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Вы можете ознакомиться и скачать презентацию на тему Polymerase chain reaction. Доклад-сообщение содержит 54 слайдов. Презентации для любого класса можно скачать бесплатно. Если материал и наш сайт презентаций Mypresentation Вам понравились – поделитесь им с друзьями с помощью социальных кнопок и добавьте в закладки в своем браузере.

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POLYMERASE CHAIN REACTION

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POLYMERASE CHAIN REACTION

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Contents Polymerase Chain Reaction PCR Reaction Components Standard PCR Reaction Avoiding Contamination Thermal Cycling Profile for Standard PCR Gel Electrophoresis PCR: Three phases Variants of PCR Polymerase Chain Reaction: Uses

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 Coping Machine for DNA Molecule  Coping Machine for DNA Molecule  Invented by Kary Mullis and his colleagues in the 1983

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Polymerase Chain Reaction PCR: Technique for in vitro (test tube) amplification of specific DNA sequences via the temperature mediated. DNA polymerase enzyme by simultaneous primer extension of complementary strands of DNA. PCR: This system for DNA replication that allows a "target" DNA sequence to be selectively amplified, several million-fold in just a few hours.

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PCR

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PCR reaction components

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PCR reaction components DNA template Two primers Four normal deoxynucleosides triphosphates Buffer system DNA polymerase I

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DNA Template Integrity High molecular weight Purity Pure Amount Human genomic DNA should be up to 500ng Bacterial DNA 1-10ng Plasmid DNA 0.1-1ng

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Primers Typical primers are 18-28 bases in length, Having 40- 60% GC composition, Have a balanced distribution of G/C and A/T rich domains, The calculated Tm for a given primer pair should be balanced (difference no more than 5 °C), Primer concentration between 0.1 and 0.6 µM are generally optimal, Contain no internal secondary structure, Have a cytosine and guanine at the 3'-end because they form three hydrogen bonds with the matrix molecules, making a more stable hybridization

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Four Normal Deoxynucleosides Triphosphate Final concentration of dNTPs should be 50-500 µM (each dNTP). Usually included at conc. of 200 µM for each nucleotide. Always use balanced solution of all four dNTPs to minimize polymerase error rate.

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The standard PCR buffer contains: Tris-HCl 10mM (10-50mM) for dissolution of nucleic acids рH 8.3 (рH 8.3-8.8 at 20C°) KCl 50mM promotes specificity of hybridization MgCL2 1.5mM (0.5-10mM) for stabilizing of complex between primers and matrix and for increasing of exit the special product of PCR Gelatin or Bovine Serum Albumin 100 µg/ml frequent unfreezing-freezing at the temperature -20C

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DNA Polymerase The most widely characterized polymerase is that from Thermus aquaticus (Taq), Thermophilic bacterium lives in hot springs and capable of growing at 70 -75 C°, Consist of a single polypeptide chain has a molecular weight of 95 Kd, and has an optimum polymerization temperature of 70 – 80 C° (72 C°). 0.5 – 2 units/50µl reaction. Too little will limit the amount of products, while too much can produce unwanted non specific products.

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Enhance The Specificity and or Efficiency of a PCR

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Calculation of Melting Temperature

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STANDARD PCR REACTION

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PCR

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AVOIDING CONTAMINATION

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Sample Handling Use sterile techniques and always wear fresh gloves, Always use new or sterilized glassware, plasticware and pipettes to prepare the PCR reagents and template DNA, Autoclave and sterilize all reagents and solution, Have your own set of PCR reagent and Solution (store in small aliquots), Positive and negative control should be included.

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Laboratory Facilities Set up physically separated working places for: Template preparation Setting up PCR reactions Post PCR analysis Use PCR only pipettes, micro-centrifuges and disposable gloves Use aerosol resistant pipette tips PCR reaction under a fume hood equipped with UV LIGHT.

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Working with RNA Do not touch a surface after putting the gloves to avoid reintroduction of RNAse to decontaminated material. Designate a special area for RNA work only. Treat surface or benches and glassware with commercially available RNAse inactivating agents.

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Polymerase Chain Reaction

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Thermal Cycling Profile for Standard PCR Initial Denaturation: Initial heating of the PCR mixture at 94- 95C within 2 min. is enough to completely denature complex genomic DNA. Each cycle includes three successive steps: Denaturation, annealing and extension. Post extension and holding: Cycling should conclude with a final extension at 72 C° for 5 -15 minute to promote completion of partial extension products and then holding at 4 C°.

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Each cycle includes three successive steps:

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PCR

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Exponential Amplification

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Number of Cycles The number of cycles required for optimum amplification varies depending on the amount of the starting material. Most PCR should, therefore, include only 25 – 35 cycles. As cycle increases, nonspecific products can accumulate. After 20- 40 cycles of heating and cooling build up over a million copies of original DNA molecules.

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GEL ELECTROPHORESIS

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Agarose Gel Electrophoresis It is a method used in biochemistry and molecular biology to separate DNA, or RNA molecules based upon charge, size and shape. Agarose is a polysaccharide derivative of agar.

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Gel Tray/ Loading

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» Factors, affect the mobility of molecules in gel Charge Size Shape Buffer conditions Gel concentration and Voltage

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PCR: Three Phases Exponential: Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise. Linear: The reaction components are being consumed; the reaction is slowing, and products are starting to degrade. Plateau: The reaction has stopped; no more products are being made and if left long enough; the PCR products will begin to degrade.

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PCR Phases

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Polymerase Chain Reaction Advantages of PCR Useful non- invasive procedure. Simplicity of the procedure. Sensitivity of the PCR Disadvantages of PCR False positive results (cross contamination). False negative results

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Variant PCR Reverse transcriptase-PCR. Nested-PCR. Hot-start PCR. Quantitative PCR. Multiplex-PCR. Mutagenesis by PCR. Allele specific PCR. …..

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Reverse Transcriptase - PCR RT-PCR, one of the most sensitive methods for the detection and analysis of rare mRNA transcripts or other RNA present in low abundance. RNA cannot serve as a template for PCR. RNA must be first transcribed into cDNA with reverse transcriptase from Moloney murine leukemia virus or Avian myeloblastosis virus, and the cDNA copy is then amplified.

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RT- PCR

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Nested PCR Nested PCR is a very specific PCR amplification. Nested PCR use two pairs (instead of one pair) of PCR primers are used to amplify a fragment.

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Nested - PCR

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Hot - Start PCR Hot Start PCR significantly improves specificity, sensitivity and yield of PCR. The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95˚C) before adding the polymerase. Specialized enzyme systems can be used.

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Hot - Start PCR

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Real Time PCR Traditional PCR has advanced from detection at the end-point of the reaction to detection while the reaction is occurring (Real-Time). Real-time PCR uses a fluorescent reporter signal to measure the amount of amplicon as it is generated . This kinetic PCR allows for data collection after each cycle of PCR instead of only at the end of the 20 to 40 cycles.

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Real Time PCR

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Infectious Diseases/ Cancer Detection of infectious agents, such as Pathogenic bacteria, Viruses or Protozoa. Cancer Detection of malignant diseases by PCR, Recurrence of hematological cancers has also been evaluated and Detection of micro-metastasis in blood, lymph nodes and bone marrow.

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Genetic Desease Single point mutations can be detected by modified PCR techniques such as the ligase chain reaction (LCR) and PCR-single-strand conformational polymorphisms (PCR-SSCP) analysis. Detection of variation and mutation in genes using primers containing sequences that were not completely complementary to the template.

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Prenatal Diagnosis Prenatal sexing: Often required in families with inherited sex-linked diseases. Prenatal Diagnosis of diseases: Prenatal diagnosis of many of the inborn errors of metabolism is possible by DNA markers.

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Research PCR is used in research laboratories in DNA cloning procedures, Southern blotting, DNA sequencing, recombinant DNA technology. Major role in the human genome project.