Recombinant DNA Recombinant DNA Technology………..Technology………..

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Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

IntroductionIntroduction

• The Polymerase Chain Reaction The Polymerase Chain Reaction (PCR) provides an extremely (PCR) provides an extremely sensitive means of amplifying sensitive means of amplifying small quantities of DNA.small quantities of DNA.

• Kary Mullins won the Nobel Kary Mullins won the Nobel Prize in 1993 for development of Prize in 1993 for development of this novel technique.this novel technique.

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

IntroductionIntroduction

• The technique was made The technique was made possible by the discovery of Taq possible by the discovery of Taq polymerase, the DNA polymerase, the DNA polymerase that is used by the polymerase that is used by the bacterium bacterium Thermus auquaticusThermus auquaticus , , discovered in hot springsdiscovered in hot springs..

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

IntroductionIntroduction

• The use of thermostable DNA The use of thermostable DNA polymerases was an important polymerases was an important breakthrough since this enzyme breakthrough since this enzyme does not denature at the does not denature at the temperatures( 95temperatures( 9500C) used to C) used to cause denaturation of the DNAcause denaturation of the DNA..

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

IntroductionIntroduction

• The most well-known of these The most well-known of these thermostable DNA polymerases thermostable DNA polymerases is is TaqTaq..

• This enzyme has a molecular This enzyme has a molecular size of 94kD and an optimum size of 94kD and an optimum reaction temperature of 75-80reaction temperature of 75-80ooC.C.

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

IntroductionIntroduction

Amplification means making Amplification means making multiple identical copies multiple identical copies (replicates) of a DNA sequence.(replicates) of a DNA sequence.

PCR method of DNA amplification PCR method of DNA amplification has proved very important in has proved very important in recombinant DNA technology and recombinant DNA technology and is used in a range of applications is used in a range of applications in medicine and forensic sciencein medicine and forensic science . .

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

What is PCR?What is PCR?

• It is used to amplify a specific DNA It is used to amplify a specific DNA (target) sequence lying between (target) sequence lying between known positions (flanks) on a known positions (flanks) on a double-stranded DNA molecule.double-stranded DNA molecule.

• The amplification process is The amplification process is mediated by oligonucleotide mediated by oligonucleotide primers that, typically, are 20-30 primers that, typically, are 20-30 nucleotides long.nucleotides long.

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

What is PCR?What is PCR?

• The primers are single-stranded The primers are single-stranded DNADNA..

• Primers anneal to the flanking Primers anneal to the flanking regions by complementary-base regions by complementary-base pairing (Gpairing (G==C and A=T) using C and A=T) using hydrogen bonding.hydrogen bonding.

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

• The amplified product is known The amplified product is known as an as an ampliconamplicon..

• Generally, PCR amplifies Generally, PCR amplifies smallish DNA targets 100-1000 smallish DNA targets 100-1000 base pairs (bp) long.base pairs (bp) long.

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

Material required in PCR Material required in PCR process:process:

• Thermal cycler (PCR machine, Thermal cycler (PCR machine, available in different specificity available in different specificity and models).and models).

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

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PCR mix for a reaction has the PCR mix for a reaction has the following: following:

sample DNA with a target sequence sample DNA with a target sequence thermostable DNA polymerase (Taq thermostable DNA polymerase (Taq

is commonly used)is commonly used) two oligonucleotide primers which two oligonucleotide primers which

are complementary to the sequence are complementary to the sequence flanking the target sequence. flanking the target sequence.

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

deoxynucleotide triphosphates deoxynucleotide triphosphates (dNTPs)(dNTPs)

reaction buffer containing reaction buffer containing magnesium ions and other magnesium ions and other components. components. Homework:Function of Homework:Function of Magnesium for Quiz!!!Magnesium for Quiz!!!

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

Steps in PCR reactions:Steps in PCR reactions:

• 1.    Heat denaturation1.    Heat denaturation

A DNA molecule carrying a A DNA molecule carrying a target sequence is denatured by target sequence is denatured by heat at 90-95heat at 90-95ooC. The two strands C. The two strands separate due to breakage of the separate due to breakage of the hydrogen bonds holding them hydrogen bonds holding them together.together.

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

• 2.   Primer annealing2.   Primer annealing

Primers are added to the Primers are added to the dissociated target strands and dissociated target strands and incubated together first at a incubated together first at a temperature that allows the primers temperature that allows the primers to hybridize to the target strands to hybridize to the target strands (usually somewhere between 40 and (usually somewhere between 40 and 55 55 ooC).C).

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

• 3. Primer extension3. Primer extension

DNA polymerase is then added and DNA polymerase is then added and complementary strands are complementary strands are synthesized at a temperature of 60-synthesized at a temperature of 60-7575ooC. The polymerase causes C. The polymerase causes synthesis of new material in the 5' synthesis of new material in the 5' to 3' direction away from each of the to 3' direction away from each of the primers.primers.

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

This allows a first round of This allows a first round of synthesis to occur on each of the synthesis to occur on each of the DNA templates. DNA templates.

Following primer extension, the Following primer extension, the mixture is heated (again at 90-mixture is heated (again at 90-9595ooC) to denature the molecules C) to denature the molecules and separate the strands and the and separate the strands and the cycle repeatedcycle repeated

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

Each new strand then acts as a Each new strand then acts as a template for the next cycle of template for the next cycle of synthesis. synthesis.

Amplification (replication) Amplification (replication) proceeds at an exponential proceeds at an exponential (logarithmic) rate (amount of (logarithmic) rate (amount of DNA produced doubles at each DNA produced doubles at each cycle).cycle).

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

A thermal cycler (a machine that A thermal cycler (a machine that automatically changes the automatically changes the temperature at the correct time temperature at the correct time for each of the stages and can be for each of the stages and can be programmed to carry out a set programmed to carry out a set number of cycles) is used for a number of cycles) is used for a PCR reaction.PCR reaction.

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

The amplified product can be The amplified product can be detected using gel detected using gel electrophoresis to view the band electrophoresis to view the band containing DNA fragments of a containing DNA fragments of a particular size containing the particular size containing the gene of interest in the original gene of interest in the original starter DNA sample.starter DNA sample.

Diagrammatic steps in PCR process Diagrammatic steps in PCR process (Read summary):(Read summary):

• Three major steps in PCR:Three major steps in PCR:

1.1. Template denaturation Template denaturation 2.2. Primer annealing Primer annealing 3.3. Primer extension Primer extension

Home work for next class!!Home work for next class!!Summarize the steps in PCR processSummarize the steps in PCR process

•• The oligonucleotides serve as primers for The oligonucleotides serve as primers for DNA polymerase and the denatured strands DNA polymerase and the denatured strands of the large DNA fragment serves as the of the large DNA fragment serves as the template.template.

•• This results in the synthesis of new DNA This results in the synthesis of new DNA strands which are complementary to the strands which are complementary to the parent template strands.parent template strands.

•• After each cycle the newly synthesized DNA After each cycle the newly synthesized DNA strands can serve as templates in the next strands can serve as templates in the next cycle.cycle.

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

Diagrammatic steps in PCR processDiagrammatic steps in PCR process

•• PCR amplification is achieved by PCR amplification is achieved by using oligonucleotide primers.using oligonucleotide primers.

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

Diagrammatic steps in PCR processDiagrammatic steps in PCR process

••

Fig. 3.8 The Polymerase Chain Fig. 3.8 The Polymerase Chain ReactionReaction

Go to Animation courtesy of the Go to Animation courtesy of the following websites:following websites:

References References http://faculty.plattsburgh.edu/donald.slish/PCRmov.html (Animation)http://faculty.plattsburgh.edu/donald.slish/PCRmov.html (Animation) http://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.htmlhttp://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.html http://www.people.virginia.edu/~rjh9u/pcranim.html ( PCR Animation)http://www.people.virginia.edu/~rjh9u/pcranim.html ( PCR Animation) http://www.escience.ws/b572/L3/L3.htm (PCR Animation)http://www.escience.ws/b572/L3/L3.htm (PCR Animation) http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect4.htmlhttp://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect4.html http://en.wikipedia.org/wiki/Polymerase_chain_reactionhttp://en.wikipedia.org/wiki/Polymerase_chain_reaction http://www.escience.ws/b572/L3/L3.htmhttp://www.escience.ws/b572/L3/L3.htm http://allserv.rug.ac.be/~avierstr/principles/pcrani.htmlhttp://allserv.rug.ac.be/~avierstr/principles/pcrani.html