pcr, rapd dan rflp

Download Pcr, rapd dan rflp

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  1. 1. PCR, RAPD dan RFLP
  2. 2. Polymerase Chain Reaction PCR
  3. 3. The polymerase chain reaction(PCR) is to used to amplify a sequence of DNA using a pair of primers each complementary to one end of the DNA target sequence
  4. 4. The PCR cycle Denaturation: The target DNA (template) is separated into two strands by heating to 95 Primer annealing: The temperature is reduced to around 55 to allow the primers to anneal. Polymerization (elongation, extension): The temperature is increased to 72 for optimal polymerization step which uses up dNTPs and required Mg++.
  5. 5. The PCR Process PCR works like this: DNA and two primers are combined in a salt solution with dNTPs and a heat stable DNA polymerase enzyme The primers match some sequence in the target DNA The solution is rapidly heated to DNA denaturing temperatures (~95C) and cooled to a temperature where the polymerase can function Each thermal cycle generates copies of the sequence between the primers, so the total number of fragments amplifies in an exponential fashion: 2, 4, 8,16, 32, 64, etc.
  6. 6. PCR Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oC Temperature 100 0 50 T i m e 30x 53 35 35 5 53 5 35 5 5 5 53 35 35 53 53 5
  7. 7. PCR Melting 94 oC Temperature 100 0 50 T i m e 53 35
  8. 8. PCR Melting 94 oC Temperature 100 0 50 T i m e 35 53 Heat
  9. 9. PCR Melting 94 oC Annealing Primers 50 oC Extension 72 oCTemperature 100 0 50 T i m e 35 53 5 5 Melting 94 oC
  10. 10. PCR Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oCTemperature 100 0 50 T i m e 30x 35 53 Heat Heat 5 5 5
  11. 11. PCR Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oCTemperature 100 0 50 T i m e 30x 35 53 5 5 5 5 5 5
  12. 12. PCR Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oCTemperature 100 0 50 T i m e 30x 35 53 5 5 5 5 5 5 Heat Heat
  13. 13. PCR Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oCTemperature 100 0 50 T i m e 30x 35 53 5 5 5 5 5 5 5 5 5 5
  14. 14. Fragments of defined length PCR Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oCTemperature 100 0 50 T i m e 30x 35 53 5 5 5 5 5 5 5 5 5 5
  15. 15. DNA Between The Primers Doubles With Each Thermal Cycle 0 Cycles Number 1 3 8 2 4 1 2 4 16 5 32 6 64
  16. 16. Template Any source of DNA that provides one or more target molecules can in principle be used as a template for PCR Whatever the source of template DNA, PCR can only be applied if some sequence information is known so that primers can be designed.
  17. 17. Primers PCR primers need to be about 18 to 30 nt long and have similar G+C contents so that they anneal to their complementary sequences at similar temperatures.They are designed to anneal on opposite strands of the target sequence Tm=2(a+t)+4(g+c): determine annealing temperature. If the primer is 18-30 nt, annealing temperature can be Tm5oC
  18. 18. Primer Design Rules primers should be at least 15 base pairs long have at least 50% G/C content anneal at a temperature in the range of 50-65 degrees C Usually higher annealing temperatures (Tm) are better (i.e. more specific for your desired target) forward and reverse primer should anneal at approximately the same temperature
  19. 19. Primer Problems primers should flank the sequence of interest primer sequences should be unique primers that match multiple sequences will give multiple products repeated sequences can be amplified - but only if unique flanking regions can be found where primers can bind primers can have self-annealing regions within each primer (i.e. hairpin and foldback loops) pairs of primers can anneal to each other to form the dreaded "primer dimers"
  20. 20. Degenerate primers: an oligo pool derived from protein sequence. E.g. His-Phe-Pro-Phe-Met-Lys can generate a primer 5-CAY TTY CCN TTY ATG AAR Y= Pyrimidine N= any base R= purine
  21. 21. Specific Primers : Primers designed from already known DNA sequences (genes)
  22. 22. Random Amplified Polymorphic DNA RAPD
  23. 23. Recognizing/producing polymorphism caused by differential amplification of DNA sequence
  24. 24. History Shortly after Kary Mullis invented the Polymerase Chain Reaction (PCR) it was realized that short primers would bind to several locations in a genome and thus could produce multiple fragments Williams et al. (1990) developed Random Amplified Polymorphic DNA (RAPD) a technique using very short 10 base primers to generate random fragments from template DNAs RAPD fragments can be separated and used as genetic markers or a kind of DNA fingerprint
  25. 25. Components of a PCR and RAPD Reactions RAPD 1. Buffer (containing Mg++) - usually high Mg++ concentrations are used lowering annealing stringency 2. Template DNA 3. 1 short primer (10 bases)not known to anneal to any specific part of the template DNA 4. dNTPs 5. Taq DNA Polymerase PCR 1. Buffer (containing Mg++) 2. Template DNA 3. 2 Primers that flank the fragment of DNA to be amplified 4. dNTPs 5. Taq DNA Polymerase (or another thermally stable DNA polymerase)
  26. 26. Modifying Thermal Cycling Two modifications made to typical thermal cycling when RAPD is being done: 1. Annealing temperatures are generally very low, around 36 oC - This allows very short primers to anneal to template DNA 2. More thermal cycles are used, typically 45 - This compensates for the inefficiency which results from using such short primers.
  27. 27. RAPD Template DNA Primer binds to many locations on the template DNA Only when primer binding sites are close and oriented in opposite direction so the primers point toward each other will amplification take place
  28. 28. RAPD Template DNA Primers point away from each other, so amplification wont happen
  29. 29. RAPD Template DNA Primers point in the same direction, so amplification wont happen
  30. 30. RAPD Template DNA Primers too far apart, so amplification wont happen > 2,000 bases
  31. 31. Template DNA Primers are just the right distance apart, so fragment is amplified 100 - 1,500 bases RAPD
  32. 32. MM 2 3 4 5 6 7 8 9 10 Separated RAPD Fragments4mM MgCl2 1.2 U Taq 5 pM OPA-16 4mM MgCl2 0.6 U Taq 10 pM OPA-16 2mM MgCl2 1.2 U Taq 10 pM OPA-16 Normal concentrations are shown in yellow text. M = A size standard Lowering Magnesium ion concentration results in loss of the largest fragment visible in lanes 2-7 RAPD reactions were run in groups of 3 using the same template and primer, but varying Magnesium, polymerase and primer concentrations Which variable has the greatest impact on fragment patterns?
  33. 33. Restriction Fragment Length Polymorphism RFLP
  34. 34. Recognizing/producing polymorphism caused by differential recognition site of restriction enzyme on DNA sequence
  35. 35. AGATCT Wild-type allele Mutant allele TCTAGA A single nucleotide change can make a difference AGAGCT TCTCGA Restriction site Not a restriction site
  36. 36. RFLP-determination Differences in DNA-sequence between the two parents ( due to mutations ) Differences in restriction - enzym sites
  37. 37. Dominant vs Co-dominant Most organisms we study are diploid Two sets of chromosomes Co-dominant: the marker on both chromosomes is visible and distinguishable Dominant: the marker is present and you can not see whether is coming from both chromosomes or from only one
  38. 38. B=AB C=BB B CA B C A=AA A Dominant vs Co-dominant
  39. 39. The laboratory steps involved in RFLP detection Isolation of DNA Restriction digestion and gel electrophoresis DNA transfer by Southern blotting DNA hybridisation
  40. 40. Southern Blotting
  41. 41. Restriction sites B C A D E C A Parent 2 Parent 1 GAATTC CTTAAG GAAATC CTTTAG No EcoRI site EcoRI site
  42. 42. Restriction sites B C A D E C A Parent 2 Parent 1 probe Probe recognizes complementary sequence Probe has a color label or is radio-active probe
  43. 43. A C C A D E B C A D E C A Parent 2 Parent 1 probe Separation with gel electrophoresis; smaller fragments run faster B
  44. 44. A C C A D E B C A D E C A Parent 2 Parent 1 probe Separation with gel electrophoresis; many many fragments B
  45. 45. Question: You are using Northern blotting to analyze two mRNA samples derived from fibroblasts and hepatocytes. What will you see if you use a probe made from exon EIIIB of the fibronectin gene? What about using a probe made from the exon next to EIIIB? Detection of alternative splicing by Northern blotting Northern blotting can be used to detect specific RNAs in complex mixtures. Southern blotting detects specific DNA fragments. Western blotting (immunoblotting) detects specific proteins with antibodies. RNA RNA mixture Transfer solution

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