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PCR – polymerace chain reaction

PCR – polymerace chain reaction. Solubiosysteemit S-114.2500 Harjoitustyö Maria Sipilä 30.11.2005. Introduction. Labratorists need to multiply genes for research, producing ”better” mutation etc. Two main ways: Gene cloning in bacteria PCR. Gene cloning in bacteria.

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PCR – polymerace chain reaction

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  1. PCR – polymerace chain reaction Solubiosysteemit S-114.2500 Harjoitustyö Maria Sipilä 30.11.2005

  2. Introduction • Labratorists need to multiply genes for research, producing ”better” mutation etc. • Two main ways: • Gene cloning in bacteria • PCR

  3. Gene cloning in bacteria • Main meaning: to multiply some gene in bacteria and then separate it • Usually only plasmides are used • Needed: • Restriction enzymes • Ligase enzymes • DNA-polymerase enzymes • Reverse transcriptases

  4. How to remove genes to another cell? • Plasmid vectors • Virus vectors • Micro injection • Using electricity or chemical agent to brake cell membrane • Gene library

  5. The other way: PCR • Inventor: 1983 Kary Mullis • Nobel prize in chemistry in 1993 + Much faster than the traditional cloning + needs only slightly DNA molecules to produce a huge range of copies + mutations in primers enables to transfer genes into plasmids (makes ligase enzymes easy to attach it)

  6. PCR needs unleast some information of the gene order (or from some similar gene) to make the primers • Very susceptible to surroundings

  7. Tools for PCR • A small amount of DNA • DNA polymerace enzymes • Nucleotides • Primers • Two different kind of • Usually about 20 nucleotides

  8. The principle of PCR 1. Clean DNA 2. Tm=+95°C 3. Ta=+55-72°C 4. +72°C 5. +95°C 6. … 7. …

  9. Melting Point Temperature • Denaturation • The more there is G or C, the higher Tm • The longer the primers, the higer Tm • 0,2 µm Tm = 81,5°C + 0,41(%G + %C) – 550/n n=number of nucleotides ∆ Tm < 2°C

  10. Annealing State Temperature • Depends: • Concentration of primers • Composition of nucleotides • Normally takes only few seconds, but it is programmed to 0,5-2 minutes • Building starts from the 3’end

  11. What time does it take? • Denaturation: 30 - 60 sec • Annealing: 30 - 60 sec • Doupling: 30 - 60 sec • 25 - 35 cycles only (otherwise enzyme decay causes artifacts) • 72oC for 5 min at end to allow complete elongation of all product DNA Altogether: 7 min ( 8,5 min) * 25 (35) = 3h-5h

  12. Problems with primers • ”hairpin” structure • If 3’side is included in structure, the primer doesn’t work • Primer dimers • Only harm if the binding is formed at the 3’ends

  13. Non-spesific products • Mismatchs • Possible if: • Only few nucleotides is wrong • Ta is too low • Computer programs can calculate

  14. Allocated mutagenesis • No harm (for binding) of one or two mismatches • Primers can be designed to contain errors • Binding is not disturbed SILENT MUTATION: one base is placed by another base, witch won’t change amino acid sequence

  15. Allocated mutagenesis is used in reforming proteins • The precise base can be traced out (which cause a certain property to the protein) • Mutation in base order may lead to a better stand of heat or cold

  16. Other techniques and applications for PCR • Non-symmetric PCR: one stranded DNA (for example for sequencing) • Inverse PCR: copy some unknown piece of DNA-strand between 2 known ones • To find genetic diseases: primers for healthy and sick allele • …

  17. Sources: • Ulmanen, Tenhunen ym. Geeni Biologia, WSOY, Porvoo 2000 • http://www.mcb.uct.ac.za/pcrcond.htm • http://www.edu.fi/oph/abc/dna/pcr1.html • http://www.mcb.uct.ac.za/hybridn.htm#INTRODUCTION • Pictures: • Ulmanen, Tenhunen ym. Geeni Biologia, WSOY, Porvoo 2000 • http://www.edu.fi/oph/abc/dna/pcr1.html • http://www.biology.lsu.edu/heydrjay/1201/Chapter17/SCI_Amino_Acid_CIRCLE.jpg

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