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Pcr, rapd dan rflp
The document outlines the principles and processes of three genetic techniques: Polymerase Chain Reaction (PCR), Random Amplified Polymorphic DNA (RAPD), and Restriction Fragment Length Polymorphism (RFLP). It provides detailed information on PCR methodology, including temperature cycles and primer design, as well as how RAPD uses short primers to generate genetic markers. Additionally, it discusses RFLP's reliance on restriction enzymes for detecting genetic variations through electrophoresis and hybridization techniques.
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Pcr, rapd dan rflp
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- 2.
- 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. 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. The PCR Process PCRworks 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 (~95°C) 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. PCR Melting 94 oC Melting 94 oC Annealing Primers 50oC Extension 72 oC Temperature 100 0 50 T i m e 30x 5’3’ 3’5’ 3’5’ 5’ 5’3’ 5’ 3’5’ 5’ 5’ 5’ 5’3’ 3’5’ 3’5’ 5’3’ 5’3’ 5’
- 7. PCR Melting 94 oC Temperature 100 0 50 T im e 5’3’ 3’5’
- 8. PCR Melting 94 oC Temperature 100 0 50 T im e 3’5’ 5’3’ Heat
- 9. PCR Melting 94 oC Annealing Primers 50 oC Extension 72oCTemperature 100 0 50 T i m e 3’5’ 5’3’ 5’ 5’ Melting 94 oC
- 10. PCR Melting 94 oC Melting 94 oC Annealing Primers 50oC Extension 72 oCTemperature 100 0 50 T i m e 30x 3’5’ 5’3’ Heat Heat 5’ 5’ 5’
- 11. PCR Melting 94 oC Melting 94 oC Annealing Primers 50oC Extension 72 oCTemperature 100 0 50 T i m e 30x 3’5’ 5’3’ 5’ 5’ 5’ 5’ 5’ 5’
- 12. PCR Melting 94 oC Melting 94 oC Annealing Primers 50oC Extension 72 oCTemperature 100 0 50 T i m e 30x 3’5’ 5’3’ 5’ 5’ 5’ 5’ 5’ 5’ Heat Heat
- 13. PCR Melting 94 oC Melting 94 oC Annealing Primers 50oC Extension 72 oCTemperature 100 0 50 T i m e 30x 3’5’ 5’3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’
- 14. Fragments of defined length PCR Melting 94oC Melting 94 oC Annealing Primers 50 oC Extension 72 oCTemperature 100 0 50 T i m e 30x 3’5’ 5’3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’
- 15. DNA Between ThePrimers Doubles With Each Thermal Cycle 0 Cycles Number 1 3 8 2 4 1 2 4 16 5 32 6 64
- 16. Template •Any source ofDNA 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. Primers • PCR primersneed 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. 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. Primer Problems • primersshould 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. Degenerate primers: anoligo 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. Specific Primers :Primers designed from already known DNA sequences (genes)
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- 23. Recognizing/producing polymorphism caused by differentialamplification of DNA sequence
- 24. History Shortly afterKary 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. Components of aPCR 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. Modifying Thermal Cycling Twomodifications 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. RAPD Template DNA Primer bindsto 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. RAPD Template DNA Primers point away fromeach other, so amplification won’t happen
- 29. RAPD Template DNA Primers point in thesame direction, so amplification won’t happen
- 30. RAPD Template DNA Primers too far apart,so amplification won’t happen > 2,000 bases
- 31. Template DNA Primers are just theright distance apart, so fragment is amplified 100 - 1,500 bases RAPD
- 32. MM 2 34 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.
- 34. Recognizing/producing polymorphism caused by differentialrecognition site of restriction enzyme on DNA sequence
- 35. AGATCT Wild-type allele Mutant allele TCTAGA Asingle nucleotide change can make a difference AGAGCT TCTCGA Restriction site Not a restriction site
- 36. RFLP-determination Differences inDNA-sequence between the two parents ( due to mutations ) Differences in restriction - enzym sites
- 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. B=AB C=BB B CA BC A=AA A Dominant vs Co-dominant
- 39. The laboratory stepsinvolved in RFLP detection Isolation of DNA Restriction digestion and gel electrophoresis DNA transfer by Southern blotting DNA hybridisation
- 40.
- 41. Restriction sites B C AD E C A Parent 2 Parent 1 GAATTC CTTAAG GAAATC CTTTAG No EcoRI site EcoRI site
- 42. Restriction sites B C AD E C A Parent 2 Parent 1 probe Probe recognizes complementary sequence Probe has a color label or is radio-active probe
- 43. A C C A D E B C AD E C A Parent 2 Parent 1 probe Separation with gel electrophoresis; smaller fragments run faster B
- 44. A C C A D E B C AD E C A Parent 2 Parent 1 probe Separation with gel electrophoresis; many many fragments B
- 45. Question: You are usingNorthern 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