Содержание
- 2. POLYMERASE CHAIN REACTION
- 3. Contents Polymerase Chain Reaction PCR Reaction Components Standard PCR Reaction Avoiding Contamination Thermal Cycling Profile for
- 5. Coping Machine for DNA Molecule Invented by Kary Mullis and his colleagues in the
- 6. Polymerase Chain Reaction PCR: Technique for in vitro (test tube) amplification of specific DNA sequences via
- 7. PCR
- 8. PCR reaction components шаблон A, G, C, T Mg2+ (forward and reverse)
- 9. PCR reaction components DNA template Two primers Four normal deoxynucleosides triphosphates Buffer system DNA polymerase I
- 10. DNA Template Integrity High molecular weight Purity Pure Amount Human genomic DNA should be up to
- 11. Primers Typical primers are 18-28 bases in length, Having 40- 60% GC composition, Have a balanced
- 12. Four Normal Deoxynucleosides Triphosphate Final concentration of dNTPs should be 50-500 µM (each dNTP). Usually included
- 13. Tris-HCl 10mM (10-50mM) for dissolution of nucleic acids рH 8.3 (рH 8.3-8.8 at 20C°) KCl 50mM
- 14. DNA Polymerase The most widely characterized polymerase is that from Thermus aquaticus (Taq), Thermophilic bacterium lives
- 15. Enhance The Specificity and or Efficiency of a PCR Betadine (antiseptic) Bovine serum albumin (for stabilizing
- 16. Calculation of Melting Temperature Tm= 2 C° X (number of A and T bases)+4 C°X (number
- 17. STANDARD PCR REACTION
- 18. PCR
- 19. AVOIDING CONTAMINATION
- 20. Sample Handling Use sterile techniques and always wear fresh gloves, Always use new or sterilized glassware,
- 21. Laboratory Facilities Set up physically separated working places for: Template preparation Setting up PCR reactions Post
- 22. Working with RNA Do not touch a surface after putting the gloves to avoid reintroduction of
- 23. Polymerase Chain Reaction
- 25. Thermal Cycling Profile for Standard PCR Initial Denaturation: Initial heating of the PCR mixture at 94-
- 26. Each cycle includes three successive steps:
- 27. PCR
- 28. Exponential Amplification As amplification proceeds, the DNA sequence between primers doubles after each cycle. (The amplification
- 29. Number of Cycles The number of cycles required for optimum amplification varies depending on the amount
- 30. GEL ELECTROPHORESIS
- 31. Agarose Gel Electrophoresis It is a method used in biochemistry and molecular biology to separate DNA,
- 32. Gel Tray/ Loading
- 33. PCR Product DNA Molecular Marker Amplified fragments can be visualized easily following staining with a chemical
- 34. » Factors, affect the mobility of molecules in gel Charge Size Shape Buffer conditions Gel concentration
- 35. PCR: Three Phases Exponential: Exact doubling of product is accumulating at every cycle (assuming 100% reaction
- 36. PCR Phases
- 37. Polymerase Chain Reaction Advantages of PCR Useful non- invasive procedure. Simplicity of the procedure. Sensitivity of
- 38. Variant PCR Reverse transcriptase-PCR. Nested-PCR. Hot-start PCR. Quantitative PCR. Multiplex-PCR. Mutagenesis by PCR. Allele specific PCR.
- 39. Reverse Transcriptase - PCR RT-PCR, one of the most sensitive methods for the detection and analysis
- 40. RT- PCR
- 41. Nested PCR Nested PCR is a very specific PCR amplification. Nested PCR use two pairs (instead
- 42. Nested - PCR
- 43. Hot - Start PCR Hot Start PCR significantly improves specificity, sensitivity and yield of PCR. The
- 44. Hot - Start PCR
- 45. Real Time PCR Traditional PCR has advanced from detection at the end-point of the reaction to
- 46. Real Time PCR
- 48. Infectious Diseases/ Cancer Detection of infectious agents, such as Pathogenic bacteria, Viruses or Protozoa. Cancer Detection
- 49. Genetic Desease Single point mutations can be detected by modified PCR techniques such as the ligase
- 51. Prenatal Diagnosis Prenatal sexing: Often required in families with inherited sex-linked diseases. Prenatal Diagnosis of diseases:
- 52. Research PCR is used in research laboratories in DNA cloning procedures, Southern blotting, DNA sequencing, recombinant
- 53. Polymerase Chain Reaction
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