Types of pcr and fluorimeter (1)

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  • 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)

    1. 1. TYPES OF PCR AND FLUORIMETER ETBM: Essentials Techniques in Biochemistry and Molecular Biology
    2. 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. 3. 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
    4. 4. TYPES OF PCR  Reverse Transcriptase PCR  Real Time PCR  Nested PCR  Multiplex PCR  Inverse PCR  Touch Down PCR
    5. 5. 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
    6. 6. REAL TIME PCR The probe used Procedure
    7. 7. REAL TIME PCR    Simplifies amplicon recognition Amplification progress can be measured simultaneously The analysis can be performed without opening the tube
    8. 8. 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.
    9. 9. NESTED PCR
    10. 10. Advantages Identify error Specific PCR amplification
    11. 11. 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
    12. 12.  Multiplex PCR
    13. 13. Advantages Less in cost Less time consuming Indication of Template Quality
    14. 14. INVERSE PCR Principle:  Information of one internal sequence.  one known sequence  primers may be designed. Method:  Series of restriction digestions and ligations  Looped fragment
    15. 15. 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.
    16. 16. 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.
    17. 17. TOUCH DOWN PCR Principle:  Initial annealing temperature being higher than the optimal Tm of primers  Gradually reduced over subsequent cycles.
    18. 18. 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.
    19. 19. CONT. Advantages:  for templates that are difficult to amplify  To enhance specificity  Increases yield without lengthy optimizations
    20. 20.  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.
    21. 21. FLOUROMETER An instrument for detecting and measuring fluorescence
    22. 22. FLUORESCENCE  Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation
    23. 23. COMPONENTS 1. All fluorescence instruments contain three basic elements a source of light 2. a sample holder 3. a detector 
    24. 24. SCHEMATIC REPRESENTATION
    25. 25.     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
    26. 26. 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 
    27. 27. TYPES OF FLUOROMETER  Two types of fluorometer • Filter fluorometer • Spectro fluorometer
    28. 28. 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.
    29. 29. 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 
    30. 30. 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.
    31. 31. FLUOROMETER VS SPECTROPHOTOMETER:  A fluorometer measures fluorescence, a spectrophotometer measures absorbance or transmittance.
    32. 32. 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.
    33. 33. CONCLUSION

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