POLYMERASE CHAIN REACTION (PCR): PRINCIPLES AND APPLICATIONS DR. IRAM SIDDIQUE ASSISTANT PROFESSOR

What is PCR? The polymerase chain reaction (PCR) is a technique to amplify a piece of DNA very rapidly outside of a cell. Starting with a single molecule of DNA, 25 rounds or cycles of PCR will produce about 10 million identical DNA molecules!!

It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993.

P: POLYMERASE C: CHAIN R: REACTION

What is PCR? : Why “Polymerase”? It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.

What is PCR? : Why “Chain”? It is called “chain” because the products of the first reaction become substrates of the following one, and so on.

What is PCR? : The “Reaction” Components 1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks. 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction 5) Mg++ ions – cofactor of the enzyme 6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme

DNA DNA Sugar DNA has four nitrogen bases. Deoxyribonucleic acid 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 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 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 Example of bonding pattern. Primary strand CCGAATGGGATGC GGCTTACCCTACG Complementary strand

DNA Molecule Adenine Thymine Cytosine Guanine

PCR PCR is a technique that takes a specific sequence of DNA of small amounts and amplifies it to be used for further testing. PCR Targets: The targets in PCR are the sequences of DNA on each end of the region of interest, which can be a complete gene or small sequence.

TTAAGGCTCGA . . . . AATTGGTTAA PCR Targets The number of bases in the targets can vary. TTAAGGCTCGA . . . . AATTGGTTAA The . . . . Represents the middle DNA sequence

PCR Requirements Magnesium chloride: 0.5-2.5mM Buffer: pH 8.3-8.8 dNTPs: 20-200µM Primers: 0.1-0.5µM DNA Polymerase: 1-2.5 units Target DNA:  1 µg

PCR Cycles Review Denaturalization: 94°- 95°C Primer Annealing: 50°- 65°C Extension of DNA: 72° C Number of Cycles: 25-40

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.

PCR Primers TTAACGGCCTTAA . . . TTTAAACCGGTT AATTGCCGGAATT . . . . . . . . . .> and <. . . . . . . . . . AAATTTGGCCAA

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 Taq DNA Polymerase Taq stands for Thermus aquaticus, which is a microbe found in 176°F hot springs in Yellow Stone National Forest. Taq produces an enzyme called DNA polymerase, that amplifies the DNA from the primers by the polymerase chain reaction, in the presence of Mg.

PCR Denaturing Denaturing is the first step in PCR, in which the DNA strands are separated by heating to 95°C.

PCR Annealing Annealing is the process of allowing two sequences of DNA to form hydrogen bonds. The annealing of the target sequences and primers is done by cooling the DNA to 55°C.

Extension DNA polymerization by a thermostable DNA polymerase at 72 degrees C.

The Reaction PCR tube THERMOCYCLER

Lower temperature to 56oC Anneal with primers Denature (heat to 95oC) Lower temperature to 56oC Anneal with primers Increase temperature to 72oC DNA polymerase + dNTPs

PCR Cycles

PCR Cycles

PCR Cycles

PCR Cycles

PCR Cycles

Applications of PCR Detection of pathogens Classification of organisms DNA fingerprinting Drug discovery Genetic matching Genetic engineering Pre-natal diagnosis Classification of organisms Genotyping Molecular archaeology Mutagenesis Mutation detection Sequencing Cancer research

Some applications of PCR. Forensic medicine. Preimplantation Genetic Diagnosis (PGD). Archeology. Paternity testing.

Forensic uses of PCR PCR can be used to amplify DNA from a small amount of cells (about 1000 cells). The amplified DNA from cells can be used in DNA fingerprinting analysis to determine who was at the crime scene.

DNA fingerprinting using PCR in forensic investigations. DNA is isolated from blood at a crime scene and amplified by PCR. The amplified DNA is digested with restriction enzymes and resolved on an agarose gel. Southern blot analysis is performed to give a DNA fingerprint.

MOLECULAR IDENTIFICATION:

Detection Of Pathogens Molecular Identification: Detection Of Pathogens

Detection Of Pathogens Molecular Identification: Detection Of Pathogens Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al.1994)

Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al.1994)