Презентация Polymerase chain reaction онлайн

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  • Тип файла:
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  • Всего слайдов:
    54 слайда
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    1,2,3,4,5,6,7,8,9,10,11
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Слайды и текст к этой презентации:

№1 слайд
POLYMERASE CHAIN REACTION
Содержание слайда: POLYMERASE CHAIN REACTION
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POLYMERASE CHAIN REACTION
Содержание слайда: POLYMERASE CHAIN REACTION
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Contents Polymerase Chain
Содержание слайда: 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
№4 слайд
Содержание слайда:
№5 слайд
Coping Machine for DNA
Содержание слайда:  Coping Machine for DNA Molecule  Coping Machine for DNA Molecule  Invented by Kary Mullis and his colleagues in the 1983
№6 слайд
Polymerase Chain Reaction PCR
Содержание слайда: 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.
№7 слайд
PCR
Содержание слайда: PCR
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PCR reaction components
Содержание слайда: PCR reaction components
№9 слайд
PCR reaction components DNA
Содержание слайда: PCR reaction components DNA template Two primers Four normal deoxynucleosides triphosphates Buffer system DNA polymerase I
№10 слайд
DNA Template Integrity High
Содержание слайда: 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
№11 слайд
Primers Typical primers are -
Содержание слайда: 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
№12 слайд
Four Normal Deoxynucleosides
Содержание слайда: 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.
№13 слайд
The standard PCR buffer
Содержание слайда: 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
№14 слайд
DNA Polymerase The most
Содержание слайда: 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.
№15 слайд
Enhance The Specificity and
Содержание слайда: Enhance The Specificity and or Efficiency of a PCR
№16 слайд
Calculation of Melting
Содержание слайда: Calculation of Melting Temperature
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STANDARD PCR REACTION
Содержание слайда: STANDARD PCR REACTION
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PCR
Содержание слайда: PCR
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AVOIDING CONTAMINATION
Содержание слайда: AVOIDING CONTAMINATION
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Sample Handling Use sterile
Содержание слайда: 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.
№21 слайд
Laboratory Facilities Set up
Содержание слайда: 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.
№22 слайд
Working with RNA Do not touch
Содержание слайда: 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.
№23 слайд
Polymerase Chain Reaction
Содержание слайда: Polymerase Chain Reaction
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Содержание слайда:
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Thermal Cycling Profile for
Содержание слайда: 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°.
№26 слайд
Each cycle includes three
Содержание слайда: Each cycle includes three successive steps:
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PCR
Содержание слайда: PCR
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Exponential Amplification
Содержание слайда: Exponential Amplification
№29 слайд
Number of Cycles The number
Содержание слайда: 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.
№30 слайд
GEL ELECTROPHORESIS
Содержание слайда: GEL ELECTROPHORESIS
№31 слайд
Agarose Gel Electrophoresis
Содержание слайда: 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
Содержание слайда: Gel Tray/ Loading
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Содержание слайда:
№34 слайд
Factors, affect the mobility
Содержание слайда: » Factors, affect the mobility of molecules in gel Charge Size Shape Buffer conditions Gel concentration and Voltage
№35 слайд
PCR Three Phases Exponential
Содержание слайда: 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.
№36 слайд
PCR Phases
Содержание слайда: PCR Phases
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Polymerase Chain Reaction
Содержание слайда: 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
№38 слайд
Variant PCR Reverse
Содержание слайда: Variant PCR Reverse transcriptase-PCR. Nested-PCR. Hot-start PCR. Quantitative PCR. Multiplex-PCR. Mutagenesis by PCR. Allele specific PCR. …..
№39 слайд
Reverse Transcriptase - PCR
Содержание слайда: 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.
№40 слайд
RT- PCR
Содержание слайда: RT- PCR
№41 слайд
Nested PCR Nested PCR is a
Содержание слайда: 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.
№42 слайд
Nested - PCR
Содержание слайда: Nested - PCR
№43 слайд
Hot - Start PCR Hot Start PCR
Содержание слайда: 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.
№44 слайд
Hot - Start PCR
Содержание слайда: Hot - Start PCR
№45 слайд
Real Time PCR Traditional PCR
Содержание слайда: 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.
№46 слайд
Real Time PCR
Содержание слайда: Real Time PCR
№47 слайд
Содержание слайда:
№48 слайд
Infectious Diseases Cancer
Содержание слайда: 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.
№49 слайд
Genetic Desease Single point
Содержание слайда: 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.
№50 слайд
Содержание слайда:
№51 слайд
Prenatal Diagnosis Prenatal
Содержание слайда: 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.
№52 слайд
Research PCR is used in
Содержание слайда: 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.
№53 слайд
Polymerase Chain Reaction
Содержание слайда: Polymerase Chain Reaction
№54 слайд
Содержание слайда:
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