The polymerase chain reaction

(PCR)

Experiment Goals

• Understand how PCR technique works.

• Perform PCR experiment.

• Analyze PCR products.

What is PCR?What is PCR?

Definition• Is a powerful and sensitive technique to

amplify a piece of DNA very rapidly outside a living cell (DNA amplification in vitro).

PCR Applications

• PCR is now a common and often indispensable (main) technique used in medical and biological research labs for a variety of applications.

Structural analysis Mapping

Disease detection Sequencing

Cloning Forensic medicine

Mutation analysis Scientific research

Pre-natal diagnosis

How does PCR work?

• There are three major steps in a PCR which are repeated for 30 or 40 cycles.

• This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.

Denaturation

• The initial step denatures the target DNA (H-bonds are broken between strands of DNA with heat) by heating it to 94°C or higher.

• In the denaturation process, the two intertwined (coiled) strands of DNA separate from one another, producing the necessary single-stranded DNA template for replication by the thermo stable DNA polymerase.

• Annealing: In this step the temperature is reduced to approximately 50–60°C. At this temperature, the oligonucleotide primers attach to complementary sequences of single stranded DNA.

• Extension: Finally, the synthesis of new DNA, the DNA polymerase attaches to primer with ssDNA and extends DNA fragment, this temperature is in the range of 70–74°C.

• The next cycle begins with a return to 94°C for denaturation.

PCR

PCR Reaction Components

1) Target DNA: contains the sequence to be amplified.

2) Pair of Primers: oligonucleotides that define the sequence to be amplified.

3) dNTPs: deoxynucleotidetriphosphates: DNA building blocks.

4) DNA Polymerase: enzyme that catalyzes the reaction

5) Mg++ ions: cofactor of the enzyme

6) Buffer solution: maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme

1) Target DNA• Use of high quality, purified DNA templates

greatly enhances the success of PCR reactions.• Approximately 104 copies of the target DNA “this

means a 1–10 µg/ml of genomic templates” are required to detect a product in 25–30 cycles of PCR.

• DNA can be • a single gene • part of a gene• a non-coding sequence

DNA Quality• DNA should be intact and free of contaminants

that inhibit amplification. – Contaminants can be purified from the

original DNA source.• Heme from blood, and melanin from hair

– Contaminants can be introduced during the purification process. • Phenol, ethanol, detergents, and salts.

How Big A Target is?• Amplification products are typically in the

size range 100-1500 bp.

• Longer targets are amplifiable >25 kb.

• Requires modified reaction buffer, cocktails of polymerases, and longer extension times.

2) Pair of Primers• Primers: the short DNA molecules sequence to

be amplified.• Oligonucleotide primers• Generally 20–30 nucleotides in length, and

ideally have a GC content of 40–60%, with GC residues spaced evenly within the primer.

• Primers bind (anneal) to the DNA template and act as starting points for the DNA polymerase, – DNA polymerases cannot initiate DNA

synthesis without a primer.

3) dNTPs (deoxynucleotidetriphosphates)

– The building blocks for the newly synthesized DNA strands.

– dATP, dGTP, dCTP or dTTP

4) DNA Polymerase• DNA Polymerase is the enzyme responsible for

copying the sequence starting at the primer from the single DNA strand

• Commonly use Taq, an enzyme from the hyperthermophilic organisms Thermus aquaticus, isolated first at a thermal spring

• This enzyme is heat-tolerant

• The PCR is commonly carried out in a reaction volume of 15-100 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler.

• The thermal cycler allows heating and cooling of the reaction tubes to control the temperature required at each reaction step.

• Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube.

• Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

Running PCR

Initialization step• Prior to the first cycle, there is an initialization

step– the PCR reaction is often heated to a

temperature of 94-96°C, and this temperature is then held for 1-9 minutes

– This first hold is employed to ensure that most of the DNA template and primers are denatured,

– Also, some PCR polymerases require this step for activation

PCR: Completed Amplification Cycle

n cycles will give 2n copie.

DNA copies vs Cycle number

0

500000

1000000

1500000

2000000

2500000

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Cycle number

DN

A c

opie

s

Analyze PCR products

• 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.

Polymerase Chain Reaction Controls

• Blank reaction (Negative control reaction)– Controls for contamination– Contains all reagents except DNA template

• Positive control reaction– Controls for sensitivity– Contains all reagents and a known target-containing

DNA template

Procedure

1- Prepare master Mix

2- Program the thermocycler

3- Run the samples on thermocycler

4- Analysis of PCR products

Target DNA

• Amplification of part of the Human growth hormone gene

• Specific primers used• Forward primer: 5’-

TCCCTTCCCAACCATTCCCTTA-3’• Reverse primer: 5’-

CCACTCACGGATTTCTGTTGTGTTTC-3’• During Polymerase Chain Reaction (PCR) the

primers will be extended from the 3’-end.

1- Master Mix

PCR reaction mixture

Reagent Volume (µl) Final concentration

PCR buffer (X10) 2.0 10 mM

MgCl2 (25 mM) 1.6 2.0 mM

dNTPs (100mM) 0.1 0.1 mM

Primer 1 (F) 0.2 1.0 µM

Primer 2 (R) 0.2 1.0 µM

Taq DNA polymerase

0.25 2.0 U

DNA template 2.0 100 ng

Water 13.7

2- Program the Thermocycler

The Thermocycler Profile is:Step 1: Denaturation for 3 min. at 95oC

Step 2: 35 cycles

Melting for 60 sec. at 95oC

Annealing for 60 sec. at 57oC

Extension for 90 sec. at 72oC

Step 3: Final elongation for 10 min. at 72oC

4- Analysis of PCR products

Analyse products on 2% agarose gel containing ethidium bromide

Visualize the PCR product on UV transilluminator.

There should be a 400 bp band for the positive samples.

-ve +ve Sample

• http://www.sumanasinc.com/webcontent/animations/content/pcr.html

• http://www.youtube.com/watch?v=_YgXcJ4n-kQ

• http://www.dnalc.org/resources/animations/pcr.html

• http://www.youtube.com/watch?v=HMC7c2T8fVk&feature=related

• http://highered.mcgraw hill.com/sites/9834092339/student_view0/chapter17/pcr_reactions.html