The Polymerase Chain Reaction (PCR) file/molecular... · What is the Polymerase Chain Reaction? •...

The Polymerase Chain Reaction (PCR) Reaction (PCR)

Professor Md. Akram Hossain

MMC

12/24/2013 1Prof. Muhammad Akram Hossain, PCR

Topics

• What is the Polymerase Chain Reaction?

• History and (pre-history) of PCR

• How PCR works• How PCR works

• PCR primer design

• Applications of PCR


What is the Polymerase Chain Reaction?

• Chain reaction by DNA polymerase resulting amplification of target DNA -particular segment of DNA

• The segment may represent a small part of a large and complex mixture of DNAs:large and complex mixture of DNAs:e.g. A segment of bacteria, virus, toxin, a specific exon of a human gene.

• It can be thought of as a molecular photocopier.


A Molecular Photocopier

• A photocopier capable of duplicating a part of a sentence:

• “The next day was quite a different day. Instead of being • “The next day was quite a different day. Instead of being hot and sunny, it was cooland misty. Pooh didn’t mind for himself, but when he thought of all the honey the beeswouldn’t be making,a cold misty day always made him feel sorry for them.” A.A. Milne, 1928.

• The words in bluemust be unique for the copier to locate the correct piece of text.


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 template DNA need not be highly purified — a boiled bacterial colony.

• The PCR product can be digested with restriction enzymes, sequenced or cloned.

• PCR can amplify a single DNA molecule, e.g. from a single sperm.


The Invention of PCR

• Invented by Kary Mullis in 1983.

• First published account appeared in

• First published account appeared in 1985.

• Awarded Nobel Prize for Chemistry in 1993.


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: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.


The Basics of PCR Cycling

• 30–35 cycles each comprising:– denaturation (95°C),

30 sec.30 sec.

– annealing (55–60°C), 30 sec.

– extension (72°C),time depends on product size.


What’s in the Reaction?• Template DNA• Reaction buffer (Tris, ammonium ions (and/or

potassium ions), magnesium ions, bovine serum albumin)

• Magnesium chloride• Magnesium chloride• Cofactor for DNA polymerase• Needed for optimal activity

• 4 Nucleotides (dNTPs)• Primers• DNA polymerase (usually Taq)


PCR In Detail

• Denature, anneal, extend and repeat the cycle 30 to 35 times.

• “How does the polymerase know to stop • “How does the polymerase know to stop when it reaches the other primer?”

• PCR animation

• PCR animationof DNA sequencing


Reaction Cycle• Denaturing step

– 94-95°C– Denatures the double stranded DNA into single

strands

• Annealing step• Annealing step– 45-60°C– Allows the primers to attach to complementary strand

of DNA

• Extension step– 72°C– Optimal temperature for Taq polymerase to attach to

and extend the new strand of DNA12/24/2013 11Prof. Muhammad Akram Hossain, PCR

Reaction Cycle• These steps are repeated for 20-30 cycles

• Thus DNA increases exponentially – 2n where n = # of cycles– 30 cycles = 230 = 1x 109 copies of DNA

• Usually >30 cycles is not beneficial• Usually >30 cycles is not beneficial– Decreased enzyme activity– Decreased dNTP concentration


Reaction Cycle

5’5’5’5’

5’5’5’5’

5’5’

DenaturingDenaturing

AnnealingAnnealing

5’5’5’5’

5’5’

5’5’5’5’

5’5’

5’5’

5’5’

ExtensionExtension

Repeat CycleRepeat Cycle


How many copies?

• No target products are made until the third cycle.

• The accumulation is not strictly a doubling • 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 (~1×109).

• 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 plateau occurs when:– The reagents are depleted– The products re-anneal– The polymerase is

damaged

• Unwanted products accumulate.


So Then, it’s Easy?

• Cycling performed with three water baths.• Thermal cyclers introduced in 1986.• Early polymerases were not thermostable, • Early polymerases were not thermostable,

so had to be replenished each cycle.• The 37°C temperature caused non-specific

priming, resulting in unwanted products.• Taq (Thermus aquaticus) DNA polymerase

first described in 1988.


Thermal Cyclers•PCR cyclers available from many suppliers.

•Many block formats and multi-block systems.

•Reactions in tubes or 96-well micro-titre plates.


Can I PCR Amplify RNA?

• Not directly — the DNA polymerase requires a DNA template and will not copy RNA.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 blunt-ended restriction enzyme site.

• Lower than expected efficiency.• Lower than expected efficiency.• Products are not truly blunt-ended.• Taq polymerase adds a single non-

templated base (usually A) to the 3´ end:NNNNNNN…NNNNNNNA

ANNNNNNN…NNNNNNN


TA Cloning of PCR Products

• Take advantage of the non-templated bases.

• Linearise vector at a blunt-ended site (e.g.EcoRV).EcoRV).

• Incubate linear vector with Taq polymerase and dTTP to add non-templated Ts.

• Ligate:


Designing PCR Primers

• Primers should be ~20 bases long.• The G/C content should be 45–55%.• The annealing temperatures should be • The annealing temperatures should be

within 1°C 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.12/24/2013 21Prof. Muhammad Akram Hossain,

PCR

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 • Ala alleles are digested by AluI, producing fragments of 82 & 574 bp.


Applications of PCR

• Diagnosis or screening of microbial diseases– where culture are difficult or hazardous , e.g.

• AIDS, TB, Chlamydia, Mycoplasma, rickettssia etc.

• Sensitivity testing-– resistance gene detection

• Mutation testing, e.g. cystic fibrosis.• Forensic medicine

– Paternity testing, victim identification, Genetic profiling in legal and bio-diversity applications.

• Site-directed mutagenesis of genes.• Quantitation of mRNA in cells or tissues.


Multiplex PCR

• PCR reactions can be devised in which several targets are amplified simultaneously — often used in diagnostic applications.— often used in diagnostic applications.


RAPD-PCR• Random Amplified Polymorphic DNA PCR

• DNA fragments generated by PCR amplification– Uses short arbitrary primers 8-10 bp long– DNA synthesis at sites the primers “just happen” to

bind tobind to– Amplification of various sized fragments resulting in a

banding pattern – As few as 1 or as many as 12 primers can be used

• Advantages over RFLPs– Only takes 2-3 hours– Only needs a small amount of DNA– Very inexpensive12/24/2013 25Prof. Muhammad Akram Hossain,

PCR

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