PCR Amplification

• PCR is defined as the exponential amplification or replication of specific DNA sequences or targets
• PCR uses a thermal cycler to amplify DNA through temperature phases

Steps in PCR

• Master mix is prepared first
• TE buffer is added to adjust the sample volume
• Template DNA is added from an extract tube
• Positive and negative amplification controls are prepared
• DNA is amplified

Master Mix Components

• Determine the number of samples plus two controls, including two sample overages
• Combine reaction mix with primer mix, vortexing and pulse spinning each tube before combining
• Vortex the master mix and distribute it into each labeled tube

Reaction Mix

• dNTPs serve as the substrate for replication
• Buffer is added to maintain a specific pH
• Taq polymerase is used for its heat resistance
• MgCl and KCl are included
• Bovine serum albumin inhibits inhibitors by binding to organic materials

Primer Mix

• Contains oligonucleotides complementary to a specific sequence

PCR Process Steps

• Step 1: Denaturing occurs at 95°C
• Step 2: Annealing occurs at 55°C
• Step 3: Elongation occurs at 72°C
• Exponential growth of DNA occurs during amplification

PCR End Product Estimation

• Use the formula: Xn = Xo(1 + E)^(N-2)
• Xn is the predicted number of target molecules
• Xo is the number of starting materials
• E is the reaction efficiency
• N is the number of cycles

PCR Multiplexing

• Simultaneous amplification of multiple DNA regions in a single reaction
• Increases discrimination power
• Each region requires its own primer set
• Affects PCR reaction by increasing the amount of reagents like Taq, MgCl, KCl, and dNTPs
• Requires increased extension times
• Needs more dyes for separation and detection

Optimizing PCR: Primer Design

• Optimal primer length is 15-25 base pairs
• Primers that are too long might not bind sufficiently
• Primers that are too short might lack site specificity

Melting Temperature

• Optimal melting temperature is +/- 5°C for forward and reverse primer sets
• Optimal GC content is 40-60
• GC content affects temperature due to extra hydrogen bonds
• pH, salt concentrations, and molecule length also affect temperature

Primer Dissociation

• Primers may dissociate and fail to bind if the temperature is incorrect

Avoiding Secondary Structures

• Types of secondary structures include self-complementary hairpin structures

Preventing Primer Issues

• Avoid complementary sequences to other primers
• Have less than or equal to 3 contiguous bases
• Primers are otherwise rendered useless

Optimizing PCR: Sample Volume

• Optimal sample volume is 20-50 microL
• Too low volume leads to evaporation and accuracy issues due to DNA sticking to the sides
• Too high volume leads to inaccurate steps because thermal equilibrium is not accurate with temperature

Taq vs. AmpliTaq Gold

• HotStart PCR involves reverse transcription
• HotStart PCR modifies Taq polymerase, preventing activity until reaching a high temperature
• HotStart PCR reduces contamination and premature Taq polymerase starts, thus, it is better for forensics

Template Quality

• Degradation causes DNA to break into smaller fragments
• PCR failure can occur if DNA is degraded
• Longer amplicon lengths increase the risk of failure if degraded

Low Copy Number

• Insufficient DNA for a good profile
• 100 picograms of DNA is required as a template
• Low copy number leads to data drop out

PCR Inhibitors

• Two main types are those that block Mg from binding to Taq polymerase and those that bind to DNA, preventing Taq polymerase access
• Inhibitors result in no DNA amplification

Contamination Prevention

• Separate areas for re-amplification and post-amplification procedures and store samples
• Use a hood
• Use PPE
• Use strict contamination procedures for benches, equipment, and utensils through UV irradiation and cleaning with bleach
• Use aerosol-resistant pipette tips to prevent cross-contamination
• Maintain an elimination database

Quantification

• Quantification determines the concentration range for amplification

Reasons for Quantifying

• Determine the needed amount of DNA template for amplification
• Avoid off-scale data and artifacts, as well as interpretation problems
• Avoid stochastic effects
• Meet multiplex STR kit requirements
• Ensure quality check
• Method development

DNA Quantity in One Cell

• Basepair weight: A and T equals 617 g/mol, C and G equals 618 g/mol
• Molecular weight is 1.85 x 10^12 g/mol
• A haploid cell contains 3 picograms of DNA, while a diploid cell contains 6 picograms

DNA Content

• One nanogram of DNA contains DNA from 167 diploid cells
• One nanogram of genomic DNA equals 333 copies of each locus

Methods for Quantitation

• Absorption x XmicroG/mL determines the amount of dsDNA, ssDNA, and ssRNA in a sample

UV Quantitation

• Measuring optical density (OD) for dsDNA

UV Wavelengths

• Measuring excitation and emission measures the amounts of dsDNA, ssDNA, and ssRNA
• UV quantitation is quick, easy, and good for research

UV Limitations

• UV quantitation focuses on DNA instead of RNA
• UV quantitation is not very sensitive for small amounts of DNA
• UV quantitation requires a lot of sample and is consumed after being placed in a cuvette
• Proteins can easily impact UV quantitation results

Yield Gel

• Yield gels contain DNA in an agarose gel with an intercalating dye like EtBr
• Compare intensity to that of known samples and standards
• Better for degraded DNA

Yield Gel Problems

• EtBr is dangerous and not human-specific

Fluorescence

• Add DNA to a solution with intercalating dyes that fluoresce at a known wavelength
• Measure with a spectrofluorometer, and excitation and emission wavelength types
• Compare to a standard curve
• An example includes picogreen microtiter
• It is slightly human-specific
• Bacteria can interfere with results

Slot Blot

• Requires single-stranded DNA
• Examples include chemiluminescent assays like lumigen and lumin
• Also includes colorimetric assays like quantiblot
• Compare signal to standards
• DNA captured on a membrane through conjugated probes
• Apply a human-specific probe to hybridize to the DNA
• Human specific
• Takes time

Real-Time PCR

• Quantitative PCR is based on PCR amplification; the amount of PCR correlates with the initial concentration

Definition of Real-Time PCR

• Measurement occurs during the exponential phase of PCR
• Better precision due to unaffected results by slight PCR condition variations

Real-Time PCR Advantages

• Increased sensitivity
• Better accuracy with small quantities
• Can be automated
• Can detect inhibitors and sample quality
• Can detect multiple areas of DNA, including male, human, and mitochondrial

Taq-Man Probe Fluorescence

• Probe anneals to the target sequence between PCR primers
• The primer is labeled with reporter fluorescent dye and also non-fluorescent quencher dyes
• Fluorescent Resonance Energy Transfer (FRET)

Taq Polymerase Activity

• Taq Polymerase has 5' exonuclease activity
• NFQ dye quenches reporter dye when in close proximity by pulling energy from the reporter dye. When taq poly reaches area of probe it cuts it up and the quencher dye is no longer near reporter dye so the reporter dye can fluoresce

Detection Process

• Three phases of the PCR process: exponential, linear, plateau
• Exponential Phase: the amplicon consistently doubles with each cycle, providing the optimal measurement point of fluorescence versus cycle number
• Linear Phase: amplification efficiency slows because components fall below a critical concentration
• Plateau Phase: no more PCR product accumulates from component exhaustion, and fluorescence signal levels out

Detection Threshold

• Also known as the cycle threshold, marking where DNA is measured via fluorescence

Standard Curve Use

• Used to determine how many cycles it took to reach a predetermined DNA amount

Quantifiler Trio

• Contains 4 targets: large autosomal, small autosomal, Y-chromosome, internal positive control
• The large autosomal target has 214 bp and degrades first if DNA is degraded
• The small autosomal target has 180 bp and is better for degraded DNA
• The Y-chromosome target has 75 bp
• Internal positive control (IPC) target has 130 bp, amplifies consistently as a positive control, and indicates inhibitor presence if it doesn't work

Male: Female Ratios

• Determined by direct downstream testing
• Male:Female Ratios determined by direct downstream testing via Quantifying male DNA/quantifying male DNA -> (quantifying human DNA - quantifying male DNA)/quantifying male DNA = female DNA

Degradation

• Determined by the degree of degradation of the large autosomal target
• Determined by assessing the ratio of small target DNA concentration to large target DNA concentration

Controls Included

• Standards
• Extraction control
• Internal positive control
• Reagent blank/control
• Negative control - kit-specific buffer