Amplification Techniques In Microbiology


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Amplification Techniques In Microbiology continues to be backbone of laboratory technologies, several earlier methods are giving way to the newer technologies..In spite of several problems in implementing the newer technologies we have to thrive to implement and adopt to newer technologies.

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  • Amplification Techniques In Microbiology

    1. 1. Nucleic Acid Amplification Techniques in Microbiology Dr.T.V.Rao MD
    2. 2. DNA replication Basis of Biological Evolution <ul><li>DNA replication , the basis for biological inheritance, is a fundamental process occurring in all living organisms to copy their DNA. </li></ul>
    3. 3. DNA Replication is the Basis of all Technologies <ul><li>This process is &quot;semi conservative&quot; in that each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Hence, following DNA replication, two identical DNA molecules have been produced from a single double-stranded DNA molecule </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    4. 4. Understanding events in vivo Lead to vitro applications <ul><li>DNA replication can also be performed in vitro (outside a cell). DNA polymerases, isolated from cells, and artificial DNA primers are used to initiate DNA synthesis at known sequences in a template molecule. The polymerase chain reaction (PCR), a common laboratory technique, employs such artificial synthesis in a cyclic manner to amplify a specific target DNA fragment from a pool of DNA </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    5. 5. Har Gobind Khorana, Indian Scientist, Nobel Prize Winner <ul><li>Polymerase Chain Reaction (PCR), a process first described by Kjell Kleppe and 1968 Nobel laureate H. Gobind Khorana that allows the amplification of specific DNA sequences . The improvements provided by Mullis have made PCR a central technique in biochemistry and molecular biology. </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    6. 6. Polymerase Chain Reaction Methodology A Mile stone in Medical History <ul><li>He had the idea to use a pair of primers to bracket the desired DNA sequence and to copy it using DNA polymerase, a technique which would allow a small strand of DNA to be copied almost an infinite number of times. Cetus took Mullis off his usual projects to concentrate on PCR full-time </li></ul>
    7. 7. Dr. Kary Mullis, wins Nobel Prize in 1993 <ul><li>Kary received a Nobel Prize in chemistry in 1993 , for his invention of the polymerase chain reaction (PCR). The process, which Kary Mullis conceptualized in 1983, is hailed as one of the monumental scientific techniques of the twentieth century. </li></ul>
    8. 8. How DNA Works <ul><li>DNA usually exists as a double-stranded structure, with both strands coiled together to form the characteristic double-helix. Each single strand of DNA is a chain of four types of nucleotides: adenine, cytosine, guanine, and thymine. </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    9. 9. DNA – RNA - DNA <ul><li>In Molecular biology, the polymerase chain reaction ( PCR ) is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of a particular DNA sequence. </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    10. 10. Create primers <ul><li>To begin synthesis of a new strand, a short fragment of DNA or RNA , called a primer , must be created and paired with the template strand before DNA polymerase can synthesize new DNA. </li></ul>
    11. 11. Precise Oligonucleotide Matches the Sequences
    12. 12. Thermocycler is Back bone of PCR methodology <ul><li>The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    13. 13. Primers <ul><li>Primers (short DNA fragments) containing sequences complementary to the target region along with a Primers after which the method is named) are key components to enable selective and repeated amplification. </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    14. 14. PCR a Chain reaction <ul><li>As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations . </li></ul>
    15. 15. Taq polymerase fuels the Reaction <ul><li>T.aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase was identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR. It replaced the DNA polymerase from E.coli originally used in PCR . Taq's optimum temperature for activity is 75-80°C, with a halflife of 9 minutes at 97.5°C, and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72°C. </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    16. 16. Bacteria Of Boiling Hot Springs In Yellowstone National Park
    17. 17. Boiling hot springs in Yellowstone National Park. The orange-red coloration is caused by thriving colonies of photosynthetic cyanobacteria.
    18. 18. Taq polymerase <ul><li>Taq polymerase is a thermos table DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock in 1965. It is often abbreviated to &quot; Taq Pol &quot; (or simply &quot; Taq &quot;), and is frequently used in polymerase chain reaction (PCR), methods for greatly amplifying short segments of DNA </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    19. 19. Drawbacks of Taq polymerase <ul><li>One of Taq's drawbacks is its relatively low replication fidelity. It lacks a 3' to 5' exonuclease proofreading activity, and has an error rate measured at about 1 in 9,000 nucleotides. Some thermos table DNA polymerases have been isolated from other thermophilic bacteria and archaea, such as Pfu DNA polymerase, possessing a proofreading activity, and are being used instead of (or in combination with) Taq for high-fidelity amplification. </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    20. 20. Disadvantages of Taq Pol <ul><li>Taq Pol lacks 3’ to 5’ exonuclease proof reading activity, commonly present in </li></ul><ul><li>other polymerases. </li></ul><ul><li>Taq mis-incorporates 1 base in 10 4 . </li></ul><ul><li>A 400 bp target will contain an error in 33% of molecules after 20 cycles. </li></ul><ul><li>Error distribution will be random. </li></ul>
    21. 21. PCR - Three basic Steps <ul><li>Cut </li></ul><ul><li>Paste </li></ul><ul><li>Amplify </li></ul>
    22. 22. What does PCR need? <ul><li>Template (the DNA you are exploring) </li></ul><ul><li>Sequence-specific primers flanking the </li></ul><ul><li>target sequence, Forward & Reverse. </li></ul><ul><li>Polymerases </li></ul><ul><li>Nucleotides (dATP, dCTP, dGTP, </li></ul><ul><li>dTTP) </li></ul><ul><li>Magnesium chloride (enzyme cofactor) </li></ul><ul><li>Buffer </li></ul><ul><li>Water, mineral oil </li></ul>
    23. 23. Four Alphabets make what we are
    24. 24. DNA polymerases <ul><li>DNA polymerases are a family of enzymes that carry out all forms of DNA replication. A DNA polymerase can only extend an existing DNA strand paired with a template strand; it cannot begin the synthesis of a new strand. . </li></ul>
    25. 25. Create primers <ul><li>To begin synthesis of a new strand, a short fragment of DNA or RNA , called a primer , must be created and paired with the template strand before DNA polymerase can synthesize new DNA. </li></ul>
    26. 26. PCR Primers <ul><li>TT AA C GG CC TT AA . . . TTT AAA CC GG TT </li></ul><ul><li>AA TT G CC GG AA TT . . . . . . . . . .> </li></ul><ul><li>and </li></ul><ul><li><. . . . . . . . . . AAA TTT GG CC AA </li></ul><ul><li>TT AA C GG CC TT AA . . . TTT AAA CC GG TT </li></ul>
    27. 27. Cutting, pasting and amplifying is the basis of Reaction
    28. 28. DNA <ul><li>Example of bonding pattern. </li></ul><ul><li>Primary strand </li></ul><ul><li>CC G AA T GGG A T G C </li></ul><ul><li>GG C TT A CCC T A C G </li></ul><ul><li>Complementary strand </li></ul>
    29. 29. What does PCR need? <ul><li>Template (the DNA you are exploring) </li></ul><ul><li>Sequence-specific primers flanking the </li></ul><ul><li>target sequence, Forward & Reverse. </li></ul><ul><li>Polymerases </li></ul><ul><li>Nucleotides (dATP, dCTP, dGTP, </li></ul><ul><li>dTTP) </li></ul><ul><li>Magnesium chloride (enzyme cofactor) </li></ul><ul><li>Buffer </li></ul><ul><li>Water, mineral oil </li></ul>
    30. 30. PCR Requirements <ul><li>Magnesium chloride: .5-2.5mM </li></ul><ul><li>Buffer: pH 8.3-8.8 </li></ul><ul><li>dNTPs: 20-200µM </li></ul><ul><li>Primers: 0.1-0.5µM </li></ul><ul><li>DNA Polymerase: 1-2.5 units </li></ul><ul><li>Target DNA:  1 µg </li></ul>
    31. 31. Steps in PCR <ul><li>Denaturation 93 to 95°C 1min </li></ul><ul><li>Annealing 50 to 55°C 45sec </li></ul><ul><li>Elongation 70 to 75°C 1-2min </li></ul>
    32. 32. How does PCR work? <ul><li>Heat (94 o C) to denature DNA strands </li></ul><ul><li>Cool (54 o C) to anneal primers to template </li></ul><ul><li>Warm (72 o C) to activate Taq Polymerase, </li></ul><ul><li>which extends primers and replicates DNA </li></ul><ul><li>Repeat multiple cycles </li></ul>
    33. 33. Denaturation of DNA <ul><li>Denaturation is the first step in PCR, in which </li></ul><ul><li>the DNA strands are separated by heating to </li></ul><ul><li>95°C. </li></ul><ul><li>The Hydrogen bonds between the two strands </li></ul><ul><li>breaks down and the two strands separates. </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    34. 34. <ul><li>Annealing is the process of allowing two </li></ul><ul><li>sequences of DNA to form hydrogen bonds. </li></ul><ul><li>The annealing of the target sequences and </li></ul><ul><li>primers is done by cooling the DNA to 55°C. </li></ul><ul><li>Time taken to anneal is 45 seconds </li></ul>Annealing
    35. 35. Taq polymerase binds …. <ul><li>Taq polymerase binds to the template DNA </li></ul><ul><li>and starts adding nucleotides that are </li></ul><ul><li>complementary to the first strand. </li></ul><ul><li>This happens at 72°C as it is the optimum </li></ul><ul><li>temperature for Taq Polymerase. </li></ul>
    36. 36. Denaturing Template Heat causes DNA strands to separate Denature DNA strands 94 o C 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’
    37. 37. Annealing Primers <ul><li>Primers bind to the template sequence </li></ul><ul><li>Taq Polymerase recognizes double-stranded substrate </li></ul>3’ 5’ Primers anneal 64 o C 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’
    38. 38. Taq Polymerase Extends 3’ 5’ 3’ 5’ Extend 72 o C 3’ 5’ 3’ 5’ <ul><li>Taq Polymerase extends primer </li></ul><ul><li>DNA is replicated </li></ul>Repeat denaturing, annealing, and extending 30 cycles 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’
    39. 39. Taq Polymerase Extends 3’ 5’ 3’ 5’ Extend 72 o C 3’ 5’ 3’ 5’ <ul><li>Taq Polymerase extends primer </li></ul><ul><li>DNA is replicated </li></ul>Repeat denaturing, annealing, and extending 30 cycles 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’
    40. 40. The target product is made in the third cycle 3 ’ 5’ 3 ’ 5’ 3’ Cycle 1 Cycle 2 Cycle 3 3’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’
    41. 41. PCR Cycles Review <ul><li>Denaturation: 94°- 95°C </li></ul><ul><li>Primer Annealing: 55°- 65°C </li></ul><ul><li>Elongation of DNA: 72° </li></ul><ul><li>Number of Cycles: 25-40 </li></ul><ul><li>No target products are made until the third cycle. </li></ul><ul><li>At 30 cycles there are 1,073,741,764 target copies (~1×10 9 ). </li></ul>
    42. 42. Primers That Form Hairpins <ul><li>Primers can have self-annealing regions within each primer (i.e. hairpin and fold back loops) </li></ul><ul><li>A primer may be self-complementary and be able to fold into a hairpin: </li></ul><ul><li>5´-GTTGACTTGATA </li></ul><ul><li>||||| T </li></ul><ul><li>3´-GAACTCT </li></ul><ul><li>The 3´ end of the primer is base-paired, </li></ul><ul><li>preventing it annealing to the target DNA. </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    43. 43. Applications of PCR differentiates the results can be compared of Individual specimens
    44. 44. Advantages of PCR <ul><li>Speed </li></ul><ul><li>Ease of use </li></ul><ul><li>Sensitivity </li></ul><ul><li>Robustness </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    45. 45. Limitations of PCR <ul><li>Need for target DNA sequence information </li></ul><ul><li>Primer Designing for unexplored ones. </li></ul><ul><li>Boundary regions of DNA to be amplified must be known. </li></ul><ul><li>Infidelity of DNA replication. </li></ul><ul><li>Taq Pol – no Proof reading mech – Error 40% after 20 cycles </li></ul><ul><li>Short size and limiting amounts of PCR product </li></ul><ul><li>Up to 5kb can be easily amplified . </li></ul><ul><li>Up to 40kb can be amplified with some modifications. </li></ul><ul><li>Cannot amplify gene >100kb </li></ul><ul><li>Cannot be used in genome sequencing projects . </li></ul>
    46. 46. How to overcome Difficulties ? <ul><li>Pfu DNA Polymerase from Pyrococcus furiosus possesses 3' to 5' exonuclease proofreading activity. </li></ul><ul><li>The error rate is only 3.5% after 20 cycles </li></ul><ul><li>More amount of primer is added to avoid </li></ul><ul><li>primer dimering. </li></ul><ul><li>For unexplored genes, primers used in closely </li></ul><ul><li>related species are used. </li></ul>
    47. 47. REAL TIME ASSAYS
    48. 48. New Technologies – Real Time Assays <ul><li>The Real Time assays are proving to better technologies </li></ul><ul><li>1 Rapid </li></ul><ul><li>2 Quantitative measurement </li></ul><ul><li>3 Lower contamination rate </li></ul><ul><li>4 Higher sensitivity </li></ul><ul><li>5 Higher specificity </li></ul><ul><li>6 Easy standardization </li></ul><ul><li>Now a new gold standard for rapid diagnosis of virus infection in the acute phase samples. </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    49. 49. Real-time polymerase chain reaction <ul><li>Real-time polymerase chain reaction , also called quantitative real time polymerase chain reaction (Q-PCR/qPCR) or kinetic polymerase chain reaction , is a laboratory technique based on the polymerase chain reaction, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample. </li></ul>
    50. 50. RT - PCR <ul><li>Proving to be </li></ul><ul><li>Accurate </li></ul><ul><li>Precise </li></ul><ul><li>Easy to perform </li></ul><ul><li>RT PCR technologies are easy to transfer research Laboratory protocols to Diagnostic Laboratories </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    51. 51. OVERVIEW of RT - PCR tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR analyze results
    52. 52. Real Time Reporters <ul><li>All real time PCR systems rely upon the detection and quantization of fluorescent reporter, the signal of which increases in direct proportion of the amount of PCR product in a reaction. </li></ul>
    53. 53. REAL TIME PCR Cyber Green <ul><li>USING SYBER® GREEN </li></ul><ul><li>The simplest and economical format the reporter is the double strand DNA specific dye SYBR ® Green </li></ul><ul><li>Called as Molecular Probe. </li></ul>
    54. 54. How SYBR Green works <ul><li>SYBR green binds to double stranded DNA and upon excitation emits light </li></ul><ul><li>Thus as PCR product accumulates the fluoresce increases </li></ul>
    55. 55. Limitations of SYBER®Green <ul><li>Advantages </li></ul><ul><li>Inexpensive </li></ul><ul><li>Easy to Use </li></ul><ul><li>Sensitive </li></ul><ul><li>Disadvantages </li></ul><ul><li>SYBR green will bind to any double stranded DNA in a reaction, may result in an overestimation of the target concentration </li></ul>
    56. 56. Reverse transcription polymerase chain reaction <ul><li>Reverse transcription polymerase chain reaction ( RT-PCR ) is a variant of polymerase chain reaction (PCR), commonly used in molecular biology to generate many copies of a DNA sequence, a process termed &quot;amplification&quot;. In RT-PCR, however, RNA strand is first reverse transcribed into its DNA complement using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional or real-time PCR.. </li></ul>
    57. 57. Not to be confused with RT- PCR <ul><li>Reverse transcription PCR is not to be confused with real-time polymerase chain reaction (Q-PCR), which is also sometimes (incorrectly) abbreviated as RT-PCR. </li></ul>
    58. 58. Other Emerging Alternatives <ul><li>Two most popular alternatives to SYBR green are TaqMan® and Molecular Beacons . </li></ul><ul><li>Both technologies depend on hybridization probes relying on fluorescence resonance energy transfer.( FRET) and quantization </li></ul>
    59. 59. TaqMAN ®
    60. 60. TaqMAN ® Sequencing
    61. 61. TaqMAN ® probes
    62. 62. Documentation of Amplification <ul><li>The light emitted from the dye in the excited state is received by a computer and shown on a graph display, such as this, showing PCR cycles on the X-axis and a logarithmic indication of intensity on the Y-axis. </li></ul>
    63. 63. Molecular Beacons <ul><li>Molecular Beacons </li></ul><ul><li>Uses FRET Fluorescence Resonance Energy Transfer </li></ul><ul><li>Uses two sequence specific </li></ul><ul><li>Oligonucleotide labelled with fluorescent dyes </li></ul>
    64. 64. Molecular Beacons – RT PCR <ul><li>Molecular beacons are designed to adopt a hairpin structure while free in solution, brining the fluorescent dye and quencher in close proximity. When a molecular beacon hybridizes to a target the fluorescent dye emits light upon irradiation, and rebind to target in every cycle for signal measurement. </li></ul>
    65. 65. Loop Mediated Isothermal Amplification (LAMP) <ul><li>Loop mediated isothermal amplification is a simple, rapid, specific and cost effective nucleic acid amplification method characterized by use of 8 distinct regions on the target gene. </li></ul><ul><li>The amplification proceeds at a constant temperature using strand displacement reaction. </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    66. 66. LAMP <ul><li>Amplification and detection of gene can be completed in a single step, by incubating the mixture of samples, primers DNA polymerase with strand displacement activity and substrates at a constant temperature of 63 0 c. </li></ul>
    67. 67. LAMP <ul><li>Compared with PCR, and real time PCR, the LAMP has advantages of reaction simplicity and detection sensitivity. </li></ul><ul><li>The higher sensitivity and specificity of LAMP reaction is attributed to continuous amplification under isothermal condition employing six primers recognizing eight distinct regions of the target. </li></ul>
    68. 68. Advantages of LAMP <ul><li>LAMP functions on isothermal amplification. </li></ul><ul><li>LAMP does not require any thermal cycler and thus cane be performed even with water bath/heating block </li></ul><ul><li>LAMP method do not require sophisticated temperature control devices </li></ul><ul><li>Cost effective </li></ul>
    69. 69. Lesser False Positives in LAMP <ul><li>In LAMP both amplification and detection occur simultaneously during the exponential phase without going through the plateau phase where the non spurious amplification leads to lower sensitivity and false positivity. </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    70. 70. Loop Mediated Isothermal Amplification in Clinical Diagnosis <ul><li>LAMP technology proving to be ideal in detection of DNA or RNA of the pathogenic organisms </li></ul><ul><li>Proving to be highly efficient in diagnosis of Viral and Bacterial infections, </li></ul><ul><li>LAMP is capable of detecting the presence of pathogenic agents earlier than PCR </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    71. 71. LAMP proving an efficient Technology <ul><li>A one step single tube real time accelerated reverse transcription loop mediated isothermal amplification (RT-LAMP ) assays for rapid detection of some recently emerged viral pathogen eg West Nile, SARS, Dengue, Japanese encephalitis Chikungunya Norwalk, H5N1 highly pathogenic avian influenza., and CMV,HPV,VZV </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    72. 72. Multiplex PCR <ul><li>TaqMan probes and Molecular beacons allow multiple DNA species to be measured in the same sample ( Multiplex PCR) since fluorescent dyes with different emission spectra may be attached to different probes </li></ul>
    73. 73. Uses of Automated RT - PCR <ul><li>Several viral infections can be detected in acute phase serum samples. </li></ul><ul><li>Increasing used in for early and accurate detection of all most human viruses including </li></ul><ul><li>Measles, Mumps, Herpes simplex viruses, Rota viruses Noro virus,Influenzae virus type A and B, Respiratory Syncitical virus, SARS, Dengue Japanese Encephalitis, Hepatitis B and C, West Nile, Chikungunya,HIV, Avian flu virus, </li></ul>
    74. 74. Multiplex PCR in Real Time <ul><li>Multiplex real time quantitative RT-PCR assays have been developed for simultaneous detection identification and quantification of HBV, HCV and HIV-! In plasma and Serum samples. </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    75. 75. Real-Time PCR Method Molecular Beacons <ul><li>Molecular beacons are short segments of single-stranded DNA . The sequence of each molecular beacon must be customized to detect the PCR product of interest. </li></ul>
    76. 76. Molecular methods are necessary if the traditional methods provide poor results <ul><li>Microscopy gives false positive results - </li></ul><ul><li> - T.vaginalis, N.gonorrhoeae </li></ul><ul><li>Intracellular pathogens – viruses, M.genitalium </li></ul><ul><li>Low sensitivity – Chlamydia sp.,Neisseria sp. </li></ul><ul><li>Seropositivity is common – Chlamydia sp . </li></ul><ul><li>Subtyping is mandatory – HSV, HPV, HCV </li></ul><ul><li>Microbial growth is slow – M. tuberculosis </li></ul>
    77. 77. How are you going to measure the PCR product <ul><li>Directly </li></ul><ul><ul><li>Sybr green </li></ul></ul><ul><ul><li>Quality of primers critical </li></ul></ul><ul><li>Indirectly </li></ul><ul><ul><li>In addition to primers, add a fluorescently labeled hybridization probe </li></ul></ul>
    78. 78. Importance of controls <ul><li>Negative control (no DNA) </li></ul><ul><ul><li>checks reagents for contamination </li></ul></ul><ul><li>No reverse transcriptase control </li></ul><ul><ul><li>detects if signal from contaminating DNA </li></ul></ul><ul><li>Positive control </li></ul><ul><ul><li>checks that reagents and primers work </li></ul></ul><ul><ul><li>especially importance if trying to show absence of expression of a gene </li></ul></ul><ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul></ul>
    79. 79. Standards <ul><li>Same copy number in all cells </li></ul><ul><li>Expressed in all cells </li></ul><ul><li>Medium copy number advantageous </li></ul><ul><ul><li>correction more accurate </li></ul></ul><ul><li>Reasonably large introns </li></ul><ul><li>No pseudo gene </li></ul><ul><li>No alternate splicing in region you want to PCR </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    80. 80. Real-time PCR applications <ul><li>Quantitation of gene expression </li></ul><ul><li>Pathogen detection </li></ul><ul><li>Viral quantitation </li></ul><ul><li>Array verification </li></ul><ul><li>Drug therapy efficacy </li></ul><ul><li>DNA damage measurement </li></ul><ul><li>Quality control and assay validation </li></ul><ul><li>Genotyping </li></ul>
    81. 81. Emerging Technologies in Molecular Diagnosis
    82. 82. QIAGEN One Step RT-PCR Kit <ul><li>The QIAGEN One Step RT-PCR Kit is designed for easy and sensitive one-step RT-PCR using any RNA template. A unique enzyme combination and specially developed reaction buffer ensure efficient reverse transcription and PCR in one tube. </li></ul>
    83. 83. RT-PCR in one step The Robus™ T I Kit is base <ul><li>RobusT RT-PCR Kits perform cDNA synthesis and PCR amplification of cDNA successively in a single tube during a continuous thermal cycling </li></ul>
    84. 84. Uses and Advantages in Testing by PCR Methods <ul><li>Clinical diagnostics: detection and quantification of infectious microorganisms, cancer cells and genetic disorders </li></ul><ul><li>Capable of amplifying long targets, up to 6.0 kb </li></ul><ul><li>One-tube system allows rapid, sensitive and reproducible analysis of RNA with minimal risk of sample contamination </li></ul><ul><li>Amplifies products from a wide variety of total RNA or mRNA sources </li></ul>
    85. 85. Advantages Molecular methods <ul><li>High sensitivity and specificity </li></ul><ul><li>Detects pathogen, not immune response </li></ul><ul><li>Quick results </li></ul><ul><li>High transport toleration </li></ul>In-house (home-brew) PCR methods <ul><li>Cost effective </li></ul><ul><li>High sensitivity </li></ul><ul><li>High quality </li></ul><ul><li>Fast implementation of scientific discoveries </li></ul><ul><li>Customer friendly </li></ul>R&D is absolutely necessary
    86. 86. Advantages Molecular methods <ul><li>High sensitivity and specificity </li></ul><ul><li>Detects pathogen, not immune response </li></ul><ul><li>Quick results </li></ul><ul><li>High transport toleration </li></ul>In-house (home-brew) PCR methods <ul><li>Cost effective </li></ul><ul><li>High sensitivity </li></ul><ul><li>High quality </li></ul><ul><li>Fast implementation of scientific discoveries </li></ul><ul><li>Customer friendly </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>R&D is absolutely necessary
    87. 87. ESTABLISHMENT OF A PCR LABORATORY <ul><li>To perform PCR for the repetitive detection of a specific sequence, three distinct laboratory areas are required. The specific technical operations, reagents ,and personnel considerations </li></ul>
    88. 88. Prevention of Contamination in PCR Laboratory <ul><li>PCR contamination be considered as a form of infection . If standard sterile techniques that would be applied to tissue culture or microbiological manipulations are applied to PCR, then the risk of contamination will be greatly reduced. Above all else, common sense should prevail. </li></ul>
    89. 89. Avoiding contamination <ul><li>The single most important source of PCR product contamination is the generation of aerosols of PCR amplicons that is associated with the post-PCR analysis. Methods for eliminating this aerosol range from physical design of laboratories and use of specific pipettes to chemical and enzymatic approaches. The choice of method is often dependent on the frequency of amplification of a target amplicon and the relative amounts and concentrations of the amplicons created by the PCR. </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    90. 90. PCR laboratory Sample handling DNA preparation Clean room Stock solutions Laboratory Mixing site Thermocycler Amplification Detection Documentation QC & QA Quality control & assurance R & D ( Research and development) Alternatives: - commercial kits - robots + kits No alternative
    91. 91. Aseptic Handling of Specimen a Top Priority
    92. 92. Machines can be Operated in limited Space
    93. 93. Contamination <ul><li>PCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules of DNA. The sensitivity of PCR means that the sample used for PCR should not be contaminated with any other DNA’s that may reside in the laboratory environment. </li></ul><ul><li>Doctortvrao’s ‘e’ learning series </li></ul>
    94. 94. C an we do better ? <ul><li>Create networking between laboratories </li></ul><ul><li>Take second opinion </li></ul><ul><li>Honest dialogue with doctors </li></ul><ul><li>Education </li></ul><ul><li>Concentration into specialized laboratories </li></ul><ul><li>QC + QC + QC – to build up the trust </li></ul>
    95. 95. A Drop of Blood decides Human Destiny
    96. 96. Conclusion <ul><li>PCR is not only vital in the clinical laboratory by amplifying small amounts of DNA in infectious diseases, but it is also important for genetic predisposing for defects in Genetic disorders. </li></ul><ul><li>The PCR technology can also be employed in law enforcement, genetic testing of animal stocks and vegetable hybrids, and drug screening along with many more areas. </li></ul>
    97. 97. Dr. Kary Banks Mullis on Wisdom of Creative Nature <ul><li>Science, like nothing else among the institutions of mankind, grows like a weed every year. </li></ul><ul><li>We not only can luxuriate in its weed-like growth, but we can each of us be a creative and active part of it if we so desire. </li></ul><ul><li>There is no stopping it, nor can there be any end to it.&quot; </li></ul><ul><li>  Doctortvrao’s ‘e’ learning series </li></ul>
    98. 98. Playing well with Genes Can Change Life ?
    99. 99. Created for Awareness in Developing World on Emerging Technologies in Microbiology Dr.T.V.Rao MD Email [email_address]