Polymerase Chain Reaction

Forensic Genotyping

• Forensic samples:– Limited quantity of DNA– Poor quality (degraded)

• Need to have a mechanism to amplify the amount of DNA in the sample

• Polymerase Chain Reaction (PCR) fits the needs of Forensics perfectly:– Sensitive, rapid and not limited by quality

PCR1. Separate the Double Helix2. Bind primers (2) to sequence you want to

replicate3. DNA Polymerase copies between two

primers4. Rinse and Repeat5. Copies DNA between two primers

exponentially

PCR1. Separate the Double Helix

2. Bind primers (2) to sequence you want to replicate

Heat

Add

PCR3. DNA Polymerase copies between two

primers

4. Repeat Many Times

AddDNA Pol.

Thermal Cycles

Three main steps of temperature:1. 94 - 95 degrees C

• Denatures the double stranded DNA2. 60 - 72 degrees C

• Primer annealing temperature3. 72 degrees C

• DNA polymerase extension• Polymerase reads the sequence between

the primers to produce a copy

Exponential Growth

• Because DNA is double stranded• Add two primers• Each cycle will double the amount of DNA

– Exponentially copying the template• Theoretically:

– 3 cycles = 2 copies– 12 cycles = 1000 copies– 22 cycles = 1 million copies– 32 cycles = 1 billion copies

Forensic Kits

• Reagent kits are now available to laboratories

• Simply add DNA template to pre-mixed PCR reagents:– DNA concentration is known (quantified)– Concentration is equalized between samples

• Put mix + DNA through thermal amplification cycles – get product

PCR Needs

• You must know the sequence you are trying to amplify

• Primers: – One for each side, exact sequence correct

• Excess of four dNTPs: – 4 nucleotides

• Heat insensitive DNA Polymerase:– So that reagents can be heated and cooled

repeatedly

PrimersPrimer is a short piece of DNA:

– 20 to 30 bases long– Oligonucleotide

• Designed to match the sequence surrounding the template exactly

• Hybridize to the correct place in the genome based on complementary base pairing at the correct conditions

• Added to PCR reaction in high concentration

dNTPs

deoxyribose Nucleotide TriPhosphate:• Building blocks of new DNA strands• DNA Polymerase adds the correct dNTP

based on complementary base pairing• Just as a cell replicates it’s own DNA

during S phase• Added to PCR reaction in extreme excess

so that reaction will never run out

Heat Insensitive Polymerase

Normal DNA Polymerases will be denatured when heated to the temperature denatures double stranded DNA

• Old Method:– Added more polymerase after each 95 stage

• Now:– Use Taq Polymerase – Heat insensitive – doesn’t denature– Isolated from bacteria that lives in hot springs

Other reagents

• Tris-HCl (or 10X Buffer)– Buffers reaction to correct pH for Polymerase

• Magnesium Chloride– Salts are cofactor for Polymerase

• DNA sample• Primers• Taq Polymerase• dNTPs

Master Mix

• When setting up for many different samples

• More accurate to prepare a “Master Mix”that contains all reagents except DNA

• Then aliquot Master Mix into each tube• Finally add DNA sample• Improves:

– Accuracy of each component– Reproducibility of method

Controls

• Negative Control– Same master mix but with water or buffer– No DNA sample– Proves PCR reagents are not contaminated

• Positive Control– Master mix plus DNA of known concentration– Proves PCR reaction/conditions work

• Extraction Blank– Controls for DNA extraction reagents

DNA Template

• Normally PCR uses 1-50 ng of template• Forensic DNA samples often have very

low concentration of DNA• DNA below 100 pg (0.1 ng) will often show

allele dropout• When one allele doesn’t amplify but other

allele does – giving wrong genotype• Falsely homozygous

Thermal Cycling Conditions

• Also known as Amplification Conditions• The temperatures and amount of time at

each temp that PCR reaction is set at• Vary widely depending on:

– Length of DNA to be amplified– Primer’s ideal annealing temp– Exact Polymerase in use– Quantity and quality of DNA sample

Thermal Cycling Conditions

• Usually have 25 to 35 cycles• Example protocol:

– 95 for 5 minutes – “hot start”– 30 cycles:

• 94 for 30 seconds• 60 for 30 seconds• 72 for 1 minute

– 72 for 10 minutes – final extension– 4 degrees – to keep products from reacting

Thermal Cyclers

Machines that accurately and quickly change the temperature of the tubes

• PCR Machines• Enter the program for your amplification

conditions• Precise and accurate heating and cooling• Old Method:

– Move the tube between 3 hot water baths

Possible Problems with PCR

• Primer annealing non-specifically– At room temperature primers can start to

anneal to template – at random• Primer Dimers

– Primer anneals to other primer• Avoid both these by using “Hot Start”

Polymerase• Choose a Polymerase that only works

once reactions have been heated to 95

Designing Primers

• Most important step of PCR may be designing the primers

• Primers determine:– Exactly what DNA sequence is copied– PCR yield

• For successful PCR primers must be:– Specific to one highly conserved sequence– Possess similar annealing temps– Not interact with each other (primer dimers)

Optimal Primers• Length – 18 to 30 bases• Annealing Temp – 55 to 72 degrees• GC Content – 40 to 60%• Unique oligionucleotide sequence• Less than 800 bases between two primers• No runs of >4 of same base in a row• No hairpins (secondary structure)• No complementary sequence to other

primer (primer dimer)

Primer Design

• Should always BLAST your primers– Will examine if primers can anneal anywhere

else in genome• Programs exist to help choose:

– Primer Express– Primer3

• Important parameters to input:– Product size– Primer length– Ideal annealing temp

Multiplexing

• Adding more than one set of primers in the same reaction

• Produces more than one PCR product while only running one reaction

• Saves on amount sample used• Saves time and money• PCR is ideally suited to be multiplexed• As long as you optimize your reactions

Optimizing Multiplexing

Must optimize multiplex reactions:• Products are all of different lengths• Labeled with different fluorescent dye• Primers need similar annealing temps• Primers must not interact with any of the

other primers• Most important to optimize:

– Primer sequences – Concentration of each primer

Optimizing Multiplexing

• Our average multiplex was 9 different products per tube:– Three different colored dyes

• In commercial PCR kits markers are multiplexed together

– Already optimized– 13 markers per tube

Real Time PCR

• Analyzes the PCR product while PCR reaction is occurring

• Allows quantification of starting amount of DNA

• Two main methods:– TaqMan AssayMeasures amount of specific product– SYBR green Measures amount of any DNA produced

TaqMan Assay

• Uses a probe that binds to center of DNA sequence you are amplifying

• Probe contains both:– Reporter dye– Quencher dye – stops reporter from

fluorescence• Whenever two dyes break apart you see

fluorescence of reporter dye• Probe breaks during DNA polymerization

Polymerization and Strand Displacement

RQ

Forward primer

Reverse primer

3’5’ 3’

5’3’5’

5’

5’

Forward primer

Reverse primer

3’5’ 3’

5’5’

5’

Q

R

3’ Probe Cleavage (release of reporter dye)

Forward primer

Reverse primer

3’5’ 3’

5’5’

5’

QR

Completion of Polymerization

TaqMan probe

Fluorescence occurs when reporter dye and quencher dye

are no longer close

Figure 4.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Real Time PCR

• More DNA polymerization – more signal• Idea is the more DNA you started with, the

faster your reaction will reach a specific concentration of DNA

• Because amplification during PCR can be calculated exactly per cycle

• Set an arbitrary threshold of DNA concentration – fluorescence of probe

• See how many cycles it takes to reach

CT

Cycle Threshold• How many cycles it took for reaction to

pass arbitrary DNA concentration– Set fluorescence of probe

• Normalize fluorescence to adjust for background differences in tube

• CT or number of cycles• Used to calculate amount of starting DNA

Cycle Number

Nor

mal

ized

Flu

ores

cenc

e

threshold

CT

Exponential product growth

Linear product growth

Plateau

ΔRn

Negative control

a b c d e

Standard curve

CT

Log[DNA]

a

b

cd

e

Figure 4.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Contamination

• Easy to contaminate DNA sample• Exactly because PCR is so great at

amplifying small amounts of DNA• Need to be very careful:

– Scientist performing PCR– Police officer collecting evidence

• Everyone involved with DNA sample is often genotyped as well:– Staff elimination database

Appropriate Guidelines

• Police Officers have handout:– www.ojp.usdoj.gov/nij

• Forensic Scientists:– Lab coats– Fresh gloves at all times– Facial masks and hair nets

• Precautions are necessary when working with miniscule amounts or degraded DNA

Protect Against Contamination

• Physically separate PCR set up areas from PCR products:– Have two different benches– Different pipettes, sterile water, etc

• Wear and change disposable gloves• Use aerosol resistant pipette tips• Change tips every single time!• Irradiate benches and spray with alcohol

or bleach between uses

Advantages of PCR

• Very small amount of DNA can be used• DNA can be degraded• Reactions can be multiplexed• Non-human DNA contaminates are not a

problem– Because primers are human specific

• Commercial kits are available for labs– Only have to add DNA sample

Disadvantages of PCR

• PCR Inhibitors may block amplification:– Indigo dyes– Hemoglobin

• Amplification may fail:– Allele drop-out – false homozygous– Polymorphisms at primer binding site

• Contamination from other DNA sources:– Easy to get contamination– Difficult to separate/Identify sources

What NOT to learn…

• Skip all specifics:– Names of polymerases– Polymerase’s ideal temperature or pH– Names of thermal cyclers

• All of this has already changed• Will change quickly • Depends on budget and desires of lab

Any Questions?

Read Chapter Five