Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 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. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °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. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.
Types of pcr and fluorimeter (1)
TYPES OF PCR AND FLUORIMETER
ETBM: Essentials Techniques in Biochemistry and
PCR - POLYMERASE CHAIN REACTION
Amplification of single or a few copies of a piece of DNA
The analysis of ancient DNA from fossils
Mapping the human genome
The isolation of a particular gene
Generation of probes
Production of DNA for sequencing
PCR – AN OVER VIEW
TYPES OF PCR
Real Time PCR
Touch Down PCR
REVERSE TRANSCRIPTASE PCR
RNA strand is reverse transcribed into its cDNA
Used to compare mRNA levels among samples.
A low copy number of RNA can be detected
Also the diagnosis of genetic diseases
Measure of gene expression.
Insertion of eukaryotic genes into prokaryotes
Studying viral genomes
REAL TIME PCR
Simplifies amplicon recognition
Amplification progress can be measured
The analysis can be performed without opening the
Nested PCR is a variation of the polymerase
chain reaction (PCR), in that two pairs
(instead of one pair) of PCR primers are
used to amplify a fragment.
Step 1: Primers binds to template DNA
and PCR start.
Step 2: PCR products from the first PCR
reaction are subjected to a second PCR
Result: We can get multiple copies.
The detection of more than one template
in a mixture by addition of more than one
set of oligonucleotide primers.
Multiple primer sets within a single PCR
Amplifying multiple targets on the same
strand of DNA at the same time
Multiple amplicons need to be expressed
Different bands can be visualize by gel
of Template Quality
Information of one internal sequence.
one known sequence
primers may be designed.
Series of restriction digestions and ligations
Primed for PCR from a
single section of known
Amplified by the
Fragments of kilobases
Self ligation for circular
PCR is carried out as
usual, with primers
complementary to sections
of the known internal
Determination of insert locations.
Various retroviruses and transposons randomly
integrate into genomic DNA.
"internal" viral or transposon sequences
Design primers that will amplify a small portion of the
flanking, "external" genomic DNA.
TOUCH DOWN PCR
Initial annealing temperature being higher than the
optimal Tm of primers
Gradually reduced over subsequent cycles.
same as that of the standard PCR
Differences of the annealing temperature at the initial
(3-5 °C) above the Tm of primers used
Decreasing by 0.2 °C per cycle.
Later cycles, it is a few degrees (3-5 °C) below the
for templates that are difficult to amplify
To enhance specificity
Increases yield without lengthy optimizations
In PCR, the temperature at
which primers anneal during a
cycle determines the specificity
of annealing. The melting point
(Tm) of the coolest primer sets
the upper limit on annealing
temperature. At temperatures
just below the Tm, only very
specific base pairing between
the primer and the template
will occur. As the temperature
decreases, primer binding
becomes less specific. Nonspecific primer binding results
in the amplification of
undesired products and may
mask the actual copy number of
the gene of interest.
An instrument for detecting and measuring fluorescence
is the emission of light by
a substance that has absorbed light or
other electromagnetic radiation
All fluorescence instruments contain three basic
a source of light
a sample holder
Excitation energy is provided by a light source
Light passes through a primary (excitation)
filter before entering sample compartment
Light is absorbed by the fluorescent dye sample
After excitation of the fluorescent substance,
return to energy state occurs and light with a
longer wavelength(fluorescence) is emitted
Fluoroscent light passes through a secondary
filter (emission) which is opaque to light passing
the primary filter and is at 90 degree angle to the
primary light path
The amount of light passing through the
secondary filter is measured on a
TYPES OF FLUOROMETER
Two types of fluorometer
Filter fluorometers produce specific excitation
and emission wavelengths by using optical filters.
The filter blocks other wavelengths but transmits
wavelengths relevant to the compound.
The light passes through the sample to be
measured, and a certain wavelength is absorbed
while a longer wavelength is emitted.
The emitted light is measured by a detector. By
changing the optical filter, different substances
can be measured
Spectrofluorometers use high intensity light sources to
bombard a sample with as many photons as possible.
This allows for the maximum number of molecules to be in
the excited state at any one point in time.
The light is either passed through a filter, selecting a fixed
wavelength, or monochromator, which allows to select a
wavelength of interest to use as the exciting light.
The emission is collected at 90 degrees to the exciting light.
The emission too is either passed through a filter or a
monochromator before being detected by a PMT.
FLUOROMETER VS SPECTROPHOTOMETER:
A fluorometer measures fluorescence, a
spectrophotometer measures absorbance or
The principal advantage of fluorescence over
radioactivity and absorption spectroscopy is the
ability to separate compounds on the basis of
either their excitation or emission spectra, as
opposed to a single spectra.
This advantage is further enhanced by
commercial fluorescent dyes that have narrow
and distinctly separated excitation and emission