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TYPES OF PCR AND FLUORIMETER
ETBM: Essentials Techniques in Biochemistry and Molecular Biology
PCR - POLYMERASE CHAIN REACTION
Amplification of single or a few copies of a piece of DNA
Applications:
DNA fingerprintingThe analysis of ancient DNA from fossilsMapping the human genomeThe isolation of a particular geneGeneration of probesHeriditary diseasesProduction of DNA for sequencing
PCR – AN OVER VIEW Ingredients:
DNA template Primers Taq polymerase dNTPs Buffer solution Mg2+
Procedure: Initialization step Denaturation step Annealing step Extension/elongation step Final elongation
TYPES OF PCR
Reverse Transcriptase PCR Real Time PCR Nested PCR Multiplex PCR Inverse PCR Touch Down PCR
REVERSE TRANSCRIPTASE PCR
Principle: RNA strand is reverse transcribed into its cDNA Used to compare mRNA levels among samples.
Advantages: 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 PCRThe probe used
Procedure
REAL TIME PCR
Simplifies amplicon recognition
Amplification progress can be measured simultaneously
The analysis can be performed without opening the tube
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.
NES
TED
PC
R
Advantages
Identify error
Specific PCR amplification
MULTIPLEX PCR
Principle: The detection of more than one template in a mixture by addition of more than one set of oligonucleotide primers.Technique:• 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
Multiplex PCR
Advantages
Less in cost
Less time consuming
Indication of Template Quality
INVERSE PCR
Principle: Information of one internal sequence. one known sequence primers may be designed.Method: Series of restriction digestions and ligations Looped fragment
CONT.
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.
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.
TOUCH DOWN PCR
Principle: Initial annealing temperature being higher than
the optimal Tm of primers
Gradually reduced over subsequent cycles.
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.
CONT.
Advantages: 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. Non-specific primer binding results in the amplification of undesired products and may mask the actual copy number of the gene of interest.
FLOUROMETER
An instrument for detecting and measuring fluorescence
FLUORESCENCE
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation
COMPONENTS
All fluorescence instruments contain three basic elements
1. a source of light
2. a sample holder
3. a detector
SCHEMATIC REPRESENTATION
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
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
TYPES OF FLUOROMETER
Two types of fluorometer
• Filter fluorometer
• Spectro fluorometer
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.
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
SPECTROFLUOROMETER:
• 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 transmittance.
ADVANTAGES
• 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.
CONCLUSION
