Md. Md. Golam Hossain, Ph.D.

Pharmacy Discipline Khulna University

Replication
DNA

Transcription
RNA

Translation
PROTEIN

The central dogma of molecular biology

Discovery
PCR was discovered by Kary Mullis in 1983
On a long motorcycle drive Mentally visualized the process

Nobel Prize in Chemistry

1993

Polymerase Chain Reaction

Polymerase: DNA polymerase
DNA polymerase duplicates DNA Before a cell divides, its DNA must be duplicated Chain Reaction: The product of a reaction is used to amplify the same reaction Results in rapid increase in the product

polymerase chain reaction

The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. including: Selective DNA isolation, Amplification and quantification of DNA, PCR in diagnosis of diseases DNA cloning for sequencing, functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases.

A basic PCR set up requires several components and reagents.[ components include:
DNA template that contains the DNA region (target) to be amplified. Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target. Taq polymerase or another DNA polymerase with a temperature optimum at around 70 C. Deoxynucleoside triphosphates (dNTPs; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand. Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis[7] Monovalent cation potassium ions.

primer
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA. The polymerase starts replication at the 3'-end of the primer, and copies the opposite strand. In most cases of natural DNA replication, the primer for DNA synthesis and replication is a short strand of RNA

Thermal Cycling
A PCR machine controls temperature Typical PCR go through three steps
Denaturation Annealing Extension

Denaturation
Heating separates the double stranded DNA
Denaturation

Slow cooling anneals the two strands

Renaturation

Heat

Cool

Annealing

Two primers are supplied in molar excess They bind to the complementary region As the DNA cools, they wedge between two template strands Optimal temperature varies based on primer length etc. Typical temperature from 40 to 60 C

Extension

DNA polymerase duplicats DNA Optimal temperature 72C

PCR Amplification
Exponential Amplification of template DNA

Typical PCR mix

In a thin wall Eppendorf tube assemble the following
PCR components Template DNA (5-200 ng) 1 mM dNTPs (200 uM final) 10 X PCR buffer 25 mM MgCl2 (1.5 mM final) 20 uM forward primer (20 pmoles final) 20 uM reverse primer (20 pmoles final) 5 units/uL Taq DNA polymerase (1.5 units) Water Amount variable 10 uL 5 uL 3 uL 1 uL 1 uL 0.3 uL Variable

Final Volume

50 uL

Steps in PCR
1. Annealing step: The reaction temperature is lowered to 5065 C for 2040 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. The polymerase binds to the primer-template hybrid and begins DNA synthesis. 2. Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 7580 C, and commonly a temperature of 72 C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute.

3. Final elongation: This single step is occasionally performed at a temperature of 7074 C for 515 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
4. Final hold: This step at 415 C for an indefinite time may be employed for short-term storage of the reaction

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 polymerasemore is less.)

Thank you

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 more is less.) 4 Optimising the Annealing Temperature Primers have a calculated annealing temperature (e.g. 54C). Temperature must be confirmed practically. Temperature steps of 2C above and below. Use gradient cycler. Optimising the Mg2+ Concentration The fidelity of the PCR depends on [Mg2+].