Pcr -polymerase Chain Reaction

Polymerase Chain Reaction (PCR)

Nandana N Kumara Wayamba University of Sri Lanka

DNA DNA is a nucleic acid that is composed of two complementary nucleotide building block chains. The nucleotides are made up of a phosphate group, a five carbon sugar, and a nitrogen base.

DNA DNA has four nitrogen bases. • Two are purines ( 2 ringed base ) – Adenine ( A ), Guanine ( G )

• Two are pyrimidines ( 1 ringed base ) – Cytosine ( C ), Thymine ( T )

DNA These four bases are linked in a repeated pattern by hydrogen bonding between the nitrogen bases. The linking of the two complementary strands is called hybridization.

DNA A purine always links with a pyrimidine base to maintain the structure of DNA. Adenine ( A ) binds to Thymine ( T ), with two hydrogen bonds between them. Guanine ( G ) binds to Cytosine ( C ), with three hydrogen bonds between them.

DNA Example of bonding pattern. • Primary strand CCGAATGGGATGC GGCTTACCCTACG • Complementary strand

DNA Molecule Adeni ne Thymi ne Guani ne Cy tosi ne

What is the Polymerase Chain Reaction? • It’s a means of selectively amplifying a particular segment of DNA. • The segment may represent a small part of a large and complex mixture of DNAs: e.g. a specific exon of a human gene.

• It can be thought of as a molecular photocopier.

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 — ex: 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.

Gene Analysis Prior to PCR? • Southern blotting (1975) permitted rudimentary mapping of genes in unrelated individuals (RFLPs, insertions & deletions). • DNA sequencing (1978) required genes to first be cloned into plasmid or λ vectors. • Gene library construction and screening could take many months and libraries had to be made for each individual analyzed.

The Invention of PCR • Invented by Kary Mullis in 1983. • 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: – 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 Each cycle comprise: denaturation (95°C), - 30 sec. annealing (55–60°C), - 30 sec. extension (72°C), - time depends on product size.

What’s in the Reaction mixture? • Template DNA • Reaction buffer (Tris, ammonium ions (and/or potassium ions), magnesium ions, bovine serum albumin) • Nucleotides (dNTPs) • Primers • DNA polymerase (usually Taq)

PCR Primers Primers range from 15 to 30 nucleotides, are single-stranded, and are used for the complementary building blocks of the target sequence.

PCR Primers • A primer for each target sequence on the end of your DNA is needed. • This allows both strands to be copied simultaneously in both directions. • DNA is copied from 3’ end to the 5’ end

PCR Primers 3’TTAACGGCCTTAA . . . TTTAAACCGGTT5’ AATTGCCGGAATT . . . . . . . . . .> and <. . . . . . . . . . AAATTTGGCCAA 5’TTAACGGCCTTAA . . . TTTAAACCGGTT3’

PCR Primers The primers are added in excess so they will bind to the target DNA instead of the two strands binding back to each other.

PCR In Detail • Denature, anneal, extend and repeat the • cycle 30 to 35 times. • How does the polymerase know to stop? “when it reaches the other primer” Most textbooks do not fully explain PCR. • PCR animation at Dolan DNA Learning Center,

How many copies? • No target products are made until the third cycle. • The accumulation is not strictly a doubling at each cycle in the early phase. • If there were 2 DNA molecules at the beginning, at 30 cycles there will be 230 copies. (1073741824)

How many cycles? • Increasing the cycle number above ~35 has little positive effect. • 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, 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.

So, I Can Just Go Ahead? • Not so fast. • The PCR technique and the use of Taq DNA polymerase in PCR are both patented. • Even academic and public organisations must pay license fees • levy paid on enzyme and thermal cycler purchases.

Has It Worked? • • • • •

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.

Optimising the PCR Reaction • • • • • •

Annealing temperature of the primers The concentration of Mg2+ in the reaction The extension time The denaturing and annealing times The extension temperature The amount of template and polymerase

Optimising the Annealing Temperature • Primers have a calculated annealing temperature (e.g. 54°C) • Temperature must be confirmed practically. • Temperature steps of 2°C above and below • Use gradient cycler

Optimising the Mg2+ Concentration • The fidelity of the PCR depends on [Mg2+]. • Vary [Mg2+] in steps of 0.5 mMol. • Sometimes a compromise between yield and specificity.

Fidelity of the Reaction • Taq DNA polymerase lacks the 3´→5´ proof-reading activity commonly present in other polymerases. • Taq mis-incorporates 1 base in 104. • A 400 bp target will contain an error in 33% of molecules after 20 cycles. • Error distribution will be random.

Do Errors Matter? • Yes, if you want to clone the amplified DNA — an individual molecule may harbour several mutations. • No, if you want to sequence the amplified DNA or cut it with restriction enzymes. • Use a proof-reading thermo-stable enzyme rather than Taq.

How Big A Target? • Amplification products are typically in the size range 100-1500 bp. • Longer targets are amplifiable — >25 kb. • Requires modified reaction buffer, ocktails of polymerases, and longer extension times.

Can I PCR Amplify RNA? • Not directly — the DNA polymerase requires a DNA template and will not copy 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 bluntended restriction enzyme site. • Lower than expected efficiency. • Products are not truly blunt-ended. • Taq polymerase adds a single nontemplated base (usually A) to the 3´ end: NNNNNNN…NNNNNNNA ANNNNNNN…NNNNNNN

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

Taq DNA polymerase • • • •

Extracted from Thermus aquaticus Thermo-stable polymerases Not permanently destroyed at 94ºC Optimal temperature is 72ºC

Problems with Taq • Does not have proof reading ability • Error rate 1 in 2 X 104 bases • Seems rare but can be recovered in cloning a single molecule • Newer polymerases have high fidelity

Thermo-stable DNA polymerases