Fig.-Denaturation of DNA template by disrupting the
hydrogen bonds between complementary bases of the DNA strands, yielding single
strands of DNA.
Step 2: Annealing
· The reaction is cooled to enable the
primers to attach to a specific location on the single-stranded template DNA by
way of hydrogen bonding.
· The temperature depends on the
characteristics of the primer, but is usually between 50 and 65⁰C.
· The two separated strands of DNA are
complementary and run-in opposite directions (from one end – the 5’ end – to
the other – the 3’ end). As a result, there are two primers – a forward primer
and a reverse primer.
· This step is essential because the primers
serve as the starting point for DNA synthesis, by providing a short region of
double stranded DNA for the polymerase enzyme to work with. Only once the
primer has bound can the polymerase enzyme attach and start making the new
complementary strand of DNA from the loose DNA bases, in the extending step.
· The annealing step usually takes about
10-30 seconds.
Fig.-Then
the temperature is lowered to a temperature between 46-60 °C. The exact
temperature depends on the experiment, and is known as
the annealing temperature. At this point, the primers will
attach, or anneal, to their binding positions on the single strands of the
template DNA.
Step 3: Extending
· The heat is increased to 72⁰C to enable
the new DNA to be made by a special Taq DNA polymerase enzyme which adds DNA
bases.
· Taq DNA polymerase is an enzyme taken from
the bacteria Thermus aquaticus (“Taq”):
· This bacterium normally lives in hot
springs so can tolerate temperatures above 80⁰C, but its optimum temperature is
72⁰C.
· The bacteria’s DNA polymerase is very
stable at high temperatures, which means it can withstand the temperatures
needed to break the strands of DNA apart in the denaturing stage of PCR.
· DNA polymerase from most other organisms
would not be able to withstand these high temperatures. For example, human
polymerase works ideally at 37˚C (body temperature).
· At 72⁰C, the Taq polymerase begins to
build the complementary strand. It attaches to the primer and then adds DNA
bases to the single strand one-by-one in the 5’ to 3’ direction.
· The result is a brand-new strand of DNA
and a double-stranded molecule of DNA.
· The duration of this step depends on the
length of DNA sequence being amplified. It usually takes around one minute to
copy 1,000 DNA bases.
Fig.-optimal temperature
for the polymerase enzyme (1), from which PCR takes its name, to
start working. The polymerase enzyme builds DNA strands, and it will extend the
DNA from the primer along the DNA template, creating a new DNA strand, which
combines with the single-stranded template to form a double strand. The
polymerase enzyme uses dNTPs (2), free DNA nucleotide bases as
the building blocks for the new strand.
General Guidelines for
primers
1. Length:
´ Shorter primers have a tendency to go and anneal to
the non-target sequence of the DNA template.
´ Short primer may offer sufficient for a simple
template such as a small plasmid
´ But a long primer may be required when using
eukaryotic genomic DNA as template. In practice, 20-30 nucleotides is generally
satisfactory.
2. Mismatches:
´ Do not need to match the template completely.
´ Often beneficial to have C or Gas the 3' terminal
nucleotide which makes the binding of the 3' end of the primer to the template
more stable.
3. Melting Temperature Tm:
´ Melting temperature is the temperature at which one
half of the DNA duplex will dissociated and become single stranded. Typically, the
annealing temperature is about 3-5 degrees Celsius bellow the Tm of the primers
used.
´ Primers with melting temperatures in the range of
52-58°C generally produce the best results. Primers with melting temperatures
above 65°C have a tendency for secondary annealing.
´ Tm can be calculated from the following formula:
´
Tm= (4 x [G+C]) + 2 x [A+T])
4. Internal Secondary Structure:
´ Should be avoided in order to prevent the primer to
fold back on itself and not be available to bind to the template.
5. Primer-Primer Annealing:
´ Also, important to avoid the two primers being able to
anneal to each other. Extension by DNA polymerase of two self-annealed primers
leads to formation of a primer dimer.
6. G/C content:
´ Ideally a primer should have a near random mix of
nucleotides, a 50% G/C content.
Analysis
of Product in PCR
There are two main
methods of visualizing the PCR products: (1) staining of the amplified DNA
product with a chemical dye such as ethidium bromide, which intercalates
between the two strands of the duplex or (2) labeling the PCR primers or
nucleotides with fluorescent dyes (fluorophores) prior to PCR amplification.
The latter method allows the labels to be directly incorporated in the PCR
product. The most widely used method for analyzing the PCR product is the use
of agarose gel electrophoresis, which separates DNA products on the basis of
size and charge. Agarose gel electrophoresis is the easiest method of
visualizing and analyzing the PCR product. It allows for the determination of
the presence and the size of the PCR product (Figure). A predetermined set of
DNA products with known sizes are run simultaneously on the gel as standardized
molecular markers to help determine the size of the product.
1.Agarose gel electrophoresis: Tells:
) DNA(2)
