6-Polymerase Chain Reaction (PCR)

6-Polymerase Chain Reaction (PCR)

• PCR is a test tube method to multiply DNA.
• It requires a DNA template (the sample to be amplified).
• It needs free 3'-OH primers to initiate the polymerase reaction.
• The primers are complementary to the ends of the sequence for amplification.
• The reaction needs substrates (dNTPs), excess, thermostable DNA polymerase (Taq polymerase).
• An optimum temperature is needed, provided by a thermal cycler.
• The reaction needs an optimum pH, in an enzyme buffer.
• The reaction volume of deionized water must be free of ions, to prevent DNA precipitation.

PCR Process (Procedure)

• Denaturation: The DNA template strands are heated to become single-stranded.
• Annealing: The DNA is cooled to allow primers to bind to the complementary strand. The temperature for this step depends on the primer's and target DNA's size, GC content, and homology.
• Primer extension: DNA polymerase extends the primers in the presence of Mg2+, and the temperature is optimal for the enzyme used. The most common enzyme is Taq polymerase.

PCR Cycles and Result

• The three steps (denaturation, annealing, and extension) are repeated 28-35 times.
• PCR products are amplified exponentially.
• Contribution of amplification beyond the target sequence becomes linear over time.
• 25 cycles in a thermal cycler produce a significant amplification of the target sequence, approximately 2 to the 25th power
• PCR products can be visualized using a gel stained with nucleic acid-specific fluorescent dye compounds (e.g., ethidium bromide or SYBR green).
• Radio-labeled DNA using radio-labeled dNTPs can also be used with X-ray examination.
• Early PCR experiments used E. coli DNA polymerase.

Heat Sensitivity in PCR

• E. coli DNA polymerase is heat-sensitive.
• Its activity is destroyed during the 95°C denaturation step, requiring the addition of a new aliquot of the enzyme in each cycle or manual temperature control.

Automated PCR

• The development of thermal cyclers automated the process, allowing rapid switching between temperatures.
• Researchers can set up the reactions and return hours later to obtain products.

DNA Sequencing

• DNA sequencing characterizes cloned or amplified genes.
• Methods for DNA sequencing include manual methods (Sanger, dideoxy) and automated systems.

Manual DNA Sequencing (Sanger Method)

• Single-stranded DNA is mixed with radioactively labelled primer and DNA polymerase to initiate the DNA synthesis reaction.
• Complementary primers are used, targeting a region outside the cloning site.
• Replicating terminators (dideoxynucleoside triphosphates or ddNTPs) with no 3' -OH are added to separate reaction mixtures.
• These, lacking a 3'-OH, prevent bonding with the next nucleotide, creating different lengths of synthesized strands.
• The resulting fragments are separated via gel electrophoresis.
• The sequence is determined by reading the radioactive bands.
• The smallest fragments (those with the shortest chain that were terminated) represent the 5' end of the synthesized sequence.

Automated DNA Sequencing

• Fluorescent markers replace radioactive markers in automated sequencing.
• Combined sequencing reactions.
• Polyacrylamide gel is used to resolve samples.
• The dye molecules are excited with a laser to read the DNA sequence.
• Early automated systems produced 4800 bases per day.
• Current systems incorporate arrays of tiny capillaries as "lanes."

This quiz covers the polymerase chain reaction (PCR) process, detailing the necessary components and steps involved in amplifying DNA. Learn about denaturation, annealing, and primer extension, as well as the roles of enzymes and temperature in this essential laboratory technique.