PCR

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A technique in MB,A technique in MB,

A tool for gene A tool for gene amplification

History

1985 - Kary Mullis.1985 - Kary Mullis. 1993 -Nobel Prize in Chemistry.1993 -Nobel Prize in Chemistry. Development of a technique/ process by which Development of a technique/ process by which

DNA could be artificially DNA could be artificially multiplied through through repeated cycles of duplication. of duplication.

Essential components

• Template ? Needs amplification• Primers = determine the beginning and end of

the region to be amplified • DNA polymerase• Buffer = suitable env.

Electrophoresis

Components?

Principle?

PCR Process

Template = DNA .Template = DNA . Enzyme = Enzyme = DNA polymerase. Primers = = Bind with an end of the desired DNA with an end of the desired DNA

segment. segment. Soln Soln heated - break bonds between strands of DNA. onds between strands of DNA. Soln Soln cools, the primers bind to the separated strands, , the primers bind to the separated strands,

and DNA polymerase builds a new strand by joining and DNA polymerase builds a new strand by joining the free nucleotide bases to the primers.the free nucleotide bases to the primers.

Any problem?

When this process is repeated, a region of When this process is repeated, a region of DNA between the primers is selectively DNA between the primers is selectively replicated = replicated = New strands formed..

Further repetitions of the process can produce Further repetitions of the process can produce billions of copies of DNA in several hours. billions of copies of DNA in several hours.

DNA polymerase

occurs naturally in living organisms occurs naturally in living organisms Fx = duplicate DNA during cell division.Fx = duplicate DNA during cell division. ? = binds to a single DNA strand and creating the ? = binds to a single DNA strand and creating the

complementary strand.complementary strand. Problem : at high temp DNA polymerase is Problem : at high temp DNA polymerase is

destroyed, so the enzyme had to be replenished after destroyed, so the enzyme had to be replenished after the heating stage of each cyclethe heating stage of each cycle

Inefficient = time consuming, large amounts of DNA Inefficient = time consuming, large amounts of DNA polymerase, and continual attention throughout the polymerase, and continual attention throughout the process.process.

Discovery of Taq polymerase. Discovery of Taq polymerase. Hot Springs Hot Springs Thermophilic and thermostable.Thermophilic and thermostable. Efficient = X add bacteria at each cycle. Efficient = X add bacteria at each cycle. Commercially available by genetically Commercially available by genetically

modified bacteriamodified bacteria

Primers

Short artificial DNA strandsShort artificial DNA strands Nucleotides arranged in a specific orderNucleotides arranged in a specific order Match the beginning and end of the DNA Match the beginning and end of the DNA

fragment to be amplifiedfragment to be amplified Fx : Fx : They anneal to the DNA template and They anneal to the DNA template and

begins the synthesis of the new DNA strand.begins the synthesis of the new DNA strand.

Closer look

Denaturing of target material: original of target material: original template is melted. (90-96 C). Separate strandstemplate is melted. (90-96 C). Separate strands

Hybridization: primers bind to their : primers bind to their complementary bases to single DNA strands. complementary bases to single DNA strands. Primers anneal (45-55C)Primers anneal (45-55C)

Elongation/ DNA synthesis by RNA by RNA polymerase : polymerase makes two new polymerase : polymerase makes two new strands ( 72 C), strands ( 72 C), doubling the amount of DNA the amount of DNA present. present.

Cycles

Melting phase – High Melting phase – High temp temp

Annealing phase – Low Annealing phase – Low temptemp

Elongation phase –Elongation phase –Optimized tempOptimized temp

End product

This provides This provides 2 new templates for the next new templates for the next cycle. cycle.

The DNA is again melted, primers anneal, and The DNA is again melted, primers anneal, and the Taq makes the Taq makes 4 new strands: new strands:

Analysis of product

Agarose gel electrophoresis NA (-ve) migrate in an electric field. NA (-ve) migrate in an electric field. Rate of migration is inversely proportional to Rate of migration is inversely proportional to

the the size of the nucleic acid fragment. of the nucleic acid fragment. Small pieces fast, larger pieces slowly Small pieces fast, larger pieces slowly

By comparison with the By comparison with the migration rates of migration rates of standards of known standards of known sizes, it is possible to sizes, it is possible to estimate the size of an estimate the size of an uncharacterized uncharacterized fragment of DNAfragment of DNA

Advantages

Simple – reaction tubes, reagents, heat – reaction tubes, reagents, heat Rapid – Each cycle 1-3 mins. Each cycle 1-3 mins. Analysis using Analysis using miniscule amounts. Sensitive, accurate, high reliability – Looks directly – Looks directly

at DNA rather than searching for clues. at DNA rather than searching for clues. Unlimited quantities Wide range of specimens even old ones. post mortem even old ones. post mortem

studies and archaeological studies.studies and archaeological studies. No need of cloningNo need of cloning

Limitations

Reaction is Reaction is sensitive to the levels of divalent to the levels of divalent cations cations

Conditions for each particular application must Conditions for each particular application must be worked out = be worked out = Eg. Wrong annealing time, no binding. Requires Requires optimisation.

Primer design = must be very specific for the = must be very specific for the template to be amplified. template to be amplified.

CONTAMINATION = erroneous results. = erroneous results. Closed environment.Closed environment.

Applications – Human Health

Characterize, analyze, synthesize NA.Characterize, analyze, synthesize NA.Detection of infectious disease organisms –

and taxonomic classifications.Detection of variations and mutations in genes

– Screening of genetic disorders and can provide classification

Faster and reliable diagnostic procedure

Can detect bacterial infections even when C Can detect bacterial infections even when C &S fails. Faster.&S fails. Faster.

Certain diagnosis is based on the signs and Certain diagnosis is based on the signs and symptoms . More specific.symptoms . More specific.

Multiple detection. Eg. can detect 3 different Multiple detection. Eg. can detect 3 different STD on a single swab.STD on a single swab.

• DNA fingerprinting - Layout of DNA Layout of DNA fragments. Ffragments. Forensic technique used to identify orensic technique used to identify a person by analysis of DNA from samples eg a person by analysis of DNA from samples eg blood, semen, saliva, hair, etc. blood, semen, saliva, hair, etc.

• Paternity testings - Genetic relationships can Genetic relationships can be determined from two or more genetic be determined from two or more genetic fingerprintsfingerprints

Cloning genes

The process of isolating a gene from one The process of isolating a gene from one organism and then inserting it into another organism and then inserting it into another organism (GMO). organism (GMO).

PCR amplifies the gene = inserted into a PCR amplifies the gene = inserted into a vectorvector

DNA can then be transferred into A GMO for DNA can then be transferred into A GMO for analysisanalysis