POLYMERASE CHAINREACTION

WHAT IS PCR?

PCR is a technique that takes specific sequence of DNA of small amount and amplifies it to be used for further testing. PCR can make billions of copies of a target

sequence of DNA in a few hours

WHY “POLYMERASE”?

It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.

WHY “CHAIN”? It is called “chain” because the products of

the first reaction become substrates of the following one, and so on.

SHORT HISTORY OF PCR

In 1983 Dr. Kary Banks Mullis developed PCR. He receive a Noble Prize in Chemistry in 1993 for his invention of the polymerase chain reaction. It is hailed as one of the monumental scientific techniques of the twentieth century.

In 1985 the first publication of PCR by Cetus Corporation appears in Science. Cetus Corporation was one of the first biotechnology companies.

1986: Purified Taq polymerase is first used in PCR. TAQ Polymerase is a DNA Polymerase found in bacteria that live in thermophilic conditions, such as hot water springs. It is used to synthesize a new DNA strand from a template.

SHORT HISTORY OF PCR

1988: PerkinElmer introduces the automated thermal cycler. Also known as a thermocycler, PCR machine or DNA amplifier. It is a laboratory apparatus most commonly used to amplify segments of DNA via the polymerase chain reaction (PCR).

PCR - before the thermocyclerPCR - before the thermocycler

95º C5 min95º C5 min

35 times35 times

55º C3 min55º C3 min

72º C5 min72º C5 min

DNA REPLICATION VS. PCR PCR is a laboratory version of DNA

Replication in cells The laboratory version is commonly called “in vitro”

since it occurs in a test tube while “in vivo” signifies occurring in a living cell.

DNA REPLICATION IN CELLS (IN VIVO) DNA replication is the copying of DNA It typically takes a cell just a few hours to copy all

of its DNA DNA replication is semi-conservative (i.e. one

strand of the DNA is used as the template for the growth of a new DNA strand)

This process occurs with very few errors (on average there is one error per 1 billion nucleotides copied)

More than a dozen enzymes and proteins participate in DNA replication

POLYMERASE CHAIN REACTION

Polymerase chain reaction enables large amounts of DNA to be produced from very small samples (0.1ml)

There is a repeating cycle of:separation of double DNA strandssynthesis of a complementary strand for each

Its purpose is to amplify a lot of double-stranded DNA molecules (fragments) with same (identical) size and sequence by enzymatic method and cycling condition.

COMPONENTS DNA template - the sample DNA that contains the

target sequence. At the beginning of the reaction, high temperature is applied to the original double-stranded DNA molecule to separate the strands from each other.

DNA polymerase - a type of enzyme that synthesizes new strands of DNA complementary to the target sequence. The first and most commonly used of these enzymes is Taq DNA polymerase (from Thermis aquaticus), whereas Pfu DNA polymerase (from Pyrococcus furiosus) is used widely because of its higher fidelity when copying DNA. Although these enzymes are subtly different, they both have two capabilities that make them suitable for PCR: 1) they can generate new strands of DNA using a DNA template and primers, and 2) they are heat resistant.

COMPONENTS

Primers - short pieces of single-stranded DNA that are complementary to the target sequence. The polymerase begins synthesizing new DNA from the end of the primer.

Nucleotides (dNTPs or deoxynucleotide triphosphates) - single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands.

BASIC REQUIREMENTS FOR PCR

DNA sequence of target region must be known.

Primers - typically 20-30 bases in size.These can be readily produced by commercial companies. Can also be prepared using a DNA synthesizer

Thermo-stable DNA polymerase - eg Taq polymerase which is not inactivated by heating to 95C

DNA thermal cycler - machine which can be programmed to carry out heating and cooling of samples over a number of cycles.

CONDITION

Denaturation of ds DNA template

Annealing of primers

Extension of ds DNA molecules

DENATURATION

Temperature: 92-94C Double stranded DNA melts single

stranded DNA

92C

3’5’

3’ 5’

+

5’3’

5’ 3’

ANNEALING

Temperature: ~50-70C (dependant on the melting temperature of the expected duplex)

Primers bind to their complementary sequences

5’3’

5’ 3’

Forward primer Reverse primer

EXTENSION

Temperature: ~72C Time: 0.5-3min DNA polymerase binds to the annealed

primers and extends DNA at the 3’ end of the chain

Taq

5’3’

Taq5’

CYCLING

PRODUCTS OF EXTENSION

3’5’

3’ 5’

3’5’

3’ 5’

Taq

Taq

OVERALL PRINCIPLE OF PCR

DNA – 1 copy

Known sequence Sequence of interest Known sequence

PCR

ADVANTAGES

Much faster than using vectors Only a little bit of target DNA is needed

DISADVANTAGES

Are to synthesize primers, we need to know the sequence flanking the DNA segment of interest

Only applies to short DNA fragments, mostly less than 5 kb

WHAT IS IT USED FOR?

Medical and biological research Cloning Diagnosis of hereditary diseases Identification of fingerprints Diagnosis of infectious diseases