1. TYPES OF PCR AND FLUORIMETER
ETBM: Essentials Techniques in Biochemistry and
Molecular Biology

2. PCR - POLYMERASE CHAIN REACTION

Amplification of single or a few copies of a piece of DNA

Applications:
DNA fingerprinting
 The analysis of ancient DNA from fossils
 Mapping the human genome
 The isolation of a particular gene
 Generation of probes
 Heriditary diseases
 Production of DNA for sequencing


3. PCR – AN OVER VIEW
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Ingredients:
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DNA template
Primers
Taq polymerase
dNTPs
Buffer solution
Mg2+
Procedure:
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Initialization step
Denaturation step
Annealing step
Extension/elongation step
Final elongation

5. TYPES OF PCR
 Reverse
Transcriptase PCR
 Real Time PCR
 Nested PCR
 Multiplex PCR
 Inverse PCR
 Touch Down PCR

6. REVERSE TRANSCRIPTASE PCR

Principle:
RNA strand is reverse transcribed into its cDNA
 Used to compare mRNA levels among samples.
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Advantages:
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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

7. REAL TIME PCR
The probe used
Procedure

8. REAL TIME PCR


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Simplifies amplicon recognition
Amplification progress can be measured
simultaneously
The analysis can be performed without opening the
tube

9. NESTED PCR
Principle:
 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.
Technique:
•
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
run.
•
Result: We can get multiple copies.

10. NESTED PCR

11. Advantages
Identify
error
Specific
PCR amplification

12. MULTIPLEX PCR
Principle:
The detection of more than one template
in a mixture by addition of more than one
set of oligonucleotide primers.
Technique:
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Multiple primer sets within a single PCR
mixture
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
electrophoresis

13. 
Multiplex PCR

14. Advantages
Less
in cost
Less
time consuming
Indication
of Template Quality

15. INVERSE PCR
Principle:
 Information of one internal sequence.
 one known sequence
 primers may be designed.
Method:
 Series of restriction digestions and ligations
 Looped fragment

16. CONT.
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Primed for PCR from a
single section of known
sequence
Amplified by the
temperature-sensitive DNA
polymerase
Target DNA
Fragments of kilobases
Self ligation for circular
DNA
PCR is carried out as
usual, with primers
complementary to sections
of the known internal
sequence.

17. CONT.
Advantages:
 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.

18. TOUCH DOWN PCR
Principle:
 Initial annealing temperature being higher than the
optimal Tm of primers

Gradually reduced over subsequent cycles.

19. CONT.
Method:
 same as that of the standard PCR
 Differences of the annealing temperature at the initial
cycles
 (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
primer Tm.

20. CONT.
Advantages:
 for templates that are difficult to amplify

To enhance specificity
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Increases yield without lengthy optimizations

21. 
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.

22. FLOUROMETER
An instrument for detecting and measuring fluorescence

23. FLUORESCENCE
 Fluorescence
is the emission of light by
a substance that has absorbed light or
other electromagnetic radiation

24. COMPONENTS
1.
All fluorescence instruments contain three basic
elements
a source of light
2.
a sample holder
3.
a detector
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25. SCHEMATIC
REPRESENTATION

26. 
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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

27. CONT..
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
photomultiplier
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28. TYPES OF FLUOROMETER

Two types of fluorometer
•
Filter fluorometer
•
Spectro fluorometer

29. FILTER 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.

30. CONT..
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


31. SPECTROFLUOROMETER:
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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.

32. FLUOROMETER VS SPECTROPHOTOMETER:

A fluorometer measures fluorescence, a
spectrophotometer measures absorbance or
transmittance.

33. ADVANTAGES
•
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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
spectra.

34. CONCLUSION