime solving - matching suspect with evidence

• Paternity testing - who is the father

• Missing persons investigations - whose remains

• Immigration testing - are two people related

• Disaster victims - after an airplane crash

• Soldiers in war - who is the unknown soldier

• Developing convicted felons’ databases - cases solved

DNA Testing as a Reference:

• A DNA profile by itself is fairly useless because it has no context

• DNA analysis for identity only works by comparison — you need a reference sample:

  • Forensic case: Crime scene evidence compared to suspects

  • Paternity case: Child compared to alleged father

  • Mass disaster ID: Victim’s remains compared to biological evidence

  • Armed forces ID: Soldier’s remains compared to direct reference sample

Three Possible Outcomes of Evidence Examination:

• Exclusion (no match): The genotype comparison shows profile differences that can only be explained by the two samples originating from different sources

• Non-exclusion (fail to exclude): Statistical evaluation of the significance of the match is usually reported with the match report

• Inconclusive result: This finding might be reported if two analysts remain in disagreement after review and discussion of the data and it is felt that insufficient information exists to support any conclusion

BASICS OF DNA BIOLOGY AND GENETICS

Nuclear DNA in the Cell and Jargon:

• A specific region of DNA is called a locus

• Alternative forms of a locus are called alleles

• If two alleles are identical by descent at a locus, they are called homozygous and if different, heterozygous

• A genotype is the characterization of alleles at a locus

• A DNA profile is the genotypes obtained at multiple loci

DNA Structure and Composition:

• DNA molecule includes sugar backbone, phosphate groups and four nucleotide bases

• Two DNA strands form H-bonds to make a double helix

• Phosphodiester bonds form between adjacent nucleotides

• Formed between the 3’-OH group of one nucleotide and the phosphate group of another

• The DNA double helix denatures at elevated temperatures (above 95 degrees Celsius) or with chemical treatments

Restriction enzymes:

• Recognize a specific sequence of DNA (4-8bp)

• Produce a double-stranded cut (sticky or blunt)

• Direct detection of DNA copy number variation: cut DNA into pieces and analyze fragments by gel electrophoresis

• Can also detect sequence variation by the presence/absence of a restriction cut

Human Chromosome Nomenclature:

• Variation in chromosome size and G-banding results in a karyotype

• TH01:

  • Tyrosine Hydroxylase gene, intron 01

• D16S539:

  • D: DNA

  • 16: Chromosome 16

  • S: Single copy sequence

  • 539: 539th locus described on chromosome 16

Characteristics of DNA for Forensic Applications:

• Each person’s DNA is the same in every cell

• An individual’s DNA profile remains the same throughout life

• Half of an individual’s DNA is inherited from the mother and half from the father

• Each person has a unique DNA profile

• Population variation:

  • ~99.7% of the 6.4 billion bp are the same between people

  • This 0.3% is still ~10 million differences and make us unique

Types of DNA Polymorphisms:

• Sequence polymorphism

• Length polymorphism

• DNA markers are detectable variants of DNA which are useful when polymorphic

Genetic Variability:

• In DNA typing, multiple markers are examined to create a DNA profile

• The higher the number of markers examined, the greater the chance that two unrelated individuals will have different DNA profiles

• If each locus is inherited independently from one another, then the DNA profile frequency can be calculated using the product rule by multiplying each genotype frequency together

  • More alleles = a higher power to distinguish between individuals

• Full CODIS DNA profiles can sometimes be obtained from as little as 50 pg of DNA

• Assuming a DNA extraction is 100% efficient from the biological sample collected:

  • There is ~6.6 pg in DNA diploid cell

  • 50pg DNA/(6.6pg DNA/diploid cell) = ~ 8 diploid cells

HISTORICAL METHODS IN DNA TYPING

Types of Technology and Markers:

• Non-DNA based:

  • Blood group testing

  • Forensic protein profiling

• DNA-based:

  • RFLP (length) multi-locus markers and single-locus markers

  • PCR based (sequence): Reverse dot blot

  • PCR based (length): AFLPs, silver-stained STRs, fluorescently detected STRs

• Mitochondrial DNA sequencing

Blood Group Typing:

• Advantages:

  • Rapid, simple tests

  • Only test available for many years

• Limitation:

  • Poor power of discrimination (~ 1 in 10) with such few alleles

Basics of RFLP-Based DNA Testing:

• Detect:

  • A variable number of tandem repeats (VNTRs)

  • Repeating units up to 0.5-20 kb

  • ~30,000 in human genome

• Process:

  • Cut DNA with restriction enzymes

  • Separate fragments differing in length by gel electrophoresis

  • Detect length-based differences (polymorphisms) in DNA fragments of interest with a radioactive probe

  • Strip membrane and re-probe as necessary

• Multi-probe results are more complicated to interpret:

  • “DNA fingerprint” developed by Jeffreys were unique to the individual (high power of discrimination between samples)

  • Mixtures from individuals in a single sample were impossible to interpret

• Single-Locus Probe RFLP:

  • Repeated probing of the same membrane to yield a series of autoradiographs

• Advantages:

  • Excellent powers of discrimination (~1 in millions or greater with four loci)

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

• Limitations:

  • Limited sensitivity (>50 ng to 500 ng required)

  • Time-consuming process that cannot be automated (days to weeks)

  • Not suitable with degraded DNA samples due to high molecular weight needed

  • The need for binning introduces complications and sometimes difficulties of interpretation

  • Limited number of validated loci (4 to 6 loci commonly used) which meant that these VNTRs were of limited value in distinguishing between siblings

Invention of PCR:

• 1985: Kary Mullis invented PCR (awarded the Nobel Prize in 1993)

• First publication of PCR by Cetus Corporation appears in Science

• Amplify specific DNA loci from small amounts of starting material

• Advantages:

  • Very small amounts of DNA template may be used even as little as from a single cell

  • DNA degraded to fragments only a few hundred base pairs in length can serve as effective templates for amplification

  • Large numbers of copies of specific DNA sequences can be amplified simultaneously with multiplex PCR

  • Contaminant DNA, such as from fungal and bacterial sources, will not amplify because human-specific primers are used

  • Commercial kits are now available for easy PCR reaction setup and amplification

• Disadvantages:

  • The target DNA template may not amplify due to the presence of PCR inhibitors in the extracted DNA

  • Amplification may fail due to sequence mutations in the primer-binding region, AKA a “null allele”

  • Contamination from other human DNA sources besides the forensic evidence at hand or previously amplified DNA samples is possible

Amplified Fragment Length Polymorphisms (D1S80):

• AFLP/AMP-FLP = amplified fragment length polymorphism

• PCR products (400-800 bp) separated on a polyacrylamide gel and detected with silver staining

• Advantages:

  • Improved sensitivity compared to RFLP because it uses PCR

  • Many alleles which facilitates mixed-sample analysis

  • Discrete allele calling possible using allelic ladder, which also simplifies statistical interpretation

• Limitations:

  • Large allele range making it difficult to multiplex with other loci and giving rise to preferential amplification of smaller alleles

  • Poor power of discrimination as a single locus (~1 in 50)

  • Allele dropout seen with highly degraded DNA

  • Gel separation and silver-stain detection not amenable to automation or high-throughput sample processing

DQA1 Reverse Dot Blot Tests:

• First method used allele specific probes to find sequence polymorphisms in a dot-blot format

• Most common locus was HLA-DQA1 (integral membrane protein associated with immune response)

  • Most commonly, 8 alleles identified ( 1.1, 1.2, 1.3, 2, 3, 4.1, 4.2)

  • Commercial kit could not distinguish between 4.2 and 4.3 alleles

• The “C” control dot to test a sample is above the stochastic threshold

• The “S” dot is another control sample in the test

• Advantages:

  • Fast, simple method (compared to RFLP)

  • Capable of analyzing small or degraded samples because it uses PCR

  • No instrumentation needed after PCR

• Limitations:

  • Poor power of discrimination (~1 in 1000) with six loci developed

  • Mixture interpretation difficult with limited number of alleles per locus

Short Tandem Repeat Markers:

• The efforts of the Human Genome project have increased knowledge regarding the human genome, and hence there are many more STR loci available now than there were 25 years ago when the 13 CODIS core loci were originally selected

• More than 20,000 tetranucleotide STR loci have been characterized in the human genome

• STR sequences account for approximately 3% of the total human genome

• The FBI has selected 13(20) core STR loci that must be run in all DNA tests in order to provide a common currency with DNA profiles

• Cause of STRs:

  • A commonly observed replication error is “replication slippage”, which occurs at repetitive sequences when the new stands mis-pairs with the template strand (or visa versa)

  • The microsatellite polymorphism is mainly caused by this mechanism

  • If the mutation occurs in a coding region, it could produce abnormal proteins, leading to diseases

Silver-Stained STRs:

• Double-bands for each allele due to separation and detection of forward and reverse strands from each PCR product

• Advantages:

  • Sensitive due to PCR

  • Relatively rapid process (a day or two)

  • Works well with degraded DNA samples since shorter fragments can be analyzed

  • A lower start-up cost compared to fluorescent STRs

• Limitations:

  • Because only a single “colour” channel is available, multiplex amplification and detection is limited to 3-4 loci

  • Both strands of DNA are detected, leading to double bands with some loci that can complicate interpretation

Fluorescent STRs:

• PCR primers anneal to unique sequences bracketing the variable STR repeat region

• PCR product size generated

• Advantages:

  • Sensitive due to PCR

  • Relatively rapid process (a few hours to a day or two)

  • Works fairly well with degraded samples since shorter fragments of DNA can be analyzed

  • Multiplex PCR amplification and multi-colour fluorophore labeling and detection enables examining 15+ loci simultaneously. This provides powers of discrimination ~ 1 in billions or greater

  • Standardized sets of core loci are widely used with availability of commercial STR kits

  • Automated detection enables high-throughput sample processing

  • The potential number of loci is very large, which is important if siblings or other relatives are involved

• Limitations:

  • Less discrimination power per locus compared to VNTRs due to a smaller number of alleles and less heterozygosity per locus

  • The possibility of contamination from stray DNA is increased because of the PCR amplification process

  • Expensive equipment required for detection

  • Stutter products and unbalanced peak heights may occur and make the interpretation of mixtures more difficult

  • Data interpretation must account for the artifacts such as dye blob, electrophoretic spikes, ect

Lecture 2: Sample Collection and Storage, DNA Extraction

SAMPLE COLLECTION AND PRESERVATION

Evidence Collection and Preservation:

• O.J. Simpson case illustrates need for careful collection, documentation, on continuity of evidence, and validation of techniques

• Defense attacked the way evidence was collected and preserved

• Pamphlet: “What Every law Enforcement Officer Should Know About DNA Evidence”

• Prioritize sample collection

• Collect evidence samples, elimination, or known samples

• Protocols are in place to guard against errors still made in the field and in processing

• Best results with >100 cells, but DNA profiles can be recovered from as little as a single cell

Sources of Biological Evidence:

• Blood

• Semen

• Saliva

• Urine

• Hair

• Teeth

• Bone

• Tissue

The Grim Sleeper (1985-88, 2002-2007, California):

• 11 women murdered in California over a span of 15 years; DNA profiles generated from evidence linked to one individual

• Lonnie David was identified as a suspect using familial DNA testing

  • DNA profiles from the victims generated a partial profile that was similar to another profile (Christopher Franklin; son) in the California DNA database

• Law enforcement collected a discarded pizza slice (among other items) from Lonnie and DNA confirmed that profile matched the profile generated from crime scene evidence

Evidence Collected and Preservation Basics:

• Avoid contamination from collector (sneezing, coughing, not using clean gloves for each piece of evidence collected)

• Separately package evidence

• Air dry “wet” samples prior to packaging in paper. DO NOT use plastic (to avoid bacterial degradation of DNA or growth of mold)

• All samples must be carefully labeled and sealed

• Stains on unmovable surfaces: Swab with distilled water, air dry, place in envelope and take reference swab as a negative control

Unknown DNA Sample from Evidence:

• Cotton swabs commonly used to collect biological material from bloodstains or semen from sexual assault victims

• Cellulase can break down cotton swab fibers and release sperm cells that stick to cotton swab

• Cellular material can be collected from clothing using adhesive tape which cna be placed directly into a DNA extraction tube

• The amount of DNA needed has decreased dramatically in the past decade due to sensitivity of the PCR process

Reference DNA Sample from Suspect:

• To perform the Q-K comparative DNA test, a reference sample must be taken

• Blood samples may be collected (but not rapid or painless)

• Easier to collect a buccal swab from the inside of an individual’s mouth, which scrapes off some cheek cells (less invasive than drawing blood)

• Swab must be dried before storing and shipping to lab to avoid mold and bacterial growth

Storage of DNA Samples:

• DNA can be stored as non-extracted tissue, or fully extracted DNA

• DNA molecules are best stored dry (to prevent base hydrolysis) and cold to protect from DNA digesting enzymes (DNAses)

• Extracted DNA molecules can be stored:

  • Short-term in a fridge (4 degrees) or freezer (-20 degrees)

  • Long term in an ultralow freezer (-80 degrees)

Sample Characterization:

• Increasingly, cases come to court in which the presence of cellular material of a person is not disputed but the activity that caused the deposition is

• The debate centers around how did their DNA get there?

• Presumptive tests are performed to indicate whether biological fluids are present on an item of evidence

• Presumptive testing enables a sampling area to be selected from a piece of evidence

• Presumptive tests should be:

  • Simple

  • Inexpensive

  • Safe

  • Only use a small amount of material

  • Non-destructive

• Presumptive tests to identify sample source:

  • Blood stains: Serological methods (detect hemoglobin) or luminol (binds iron in hemoglobin)

  • Semen stains: Serological methods (detect acid phosphates) or direct observation of sperm (microscope)

  • Saliva stains: Serological method detect amylase

RNA TESTING OF FLUID SAMPLES

Body Fluid Identification with RNA Testing:

• Each cell type in the human body has a unique pattern of gene expression that is manifested by the presence and relative abundance of specific mRNA species, the molecular intermediate between DNA and expressed protein

• Some mRNA are selectively expressed in cells that collectively comprise a particular body fluid

• Modified extraction protocols could simultaneously isolate RNA and DNA from the same sample for different tests

• Advantages of RNA testing over conventional methods:

  • High sensitivity due to the possibility of PCR amplification

  • High specificity due to the pattern of gene expression unique tissue

  • Simultaneous DNA isolation without loss of material, if necessary

• mRNA markers have been identified for the most forensically relevant body fluids based on functional differences between the cells and tissues involved

• “Housekeeping” transcripts can be used as a positive RNA control

• Amplification of mRNA is detected via:

  • Real time PCR (probe based)

  • Capillary Electrophoresis (fluorescent-labelled amplicon) (primer based)

Co-Analysis System for Personal Identification:

• Arrangement of loci of the co-analysis system (PP16 STR markers in blue, green, and yellow dye channels)

  • mRNA markers in the red dye channel

• Results for STR and mRNA co-analysis:

  • STR profiling results (PP16, top three rows) for identity

  • Sample characterization indicates fluid type

DNA EXTRACTION METHODS

DNA Extraction:

• DNA is negatively charged

• DNA is tightly complexed with positively charged histone proteins

• Other molecules co-purify with DNA depending on the isolation method

• The general steps in any DNA extraction are:

  • Lyse cells and release DNA

  • Separate DNA from cellular material

  • Isolate DNA so that it can be used for downstream STR typing

  • Store DNA at -20 Celsius or -80 Celsius to prevent nuclease activity

• The goal is to maximize yield while minimizing contaminants and inhibitors

• The extraction process is where DNA is most susceptible to cross contamination in the lab

• Common PCR inhibitors:

  • Iron from RBCs

  • Ethanol

  • Minerals from bone

  • Jean and textile dyes

  • Melanin from hair

  • Proteins

• Nucleases degrade DNA, requiring magnesium and optimal activity at ~37 degrees celsius

• Hydrolytic cleavage of DNA is increased by high temperatures and high humidity, this can add “nicks” in the DNA template, which interfere with primer annealing or Taq polymerase

DNA Extraction Methods:

• Organic (phenol-chloroform)

• Solid-phase extraction methods:

  • Quiagen (silica bind/was/release with vacuum filtration or centrifugation)

  • DNA IQ and PrepFiler (silica bind/wash/release with magnetic bead capture)

• Chelex

• FTA paper

• Differential extraction:

  • Separation of non-sperm and sperm fractions based on absence or presence of DTT that breaks open the sperm cell coating

Lysing Cells and Releasing DNA:

• Dissolve tissue in buffer to break cells and nuclear membranes, disrupt DNA-protein complexes, bind multivalent ions

  • Urea disrupts hydrogen and hydrophobic bonds holding the cell membrane together

  • SDS is an ionic detergent that binds proteins and unfolds them to disrupt DNA-protein complexes

  • EDTA is a chelating agent that binds multivalent ions which are required for nuclease digestion of DNA

• Add Proteinase-K to digest proteins by breaking the peptide bonds to produce smaller peptides

  • proteinase-K activity is stimulated in the presence of detergents (SDS)

  • Enzymatic activity has an optimum temperature of ~65 degrees celsius for tertiary and quaternary protein structures and ~37 degrees celsius for primary and secondary structures

  • At these high temperatures, phosphodiester bonds in DNA can be broken and it should not be exposed to 65 degrees celsius for extended periods

• Separating DNA from cellular material (digested proteins)

  • Organic solvents remove proteins from nucleic acids

  • Solid support to bind DNA

  • Magnetic beads to bind DNA

  • Traditionally, organic solvents removed proteins by denaturing and precipitating them, where DNA is more soluble in the aqueous solution

  • DNA extracted via organic solvents is generally high molecular weight and double stranded, making it suitable for RFLP analysis or PCR

  • Other approaches take advantage of the ability of silica-oxide coated materials to bind DNA

Quiagen Extractions:

• Nucleic acids bind to a porous silicon-oxide coated membrane in the presence of chaotropic salts and ethanol

• Washing steps keep proteins and cellular debris in solution so they can flow through the column and be discarded

• The DNA is bound to the column on the membrane

• Under low salt conditions the DNA is eluted (released) into solution

Promega Extractions:

• Magnetic beads coated in silicon-oxide bind DNA

• Adding resin to lysate allows extraction to be performed in one tube, various solutions are used to wash the magnetic beads and a magnet holds DNA in place

• The amount of resin used is limiting to the amount of DNA captured

• The sample is incubated at 65 degrees Celsius in an elution buffer (TE) which releases DNA from the beads into solution

Chelex Extractions:

• 5% chelating resin is added directly to a sample of blood or semen

• Magnesium ions are drawn to and bound by resin

• Cells are lysed by boiling for several minutes

• A pellet of cellular debris and chelex resin is formed via centrifugation and the ssDNA is removed from the supernatant

FTA Paper Extractions:

• Blood added to FTA paper and left to dry

• Cellulose-based paper that lyses cells, binds white blood cells to paper’s matrix, protects DNA from nuclease activity, and deters bacterial growth

• The paper is stable at room temperature for several years

• A wash punch removes heme and other PCR inhibitors

• A clean punch can be added directly to PCR without quantification

• It is possible to perform chelex or solid support extractions on punches

Differential Extractions:

• Separate epithelial cells from sperm cells in sexual assault cases to facilitate STR interpretation

• A modified organic extraction that breaks open female epithelial cells

• Sperm cells are then lysed with DTT that breaks protein disulfide bridges of sperm cell walls

• Not useful for azoospermic sperm (vasectomy = no measurable levels of sperm)

Lecture 3: DNA Quantitation

Purpose of DNA quantitation:

• All sources of DNA are extracted when biological evidence from a crime scene is processed to isolate the DNA present

• 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 Standard 9.3 and FBI Quality Assurance Standard 9.4 requires human-specific DNA quantitation so that appropriate levels of human DNA can be included in the subsequent PCR amplification

• Higher quality data saves time and money!

The Importance of Quantitation:

• Estimate extraction method efficiency:

  • Methods yield different amounts and quality of DNA

• Multiplex STR typing works best with a narrow range of human DNA:

  • Typically 0.5 to 2.0 ng of input DNA for commercial STR kits

• It is especially important to dilute samples to a known concentration for use in PCR

  • Conserves DNA

  • Reduces the possibility of introducing inhibitors which may be in the extracted solution

• Too much DNA in multiplex PCR:

  • Off-scale peaks

  • Split peaks

  • Locus-to-locus imbalance

• Too little DNA in multiplex PCR:

  • Heterozygote peak imbalance

  • Allele drop-out

  • Locus-to-locus imbalance

• Normalization: The process of obtaining a DNA concentration suitable for STR analysis

Slot Blot Method:

• A primate specific oligonucleotide probe (40bp) binds to satellite sequence D17Z1

• DNA is bound to a nylon membrane and then probed

• The intensity of the signal from the probed DNA is compared to standards prepared via serial dilution

• Comparison is visual (subjective) but digital capture emerged in the early 2000s

• The test normally consumes 5 uL of DNA

• The test takes several hours, 30 samples per test, detects ssDNA and dsDNA down to 150 pg

UV Absorbance via Nanodrop/Spectrophotometer:

• Nucleic acids such as DNA and RNA absorb UV light at a maximal wavelength of 260 nm

• A linear relationship between the absorption of light and the concentration of nucleic acid in the cuvette

• We can use the Beer-Lambert equation to determine the concentration of DNA in a sample

• Poor limit of detection (~3.5 ng/uL)

• Not specific for human DNA and does not specify the type of DNA

• Contaminants such as proteins and phenol give false signals

PicoGreen Intercalating Dye Assays:

• Certain molecules bind dsDNA

• The inside of dsDNA molecules provides a hydrophobic environment that allows PicoGreen or other molecules to fluoresce differently than when they are in aqueous solution

• These molecules are excited by light and fluoresce in proportion to the amount of DNA, where stronger emissions indicate more DNA

• PicoGreen is automatable with 80 samples and 16 calibration samples in less than 30 minutes

• Uses a standard curve to convert fluorescence signal into the amount of DNA present in an unknown sample

• Not human specific and only dsDNA specific

PCR and Quantitative PCR:

• In PCR the products are analyzed after cycling is completed

• In qPCR the products are monitored as the PCR is occurring

• Once per thermal cycle, fluorescence is measured and recorded as a normalized reporter signal (Rn)

• The PCR is monitored during the exponential phase where the first significant increase in amount of PCR product correlates to the initial amount of target template

• Cycle Threshold (Ct): The number of cycles required for the fluorescent signal to exceed background levels (baseline noise)

• When PCR is close to 100% efficiency, doublind of amplicons occurs during each cycle

• Ct levels are inversely proportional to the amount of target nucleic acid in the sample

  • Plotting Ct vs log[DNA] should result in a linear relationship with a negative slope

  • Low Ct = greater amount of nucleic

• Two main types of qPCR assays:

  • Fluorogenic 5’ nuclease TaqMan assay with two primers and a fluorescent probe (very specific)

  • Inter-chelating dye SYBR green with two primers (less specific)

  • Both assays quantify DNA based on crossing a cycle threshold Ct relative to the # of PCR cycles

  • Reflect both quantity and quality (“amplifiability”) of DNA for subsequent STR typing processes

  • Both assays use a fluorescent reporter whose signal increases in direct proportion to the amount of PCR product in the reaction

TaqMan Assay:

• During PCR the TaqMan MGB probe anneals specifically to a complementary sequence between the forward and reverse primer sites

• The minor groove binder at the 3’ end of the probe enables the use of shorter probes that still have higher melting temperatures

• The probe is designed to have a higher Tm than the primers so that it remains hybridized during polymerization

• 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

• Energy from the reporter is absorbed by the quencher but re-emitted as heat rather than light

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

• Cleavage separates the reporter dye to the quencher dye which increases the fluorescent signal

SYBR Green:

• Reaction chemistry:

  • SYBR Green dye fluoresces when bound to dsDNA

• Denaturation:

  • When DNA is denatured, SYBR green dye is released, and fluorescence is reduced

• Polymerization:

  • PCR products are amplified

• Polymerization complete:

  • 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, but IPC can help

• <100 pg qPCR subject to variability and uncertainty

• In highly degraded samples, assays that amplify short target sequences will detect and measure more DNA than assays that amplify long target sequences (nicks between primers)

• Accurate quantitation assumes that each unknown sample is amplified at the same efficiency as the calibrant samples in the dilution series

qPCR for Human Quantitation:

• Designed to simultaneously quantify the total amount of amplifiable human DNA and human male DNA in a sample?

• If the sample contains enough human DNA and/or human male DNA to proceed with STR analysis

• The relative quantities of human male and female DNA in a sample that can assist in the selection of the applicable STR chemistry (Identifiler vs Y-Filer)

• If PCR inhibitors are present in a sample that may require additional purification before proceeding to STR analysis

Quantifiler Duo DNA Quantification Kit:

• The target-specific assays consist of:

  • Two primers to amplify human DNA + One TaqMan probe labeled with VIC dye for detecting the amplified human target sequence

  • Two primers to amplify human male DNA + One TaqMan probe labeled with FAM dye for detecting the human male amplified target sequence

• The Internal PCR Control assay:

  • Two primers to amplify a synthetic sequence not found in nature + One TaqMan probe labelled with NED dye for detecting the IPC DNA

• Polymerization and strand displacement

• Probe cleavage (release of reporter dye)

• Fluorescence occurs when reporter dye and quencher dye are no longer in close proximity

• Completion of polymerization

• Provides the quantity of human and male DNA in biological samples. From these values, one can calculate the ratio of male and female DNA in a mixture using the following equation:

  • Male DNA : Female DNA Ratio = Male DNA/Male DNA : (Human DNA - Male DNA)/Male DNA

• All quantities in the above equation are ng/μL

  • For example, assuming: Male DNA concentration = 2 ng/ul and the Human DNA concentration = 8 ng/ul, then the Male DNA:Female DNA ratio is 2/2:(8-2)/2 = 1:3

  • This ratio determines the extent of the mixture and is useful in determining whether to proceed with autosomal STR or Y STR analysis….. but there are other kits as well!!

Passive Reference Dyes:

• Passive reference dyes (ROX) used to normalize well-to-well fluorescence signal differences

  • variation in the optical paths between wells

  • minor differences in volumes due to pipetting errors

• Wells in the center of the thermal cycler block have a shorter light path and they emit higher fluorescence signals compared to wells on the perimeter of the thermal cycler block that have a longer light path

• During qPCR data processing, the sample fluorescence for each well are corrected using differences in the fluorescence of the passive reference to normalize the reporter signals

Microsatellites (STRs):

• Millions of micros in human genome (3%); ~ every 10,000bp

• Some microsatellite regions (or STRs) useful for identification because # of repetitive motifs varies between individuals!

Types of Short Tandem Repeats:

• Requires size-based DNA separation to resolve different alleles from one another

  • Dinucleotide (CA)(CA)(CA)(CA)

  • Trinucleotide (GCC)(GCC)(GCC)

  • Tetranucleotide (AATG)(AATG)(AATG)

  • Pentanucleotide (AGAAA)(AGAAA)

  • Hexanucleotide (AGTACA)(AGTACA)

• Currently, tetranucleotides most commonly used in forensics for technical reasons to be discussed

• Short tandem repeat (STR) = microsatellite = simple sequence repeat (SSR)

R CRITERIA FOR FORENSIC APPLICATIONS

• High discriminating power (i.e., high # of alleles)

• High percentage of heterozygotes (e.g., HE > 70%) that amplify similarly well within/between individuals

• Separate chromosomal locations to avoid linkage of loci (linkage would invalidate statistics of ‘product rule’ used)

• Narrow allele range (100 - 400 bp) to amplify well in degraded DNA samples

• Work in combination with other micros (multiplexing)

• (Relatively) low mutation rate (i.e., frequencies do not change over time)

• Low level of biological artifacts (i.e., stutter – more later!)

STR ALLELE NOMENCLATURE

• Nomenclature developed so labs can communicate with one another

• Define motifs using the first repeat you come across on the 5’ strand

  • Depending on strand you arrive with very different names and repeats

  • If STR is in protein coding region, coding strand should be used (e.g., TPOX)

  • Name according to placement near a gene or chr number

• Microvariants (alleles with a partial repeat) named by # of complete repeats and then the number of nucleotides in the partial repeat

  • e.g., TPOX allele 9.3 is (AATG)9 (ATG)

  • Standards set by International Society of Forensic Genetics (ISFG)

STRS IN FORENSIC APPLICATIONS

• Small product sizes are generally compatible with degraded DNA and PCR enables recovery of profile from small amounts of biological material

• Multiplex amplification with fluorescence detection enables high power of discrimination one test

• Commercially available in easy-to-use kit formats

• Uniform set of core STR loci provide capability for national and international sharing of criminal DNA profiles

THE CODIS STR LOCI

• 13 core STRs selected in 1997 to form basis of the National DNA Database in the United States known as CODIS (Combined DNA Index System)

  • As of January 1, 2017, the FBI required an additional 7 STR loci for uploading DNA profiles to the National DNA Index System (NDIS).

COMMERCIAL STR KITS

• STR kits vary based on:

  • STR loci amplified

  • Fluorescent dye combinations

  • DNA-strand labelled

  • Allelic ladder included in the kit

  • Primer sequence utilized for PCR amplification

MULTIPLEX PCR: REACTION SETUP

• DNA sample is added (~1 ng total based on DNA quantitation) – 10 µL possible

• PCR primers and other reaction chemicals from an STR typing kit are added – 15 µL

WHAT IS IN A STR TYPING KIT

• Kit Components:

  • Primer mix

  • PCR buffer (MgCl2 and dNTPs)

  • DNA polymerase (e.g., AmpliTaq Gold)

  • Allelic ladder

  • Positive control

• Common kits used:

  • Profiler Plus/COfiler (Applied Biosystems)

  • Identifiler (Applied Biosystems)

  • PowerPlex 16 (Promega)

• Primer mix contains fluorescently labeled oligonucleotides used to amplify specific STRs in the human genome

  • Applied Biosystems has not published their primer sequences

  • PowerPlex 16 (Promega), which amplifies 16 genomic regions, contains 32 PCR primers

ALLELIC LADDERS

• Mix common alleles together to create a reference for scoring alleles in unknown samples

• Generated using same primers used for sample amplification!

• Different genetic analyzer platforms and running conditions can lead to different mobility; therefore, crucial

• Measurement (genotype determination) is performed by comparing allele size (relative to an internal size standard) to a commercially provided STR kit allelic ladder with calibrated repeat numbers (sized according to the same internal size standard)

• An internal size standard is run with each sample and external standard to correlate sizes

STR TYPING

• Alleles designated by comparing sized peaks from allelic ladder “bins” (+/- 0.5 bp) to PCR-amplified samples if they have similar dye colours

• Internal size standard in all samples enables comparison!

ALLELIC OVERLAP: SOLUTIONS

• Multiplexing originally designed to avoid overlap of alleles, but as more loci were included in multiplexes, it was more difficult to avoid overlap

  • Use more dyes when possible (e.g., 4 vs 5)

  • Add non-nucleotide linkers to change mobility of PCR product (e.g., hexaethyleneoxide; HEO, product runs 2.5bp slower per unit), which allows continued use of validated primers (important advantage!)

  • Redesign primers and amplify varying amounts of flanking sequence

AmpFISTR Identifier Kit Innovations

• COfiler Kit

• Identifier Kit

PowerPlex16 Kit innovations

• Altering primers = be aware of potential null alleles

• PowerPlex 1.1 kit

• Powerplex 16 kit

SEX DETERMINATION: AMELOGENIN

• Sample donor sex critically important for context (e.g., sexual assault)

• Amelogenin encodes gene for tooth enamel (on X and Y)

• Primers flank a 6bp deletion in intron 1 of the gene

• PCR amplicon size varies based on the kit used

• X amplicon acts as internal positive control for reaction

• Peak height of amelogenin amplification can be used to determine relative amounts of female:male DNA in a mixed DNA sample

• Example

  • Example: X = 21,000 RFU and Y = 7,000 RFU?

  • Female (X,X) RFU = 21,000 – 7000 = 14,000

  • Female (X) RFU = 14,000 / 2 = 7,000

  • Therefore, female:male DNA ratio = 7,000:7,000 OR 1:1

• Technical issues: preferential amplification of X (smaller amplificon)?

• Problem 1: rare deletion of gene on Y-chr (6/30,000 males) yields no Y-amplification and a false result of female (Y allele dropout)

• Problem 2: rare mutations in primer binding sites (3/7,000 males) yields no X-chr amplification (X allele dropout)

Lecture 4: DNA Amplification (PCR)

THE POLYMERASE CHAIN REACTION

History and Application of PCR:

• First described by Mullins in 1985 (Cetus Corp)

• Revolutionized molecular biology, awarded with the Nobel Prize in 1993

• Allows a DNA template to be quickly and reliably amplified to greater than 10^9 copies starting with small amounts of DNA

• Previous methods extremely laborious to get enough DNA template for visualization

Advantages of PCR:

• Very small amounts of DNA template may be used

• DNA degraded to fragments only a few hundred base pairs in length can serve as effective templates for amplification

• Large numbers of copies can be amplified simultaneously

• Contaminant DNA, such as fungal and bacterial sources, will not amplify because of human-specific primers used

• Commercial kits available

Disadvantages of PCR:

• The target DNA template may not amplify due to the presence of PCR inhibitors

• Amplification may fail due to sequence changes in primer-binding region of the genomic DNA template

• Contamination from other human DNA sources besides the forensic evidence or previously amplified samples is possible without careful laboratory technique and validated protocols

PCR Cycles:

• Three distinct events:

  • Denature template

  • Primer annealing

  • DNA synthesis by a thermostable polymerase

• Denaturation of the template:

  • Heat reaction to 95-98 degrees Celsius

  • DNA becomes single stranded, to which primers can anneal

• Primer annealing:

  • Primers hybridize to complementary ssDNA as reaction cools to 45-65 degrees Celsius, dependent on the base composition of primers

  • A much greater concentration of primers than target DNA is used, they preferentially hybridize to their complementary sequences

  • Primers are “consumed” (used up) in the cycle

• DNA synthesis:

  • Extension of primers by thermostable DNA polymerase at 72 degrees Celsius

  • Time required to copy template depends on length of PCR product (~ 1 min/kb)

• The cycles are repeated ~28-30 times to give rise to millions of copies of the target DNA sequence

PCR Raw Materials:

• Template DNA

• Thermostable DNA polymerase

• Oligonucleotide primers

• Deoxynucleotide triphosphates (dNTPs)

• Magnesium ions

• Reaction buffer

• Optional additives

Template DNA:

• 0.5-10 ng of template DNA in the majority of PCR reactions

  • Assuming 3.2 X 10^9 bp weights ~ 3.3 X 10^-12 g

  • Then, 0.5-10 ng of genomic DNA corresponds to ~150-3000 copies of each desired region

  • A diploid cell contains 6.6 pg DNA, therefore 0.5 ng nuDNA = 500 pg = 76 diploid cells = 152 copies of each allele

  • 10 ng nuDNA = 10,000 pg = 1515 diploid cells = 3030 copies of each allele

  • Plasmid DNA, mtDNA, and chloroplast DNA are much smaller genomes and therefore less mass of template DNA is required to provide the same number of copies

  • Stochastic sampling occurs with low template amounts, where unequal sampling of two alleles present in a heterozygous individual happens by change

    • Results in allele imbalance, allele dropout, or complete locus dropout

    • Not an accurate reflection of the original DNA sample

Thermostable DNA Polymerase:

• Originally Taq DNA polymerase isolated from hot spring bacterium Thermus aquaticus

• Taq is used because of its thermostability

• Taq concentration of 0.05 units/ul in each reaction

• Taq can incorporate errors

• Other polymerases considered in specific situations based on error rate or efficiency

  • An example is AmpliTaq Gold

Oligonucleotide Primers:

• Must be specific to their target region, possess similar annealing temperatures, and not interact significantly themselves (hairpin structures)

• Programs: Oligo, Primer3, Primer Express

• Reverse primers are the reverse complement of the target sequence

• Reasons for limiting the size of a primer:

  • We want to be specific

  • We want to work within an optimal temperature range

• Maximum copy number of amplified target sequence is 1E+12, so we need more copies of primer than amplified product

• Trade-off between maximizing desired product produced and minimizing off-target products

• Consider primer design that maximizes non-template-dependent “A” addition to PCR products

• See lecture slides for calculation samples

Magnesium Ions:

• [Mg 2+] is a crucial factor affecting the performance of Taq and stringency of primer annealing

• They allow the template and primers to bind one another by alleviating the repulsion of negative charges

  • Cofactor in enzymatic reaction of DNA polymerization and without adequate free magnesium, Taq is inactive

  • Mg2+ neutralizes repulsion between negatively charged DNA strands (templates and primers)

• Reaction components, including chelating agents present in sample (EDTA) or proteins can reduce the amount of free magnesium

• Low [Mg 2+]: Repulsion of backbones of primer and template will be stronger, therefore melting will occur at low temperature

• High [Mg 2+]: Neutralizes repulsion of primer and template backbones, therefore melting will occur at higher temperatures

• Overall, there is a trade-off between decreasing primer binding to non-desirable regions, while allowing primers to bind to the desired targets

• Very small differences in [Mg 2+] can result in changes to quality and quantity of amplified product

• Optimal [Mg 2+] will be locus specific, but 1.5 mM concentration is standard and should never exceed 3.0 mM

Optimal Additives:

• Bovine Serum Albumin (BSA) bind potential PCR inhibitors such as protein and phenol

  • BSA can act as a chelating agent when it is in excess

Solutions to PCR Inhibitors:

• Dilute template DNA, which also dilutes the PCR inhibitor

• Add more Taq DNA polymerase to overcome the inhibitor, where some Taq binds inhibiting molecules and the remaining Taq amplifies the template

• Add optional additives (BSA) to minimize inhibition

• Purify/wash the template (spin column, ethanol precipitation) to remove inhibitors

STANDARD PROCEDURES IN THE PCR CYCLE

Denaturing Time and Temperature:

• Generally, there is a long initial denaturing step at 94 degrees celsius (5-20 mins) to completely dissociate the template strands

• 30 second denaturing steps are used within the cycles as templates are much shorter

Annealing Time and Temperature:

• Small molecules are more likely to form ionic bonds with the targeted annealing site

• Annealing time is short to allow primers to bind in the correct position while limiting the chances of primer mis-binding

• Annealing time is often kept stable at 1 minute through PCR cycles

• Optimizing annealing temperature is the first and most important step in optimizing a PCR reaction

• Primers with more hydrogen bonds (based on length or GC content) require higher temperatures to melt off the template

• The optimal annealing temperature is high enough to melt the primer off while being low enough to allow sequence-specific primer binding

Extension Time and Temperature:

• The extension temperature of Taq DNA polymerase is 72 degrees Celsius

• Taq synthesizes 1000bp per minute

• PCR products under 500bp do not require much time to complete synthesis, 30 seconds is enough

• Generally, extension runs 1 minute per cycle

• A final extension step at 60 degrees celsius runs for 45 minutes to ensure that all products are fully extended

• Additional adenosine is added to the end of PCR products to make scoring easier

Cycle Number:

• The PCR cycle number is generally around 30 because it will max out after this

  • Reagents become a limiting factor in PCR amplification

• The exponential plateau happens around 30 cycles (1E +11 to 1E +12 copies)

• The cycle number is only increased when starting template amounts are low

• The tradeoff of using too many cycles is that undesired regions may be amplified and contaminants may become present

Touchdown PCR:

• A ~50 cycle run at incrementally lower temperatures

• The initial temperature is higher than the primer melting temperature and gradually declines

• The first primer-template hybridization events are stringent, keeping undesired amplification low

Hot Start PCR:

• Taq polymerase is added at a higher temperature to minimize the effects of mis-priming

• Combats primers binding to non-target regions at room temperature, causing contamination of the PCR product

Hot Start Taq:

• Modified AmpliTaq Gold polymerase is only active at high temperatures

• The Taq polymerase must be “shocked” into activity at 95 degrees Celsius

• Eliminates the accumulation of non-specific product

PCR Controls:

• Controls provide perspective to interpret experimental results and guide troubleshooting

• Negative control:

  • Contents of the reaction mix and no DNA sample

  • No product formation should be observed other than primer-dimers

  • Product = contamination

• Positive control:

  • Includes a DNA sample with a known profile

  • If the sample fails to amplify, there is something wrong with the reaction mix

  • The Identifiler STR genotyping kit includes positive control DNA 9947A

• Primer control:

  • Specific uses for human forensic biology labs

  • Amplifying and sequencing mtDNA hypervariable regions

  • This control determines if a problem arose due to the primers used

  • Can signal degradation of primers used

Troubleshooting Scenario Practice:

• Positive and primer-control amplified, negative control did not amplify. Most samples amplified but some did not:

  • PCR inhibitors present

• Positive and negative control did not amplify but primer control did:

  • Degraded primers

• Positive, negative, and primer-control did not amplify:

  • Problems in reaction mix

• Positive, negative, and primer control amplified:

  • Contamination present in samples

• Avoid changing multiple conditions at once. Rule out the source of a problem with one troubleshooting run at a time.

Multiplex PCR:

• Amplifying many loci in one reaction

• Difficult to avoid primer-dimer formation if sequences are similar

• Primers must have similar annealing temperatures

• Primer and magnesium ion concentrations are adjusted accordingly

• Amplicons should not overlap in size if they are not labeled with different dyes

Week 6: DNA Separation & Detection

ELECTROPHORESIS

• Goal: Separate DNA (STRs) by size due to negative charge.

• STR typing requires:

1. Spatial resolution (separate STRs and loci)

2. Separate resolution (separate fluorescent dyes)

3. Sizing precision (run-run consistency; we use allelic ladders)

• Larger DNA molecules interact more frequently with the gel and are thus retarted in their migration through the gel.

Gel Electrophoresis System

• An electric field is applied such that the negatively charged DNA molecules migrate away from a negative (cathode) towards a positive charge (anode).

• DNA moves towards positive charge and separates based on size; with smaller DNA molecules traveling faster than larger DNA molecules.

• DNA molecules can be loaded into the wells of slab gels that consist of a microporous matric through which the DNA must pass through.

• The amount of sample loaded into the gel is dependent on the width of the tooth in a comb and the depth the comb sits in the gel.

• Gel is submerged in a tank filled with electrophoresis buffer (eg. Tris-acetate-EDTA) and DA samples are loaded into the top of the well.

• The number of samples you can run on a gel is dictated by the size of the gel and the number of the wells you have.

Loading Dyes And Gels

• Sample are mixed with a loading dye

  • Tracking dye (eg: bromophenol blue) monitors migration.

  • Sucrose that binds DNA, increases its viscosity, and pulls the DNA down into well.

• There are two types of gels used in molecular biology; agarose and polyacrylamide gels (PAGE), which differ based on the pore size.

  • Agarose = larger size which resolves large DNA

  • PAGE = smaller pore size which resolves smaller DNA molecules (<1000 bp)

• Therefore, PCR-amplified sTR alleles (100-400 bp) are better resolved by PAGE.

• However, PAGE is time consuming, not easily automated and acrylamide is neurotoxin.

Native vs Denaturing Conditions

• DNA can run through the pores of a gel as:

  • ds DNA in native/non-denaturing conditions, or as ssDNA under denaturing conditions

• Generally, better resolution between closely sized DNA molecules occurs under denaturing conditions because a ssDNA molecule is more flexible as it moves through the matrix.

• Chemicals such as formamide or urea can be added to the DNA sample where they interact with NDA and interfere with the formation of hydrogen bonds between complementary ssDNA molecules

Capillary Electrophoresis (CE)

• The general component of CE include sample injection, separation, and detection of STR alle.s

• Caps are made of glass or fused silica with a diameter of ~50 um (length 36-80 cm).

• Caps contain viscous polymer rather than gel matrix to separate DNA. New polymer added for each run.

• Longer capillary = higher resolution.

• *Sample tray moves automatically beneath the cathode end of the capillary to deliver each sample in succession.

• Capillary Electrophoresis Advantages:

  • Fully automated, no need to individually load samples, don’t need to pour gels (electrokinetic injection of samples)

  • Only a subset of the amped sample used, can be re-tested.

  • Faster: higher voltages due to enhanced heat dissipation (300 V/cm vs 10 V/cm)

  • Electronic output: no need for gel pictures nor scanning gel.

  • No lane tracking, enclosed cap mea no lane bleed through, less cross-contamination

• Capillary Electrophoresis disadvantages:

  • Each cap can only process one sample at a time, sequential injections (so 4, 16, or 96 caps).

  • Expensive to buy and maintain.

  • Salts, unwanted DNA can out-compete PCR products for sample injection.

Electrokinetic Injection

• Voltage is applied to a liquid sample to introduce DNA into the capillary.

• DNA molecules are negatively charged, and positive voltage draws DNA into the capillary.

• CE injected DNA is extremely sensitive to contaminating small molecules with a negative charge (eg; chloride from PCR) that “out-compete” the larger DNA molecules (the solution is PCR purification).

FLUORESCENT DETECTION

• Most platforms are based on fluorescence detection that excites dye molecules and then detects light emitted.

• Fluorescent dye is attached to the premier unlike DNA sequencing (FL-ddNTPS)

• Dyes are incorporated into amplicon via PCR. Two or more dyes can be separated using optical filters.

• DNA is visualized with a charge-coupled device (CCD)

• How it works:

  • Unlabeled DNA → intercalator inserts between base pairs on double stranded DNA→ DNA is then labeled with intercalating dye (SYBR green) → fluorescent dNTPs are incorporated into both strands of PCR product → fluorescent dye labeled primer is attached → the one strand of PCR product is labeled with fluorescence dye.

Fluorescence

• Argon ion lasers (488 or 514.5 mm) excite fluorophore (dye) attached to amplified DNA.

• Fluorophore absorbs laser photon energy and emits light at lower energy (higher wavelength (𝜆))

• Filters are used to collect only emitted light at a specific 𝜆.

• Filters detect multiple fluorophores at once using fluorophore separation algorithms called a matrix.

• CCD collect and amplify the signal from the fluorophore and convert it to an electronic signal (relative fluorescence unit; FRI)

Fluorescent Dyes

• Promega PowerPlex 16 (4C matrix)

  • Fluorescein (blue), JOE (green), TAMRA (yellow; black visual), ROX (red; internal size standard)

• Promega PowerPlex Fusion (5C matrix)

  • Fluorescein (blue), JOE (green), TMR-ET (yellow; black visual), CXR-ET (red), WEN (orange; ISS)

• Identifiler and Identifiler Plus (5C matrix)

  • 6FAM (blue), VIC (green), NED (yellow; black visual), PET (red), LIZ (orange; ISS)

• Dyes used depend on the system you have, the filters your instrument has, and the software you have.

SPECTRAL CALIBRATION

• Filters set to detect emission spectra of each dye; the spectra overlap to a degree.

• Spectral overlap is removed by applying a matrix where samples labeled with a single due are used to create a calibration file that shows spectral overlap between different dyes,.

• If matrix is not optimized, you can observe “pull-up” (eg; green peaks showing up under blue peaks, or vice versa).

STR Data: ABI Prism 310

• Red-labeled peaks are from the internal sizing standard GS500-ROX..

SAMPLE PREPARATION & INJECTION

• DNA from PCR is prepped in the following way (1:10) dilution:

  • 1 ul PCR product (or allelic ladder – once per plate).

  • 8.7 ul of deionized formamide (denatures DA and dilutes salts)

  • 0.3 ul of internal lane standard (eg. GS500-ROX).

  • Heat for 2 minutes at 95C, the palace on ice (“snap-cooling”). Add plate onto genetic analyzer.

• DNA injected into cap via electrokinetic injection, begins with pre-injection of samples (Eg. 15kV for 5 sec)

• Injectio is a competitive process (sample vs salts), and the amount of DNA injected is inversely proportional to the ionic strength of the sample.

DILUTION OF DNA SAMPLES

• Following PCR, a small portion of the sample is transferred for analysis and diluted in formamide.

• This aliquot of the sample is mixed with a molecule size marker (termed an internal size standard) that permits calibration of sizing measurements.

• Samples plates spun down via centrifuge:

  • Samples plates are spun to remove bubbles that would interfere with the injection (loading) process onto the capillary electrophoresis instrument.

• ABI 3130xl: Data Collection

  • Data analysis is performed on an Applied Biosystems (ABI) 3130xl capillary electrophoresis instrument.

SAMPLE PROCESSING SUMMARY

• Replace capillary, refill syringe w polymer solution, fill buffer vials → performed only once per batch of ~96 samples

• Prepare samples (denature, cool, ad mix with size standard)

• Prepare sample sheet and injection list → allelic ladder every tenth injection.

• Automated sample injection, Electrophoresis and Data collection.

• Size DNA fragments → GeneScan software Genotype STR alleles → GeneScan software

• Perform Data analysis → manually inspect the data

• ELECTROPHORESIS and DETECTION steps are simultaneous.

STR GENOTYPES VS STR PROFILES

• An STR genotype is the allele (homozygote) or alleles (heterozygote) present for a particular locus.

• An STR profile is preceded by combining all the STR genotypes (CODIS 13/20).

• Individuals will differ from one another in terms of their STR profile, but not necessarily at a single STR genotype.

• Steps involved in STR Genotyping:

  • Data collection → colour separation → peak identification → peak sizing → comparison to allelic ladder → genotype assignment to alleles → peak editing to remove artifacts calls → data review by analyst → confirmation of results by second analysts

DATA COLLECTION

• Four dimensions of data are collected by the CCD detector:

  • capillary position (x-axis)

  • wavelength of light across spectrum (y-axis)

  • intensity of light at specific wavelengths (z-axis)

  • time (t-axis)

• The analysis software then synthesizes an electropherogram of the STR PCR products for each sample by connecting thousands of CCD frames

PEAK DETECTION THRESHOLD

• 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 instrument

• Detection thresholds typically vary from 50 RFU to 200 RFU

• Analytical Threshold 50 RFUs LOD

  • Peak not considered reliable

• In between threshold

  • Peak reliable, but only used for exclusions

• Interpretation Threshold (stochastic) 150 RFUs LOQ

  • Peak reliable, can be used for inclusions

SIZING ALGORITHM AND INTERNAL STANDARDS

• How accurate are peak sizes that fall near edge of the region define by internal size standard?

• Local Southern Method

  • DNA Fragment peaks are sized based on the curve produced from the points on the internal standard

OVERVIEW OF STR TYPING

COMPARISON OF ALLELIC LADDER TO SAMPLES TO CONVERT SIZE INTO ALLELE REPEAT NUMBER

MICROVARIANT “OFF-LADDER” ALLELES

• Defined as alleles that have a form of sequence variation compared to more commonly observed alleles

• Do not size the same as common alleles (“off-ladder”)

• Alleles with partial repeat units are designated by the number of full repeats and then a decimal point followed by the number of bases in the partial repeat

• Example: TH01 9.3 allele: [TCAT]4 -CAT [TCAT]5

• Microvariants are common and sequence variation can occur in flanking regions as well!

Microvariant Allele calculations

• Relative size difference between the sample alleles and ladder alleles can be used to determine whether a microvariant exists

• Sample Allele 25 = -0.12nt

• Off-ladder allele = +0.87nt

• Relative peak shift = 0.99nt

• Therefore, off-ladder allele is 1 nt larger than ladder allele 28 and is designated 28.1

FACTORS AFFECTING GENOTYPING

• Matrix file

• Internal size standard

• Allelic ladder sample

• Degraded dna

• Micture dna

NON-ALLELIC PEAKS

• Not all data represents alleles from the sample!

• Non-allelic peaks may be:

  • PCR artifacts (e.g. stutter, non-template dependent nucleotide addition, and non-specific amplification products),

  • analytical artifacts (e.g. spikes and raised baseline),

  • instrumental limitations (e.g. incomplete spectral separation resulting in pull-up or bleed-through),

  • or may be introduced into the process (e.g. disassociated primer dyes resulting in a dye blob)

STUTTER PRODUCTS

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

• E.g., Y-STR (single source) should have one peak; used CTT repeat to exaggerate the results!

• Each successive stutter product is less intense (allele > repeat-1 > repeat-2 > repeat-3)

• Stutter less pronounced with larger repeat unit sizes (di- > tri- > tetra- > penta-nucleotides)

Stutter Product Information

• Repeat unit bulges out when strand slippage occurs during replication

• Forward stutter is RARE

  • Typically <2% of allele in tetranucleotide repeat STR loci

• Reverse stutter

  • Typically 5-15% of allele in tetranucleotide repeat STR loci

• Rpeat unit deletion

  • Caused by slippage on the copied (bottom strand)

• Repeat unit insertion

  • Caused by slippage of the copying (top) strand

STUTTER PEAK IMPLICATIONS

• Difficult to discern stutter from alleles when numerous sources (mixtures) contribute to DNA profile because stutter has same size as potential alleles; especially problematic if you have a minor contributor of DNA

• DNA labs normally quantify stutter in % relative to taller allele peak height

• What might tip you off that you have a minor contributor and not just stutter (hint: remember you are using 16 loci)?

STUTTER TRENDS AND PRINCIPLES

• Quantity of stutter depends on locus as well as PCR conditions and polymerase used

• Quantity of stutter is greater for longer alleles within a locus

• Typically, less than 15% of corresponding allele peak height

• Quantity of stutter is less if the sequence of core repeats is interrupted (e.g. compound repeat)

• Stutter amount increases when amplifying low levels of DNA template due to stochastic effects

STUTTER FOMRATION SOLUTIONS

• Longer (bp) repeat motifs (not longer # of repeats) have less stutter as do; so pentas (e.g. Penta A-G) normally have around 1% stutter (two pentas used in PowerPlex16)

• STRs with imperfect (compound) repeats have less stutter than STRs with simple repeats

• STRs with interrupted motifs (e.g., TH01 allele 9.3) have less stutter that STRs with un-interrupted motifs

• Thermocycling conditions can also influence stutter intensity (lowering annealing and extension temperature)

• Use Taq DNA polymerases that extend template faster; therefore, less time for loop formation (slippage)

NON-TEMPLATE ADDITION

• Taq DNA polymerase will often add an extra nucleotide to the end of a PCR product; most often an “A” (termed “adenylation”, or “A+”)

• If forward primer is dye-labelled, adenylation is influenced by sequence of the 5’-end of reverse primer (e.g., “G” can be put at the end of a primer to promote non-template addition)

• A+ enhanced with extension “soak” at end of PCR cycle (15- 60 min @ 60 or 72 oC) – to give polymerase more time

• Excess amounts of DNA template in the PCR can result in incomplete adenylation (not enough polymerase at end)

• Allelic and PCR products must be in the same form for easier interpretation by the analyst!

NON-TEMPLATE ADDITION SOLUTIONS

• It is best if there is not a mixture of “+/- A” peaks to improve the likelihood of a correct genotype call

TRI-ALLELIC PATTERNS

• Three alleles can be observed at a locus in a single-source DNA that are not always a result of DNA sample mixtures

• Result from extra chromosome fragments being present in a sample that produce an additional PCR product

• Likely to occur within a 15-locus STR profile about once every 1,000 samples and their distribution is not even

• How would you discern a 3-allele pattern from a mixture or a chromosomal duplication?

NULL ALELLES

• Allele is present in the DNA sample but fails to be amplified due to a nucleotide change in a primer binding site

• Mutations in primer binding sites can result in primers not annealing and no product for that allele

• Flanking regions where primers anneal, not as subject to mutation as STR repeats, should not happen that often

• Null alleles are a problem because a heterozygous sample appears falsely as a homozygote

Null Alleles and Sequence Variation

• Sequence variation within or around STR regions effect PCR amplification efficiency differently!

• Null alleles are rare events because mutation rate in flanking sequences is low

Null Allele Primer Concordance Studies

Null Allele Solutions

• Primer redesign

• Drop the locus from the STR multiplex

• Degenerate primer: include a primer with the base change so multiple primers are used to amplify the template

• Re-amplify sample with lower annealing temperature (reduces primer annealing stringency)

• Reduce match stringency (e.g., moderate stringency of 25/26 alleles) to account for variation between labs when searching a national database

• Although null alleles occur, as long as the same primers and conditions are used to amplify Q and K samples, the resulting profile should be the same!

DECIPHERING ARTIFACTS FROM THE TRUE ALLELES

• Manual inspections of allele calls can be subjective/based on personal bias, need two reads of each gel and genotypes must agree prior to report