Polymerase Chain Reaction (PCR)

Topics
What is the Polymerase Chain Reaction? History and (pre-history) of PCR How PCR works Fidelity, errors and cloning Applications of PCR PCR primer design

What is the Polymerase Chain Reaction?

Its a means of selectively amplifying a particular segment of DNA. The segment may represent a small part of a large and complex mixture of DNAs: e.g. a specific exon of a human gene. It can be thought of as a molecular photocopier. A very simple and inexpensive technique for characterizing, analyzing and synthesizing any specific piece of DNA from virtually any living organism - plant / animal / virus / bacteria.

How Powerful is PCR?

PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hours. The template DNA need not be highly purified a boiled bacterial colony. The PCR product can be digested with restriction enzymes, sequenced or cloned.

Gene Analysis Prior to PCR?

Southern blotting (1975) permitted rudimentary mapping of genes in unrelated individuals (RFLPs, insertions & deletions). DNA sequencing (1978) required genes to first be cloned into plasmid or vectors. Gene library construction and screening could take many months and libraries had to be made for each individual analysed.

The Invention of PCR

Invented by Kary Mullis in 1983. First published account appeared in 1985. Awarded Nobel Prize for Chemistry in 1993.

invented by Kary Mullis while cruising in a Honda Civic on Highway 128 from San Francisco to Mendocino, "It was quiet and something just went, Click!"

Kary B. Mullis
Nobel Laureate, 1993 Chemistry

"THE SUN HAD been hot that day in Mendocino County. A dry wind had come out of the east, and nobody knew how hot it had been until, around sunset, the wind stopped. I drove up from Berkeley through Cloverdale headed to Anderson Valley. The California buckeyes poked heavy blossoms out into Highway 128. The pink and white stalks hanging down into my headlights looked cold, but they were loaded with warmed oils that dominated the dimension of smell. It seemed to be the night of the buckeyes, but something else was stirring.""My little silver Honda's front tires pulled us through the mountains. My hands felt the road and the turns. My mind drifted back to the lab. DNA chains coiled and floated. Lurid blue and pink images of electric molecules injected themselves somewhere between the mountain road and my eyes."

Opening words, Dancing Naked in the Mind Field, 1998, by Dr. Kary Mullis, Pantheon Books.

Mullis
... PCR is a chemical procedure that will make the structures of the molecules of our genes as easy to see as billboards in the desert and as easy to manipulate as Tinkertoys.

DNA

Did He Really Invent PCR?

The basic principle of replicating a piece of DNA using two primers had already been described by Gobind Khorana in 1971: Kleppe et al. (1971) J. Mol. Biol. 56, 341-346. Progress was limited by primer synthesis and polymerase purification issues. Mullis properly exploited amplification.

So Then, its Easy?

Cycling performed with three water baths. Thermal cyclers introduced in 1986. Early polymerases were not thermostable, so had to be replenished each cycle. The 37C temperature caused nonspecific priming, resulting in unwanted products.

bacteria discovered in a hot spring in Yellowstone Natural Park in 1965, lives in salty water that ranges from 70o - 75o C, thus, does DNA replication at very high temperatures.

Thermus aquaticus Enzymes

basic research demonstrated that many enzymes isolated from Thermus aquaticus function at very high temperatures, temperatures nearing 100o C, DNA denaturating temperatures.

Kary Mullis realized that repetitive rounds of DNA synthesis could be performed by using a heat-stable polymerase, Thermus aquaticus: Taq polymerase.

So, I Can Just Go Ahead?

Not so fast. The PCR technique and the use of Taq DNA polymerase in PCR are both patented. Even academic and public organisations must pay license fees levy paid on enzyme and thermal cycler purchases. Taq patent still being challenged (Promega).

3 basic steps
Denaturation: The two strands melt open to form single stranded DNA; generally carried out at 90C 90C. Annealing: Annealing of primers to each original strand for new strand synthesis is carried out between 45C - 60C. Extension at 72C: DNA polymerase adds dNTPs complementary to the template at the 3end of the primers.

Whats in the Reaction?

Template DNA Reaction buffer (Tris, ammonium ions (and/or potassium ions), magnesium ions, bovine serum albumin) Nucleotides (dNTPs) Primers DNA polymerase (usually Taq)

Denaturing
cant use helicase in vitro

DNA denaturing conditions such as high heat or low salt concentrations irreversibly denature or inactivate most polymerases dNTPs are not affected by denaturation primers are not affected by denaturation

Denaturation @ 90C

Must Denature
Separate Strands

Making Two More Strands

Must Denature
Separate Strands

Annealing @ 45-60C
...short pieces of synthetic DNA that contain complementary sequence for template (3end)

Extension @ 70C
added Forward Primer

Add Polymerase Add dNTPs, etc.

Second strand extension @70C

add Reverse primer

Add polymerase, dNTPs etc.

Primers
Reverse primer

Forward primer

Making DNA: Components

Cell ss DNA template dNTPs Primer DNA Polymerase Environment helicase, etc. present primase DNA polIII nucleus PCR ? present ? ? test tube

Polymerase Chain Reaction (PCR)

DNA from a selected region of the chromosome or genome can to be amplified a billion-fold, effectively purifying it away from a complex mixture of DNA molecules. Amplification of a DNA Segment

Long Product

REQUIREMENTS: Oligonucleotide primers which flank the sequence of interest

A repetitive three- step process : Denature--Anneal--Elongate (94-97oC) (42-55oC) (72oC)

A DNA Template (a few ng)

Long Product

A thermal-stable DNA Polymerase (TAQ) dNTPs An automated thermocycler

Polymerase Chain Reaction (PCR)

The Long Product (LP) acts as template for new synthesis Gives rise to Short Product (SP) whose 5 and 3 ends are both set by the primer annealing positions

Polymerase Chain Reaction (PCR)

SP

SP

In subsequent rounds, the Short Products accumulate in an exponential fashion

Sequential rounds

 Exponential increase in the number of copies of the gene. The doubling of number of DNA strands corresponding to the target sequences allows us to estimate the amplification associated with each cycle using the formula: Amplification = 2n, where n: number of cycles

How many copies?

No target products are made until the third cycle. The accumulation is not strictly a doubling at each cycle in the early phase. At 30 cycles there are 1,073,741,764 target copies (~1109). There are also 60 other DNA copies.

How many cycles?

Increasing the cycle number above ~35 has little positive effect. The plateau occurs when: The reagents are depleted The products re-anneal The polymerase is damaged Unwanted products accumulate.

Thermal Cyclers
PCR cyclers available from many suppliers. Many block formats and multi-block systems. Reactions in tubes or 96-well micro-titre plates.

35

Step 1. Set up the reaction mix

Sterile Water 10X Assay Buffer 10 mM dNTP Mix Template DNA (100 ng / L) Forward Primer (100 ng / L) Reverse Primer (100 ng / L) Taq DNA Polymerase (3 U / L) 38 L 5 L 3 L 1 L 1 L 1 L 1 L 50 L

Total reaction volume

Step 2. Mix the contents gently. Step 3. Layer the reaction mix with 50 L of mineral oil.

Step 4. 30 cycles of amplification in a Thermocycler.

Initial Denaturation Denaturation Annealing Extension Final Extension 95C 94C 48C 72C 72C 1 min 30 sec. 30 sec. 1 min. 2 min.

Gene size 1kb = 1:15 min 1.5kb=2:00min 2kb=3:00min

Tv=Ta=Tm-(1-5) C Tm=4C(G+C)+2C(A+T) Tm= 64.9 +41*(G+C-16.4)/(A+T+G+C)

Has It Worked?
Check a sample by gel electrophoresis. Is the product the size that you expected? Is there more than one band? Is any band the correct size? May need to optimize the reaction conditions.

Step 5. Agarose Gel Analysis

Add 5 L of Gel loading buffer to the PCR tube. Tap the mixture thoroughly and wait for a few seconds for the 2 layers to separate. Carefully pipette out 15 L of reaction mixture (avoiding mineral oil layer) and load on to 1.5 % agarose gel. Load 10 L of the ready-to-use marker provided. Run the samples at 100 V for 1-2 hours. Visualize the gel under UV transilluminator.

Lane 1: DNA Bst E II digest Ladder. Lane 2: PCR-amplified product using Taq DNAPolymerase / Taq Buffer A. Lane 3: PCR-amplified product using Deep Vent DNA Polymerase / Thermopol Buffer. Lane 4: PCR-amplified product using Taq DNA Polymerase / Taq Buffer A.

Factors affecting amplification

Sample Volume. Amount of Template DNA Primers: 15-27 bp; Primer-Dimer; 60% G+C; Tm of FP and RP dNTP concentration Taq DNA Polymerase Taq Buffer: Tris HCl, MgCl2,

Fidelity of the Reaction

Taq DNA polymerase lacks the 35 proof-reading activity commonly present in other polymerases. Taq mis-incorporates 1 base in 10000. A 400 bp target will contain an error in 33% of molecules after 20 cycles. Error distribution will be random.

Do Errors Matter?
Yes, if you want to clone the amplified DNA an individual molecule may harbour several mutations. No, if you want to sequence the amplified DNA or cut it with restriction enzymes. Use a proof-reading thermo-stable enzyme rather than Taq.

Can I PCR Amplify RNA?

Not directly the DNA polymerase requires a DNA template and will not copy RNA. mRNA can first be copied into cDNA using reverse transcriptase. cDNA is a template for PCR it need not be double-stranded.

Cloning PCR Products

Products should be ligatable into bluntended restriction enzyme site. Lower than expected efficiency. Products are not truly blunt-ended. Taq polymerase adds a single nontemplated base (usually A) to the 3 end: NNNNNNNNNNNNNNA ANNNNNNNNNNNNNN

TA Cloning of PCR Products

Take advantage of the non-templated bases. Linearise vector at a blunt-ended site (e.g. EcoRV). Incubate linear vector with Taq polymerase and dTTP to add non-templated Ts. Ligate:

Designing PCR Primers

Primers should be ~17-27 bases long. The G/C content should be 55-60%. The annealing temperatures should be within 1C of one another. The 3-most base should be a G or C. The primers must not base pair with each other or with themselves or form hairpins. Primers must avoid repetitive DNA regions.

Primers That Form Hairpins

A primer may be self-complementary and be able to fold into a hairpin: 5-GTTGACTTGATATTCTCAAG-3 5-GTTGACTTGATA | || || T 3-GAACTCT The 3 end of the primer is base-paired, preventing it annealing to the target DNA.

Primer 5ATGCTTGGGGCCCCATGC3

Primers That Form Dimers

A primer may form a dimer with itself or with the other primer.
5-ACCGGTAGCCACGAATTCGT-3(FP) | | ||| | || || 3-TGCTTAAGCACCGATGGCCA-5(RP)

Primer dimers can be an excellent, but unwanted, substrate for the Taq polymerase.

PCR Applications
Mutation testing, e.g. cystic fibrosis. Diagnosis or screening of acquired diseases, e.g. AIDS. Genetic profiling in forensic, legal and biodiversity applications. Site-directed mutagenesis of genes. Quantitation of mRNA in cells or tissues.

PCR Applications
new applications are created every day, PCR products can be used for mapping genes, PCR products can be used as probes, PCR cDNA product can be created, PCR can be used to identify genotypes, PCR can be used to sequence DNA directly.

Diagnostic application case study

Unknown ID
Patient came in with signs of viral infection TEM analysis indicated a ~30 nm icosahedral particle infection Extracted virus from infected cells biochemical characterization-failure

Adenovirus, Papillomavirus, Herpes simplex virus, Hepatitis B virus are Icosahedral viruses
Extraction of NA(ds DNA) from the infected cells PCR carried out with specific primer for each virus separately PCR amplified product were run in AGE

Unknown ID

Lane 1 2 3 4 5 6 7 Lane 1 Marker Lane 2 Adenovirus specific primer Lane 3 Papillomavirus specific primer Lane 4 Herpes simplex virus specific primer Lane 5 Hepatitis B virus specific primer Lane 6 TMV specific primer Lane 7 Marker

Exercise primer design

Help With Primer Design

Researchers agreed early on that the design of PCR primers was difficult and unreliable. Computer programs devised to take all of the design criteria into account. Primer3 program at the Whitehead Institute is the most reliable and versatile tool currently available.

Primers for a COL3A1 variant

The human COL3A1 gene has a variant at amino acid 531 of the triple helix. Ala or Thr encoded in exon 31 of the gene. AluI restriction enzyme site present in the Ala allele but absent in the Thr allele. PCR amplify the region and genotype by digestion of PCR products with AluI.

Running Primer3
Paste the DNA sequence into Primer3 with the target enclosed in square brackets. Select a mispriming library only human and rodent available at present. Select option for a 1-base 3 GC Clamp. Select PCR product size range (>600 bp). Click the Pick Primers button. Marvel at the ease and simplicity.

The COL3A1 Ala/Thr PCR

The PCR primers amplify from the start of exon 31 to just beyond exon 33 656 bp. Ala alleles are digested by AluI, producing fragments of 82 & 574 bp.

Will Other Genes Amplify Too?

The primers have been designed on the basis of the DNA sequence of a single gene. Might the primers also amplify other segments whose sequence we have not taken into account? Need to consider the sequence of the entire genome to answer this.

Virtual PCR Results

Virtual PCR searches entire genome looking for potential primer sites within 10,000 bases of one another. If found, it performs a virtual PCR reaction. Primers for Ala/Thr polymorphism in human COL3A1.

PCR In Detail
Denature, anneal, extend and repeat the cycle 30 to 35 times. How does the polymerase know to stop when it reaches the other primer? Most textbooks to not fully explain PCR. PCR animation at Dolan DNA Learning Center, CSHL, Cold Spring Harbor.