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PCR בעזרת DNA אנאליזה של. בעזרת PCR. TSH של הרצפטור ל- DNA זיהוי נוכחות. מורן גרינברג 2008. על מה נדבר?. מהו פלסמיד? מהו cDNA ? שיטות להפקת פלסמידים עקרונות ריאקצית ה - PCR סוגים שונים של PCR שיטות להפרדת מקטעי DNA אלקטרופורזה ( Electrophoresis ). מורן גרינברג 2008.
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PCR בעזרת DNA אנאליזה של בעזרת PCR TSH של הרצפטור ל- DNA זיהוי נוכחות מורן גרינברג 2008
על מה נדבר? • מהו פלסמיד? • מהו cDNA ? • שיטות להפקת פלסמידים • עקרונות ריאקצית ה- PCR • סוגים שונים של PCR • שיטות להפרדת מקטעי DNA אלקטרופורזה (Electrophoresis) מורן גרינברג 2008
Gel Electrophoresis of DNA מורן גרינברג 2008
What is Gel Electrophoresis? • •Electro = flow of electricity, phoresis, from the Greek = to carry across •A gel is a colloid, a suspension of tiny particles in a medium, occurring in a solid form, like gelatin •Gel electrophoresis refers to the separation of charged particles located in a gel when an electric current is applied •Charged particles can include DNA, amino acids, peptides, etc מורן גרינברג 2008
How does it work? •DNA is an organic acid, and is negatively charged (remember, DNA for Negative) •When the DNA is exposed to an electrical field, the particles migrate toward the positive electrode •Smaller pieces of DNA can travel further in a given time than larger pieces מורן גרינברג 2008
- + - + Basis of migration and separation? • Gel placed in apparatus and covered with TBE • Load samples in wells -- DNA (negative charge) moves toward positive electrode (red) • Gel acts as a molecular sieve • Smaller molecules run faster (further from wells) מורן גרינברג 2008
Why do gel electrophoresis? •When DNA is cut by restriction enzymes, the result is a mix of pieces of DNA of different lengths •It is useful to be able to separate the pieces -I.e. for recovering particular pieces of DNA, for forensic work or for sequencing מורן גרינברג 2008
What is needed? •Agarose-a polysaccharide made from seaweed. Agarose is dissolved in buffer and heated, then cools to a gelatinous solid with a network of crosslinked molecules •Some gels are made with acrylamideif sharper bands are required מורן גרינברג 2008
boiling •Buffer-in this case TBE •The buffer provides ions in solution to ensure electrical conductivity. •Not only is the agarose dissolved in buffer, but the gel slab is submerged (submarine gel) in buffer after hardening מורן גרינברג 2008
Agarose -- long polymer of galactose units Dissolve solid in buffer [Tris-borate + EDTA (TBE)] with heating Gel forms upon cooling (H-bonds form between polygalactose units) מורן גרינברג 2008
Intercalation of EtBr in DNA Q6: Visualization of DNA? • Visualize DNA by use of fluorescent dye – Ethidium bromide (EtBr) intercalates in DNA. • UV light irradiates gel (WEAR FACE SHIELD). EtBr stacks between the bases of the DNA helix. + + The smaller the DNA molecule, the less EtBr bound, the fainter the image seen. מורן גרינברג 2008
Agarose Gel ElectrophoresisQ5: Loading dye components and purpose? • Sample buffer (“blue juice”) • Glycerol -- for density, to make sample sink to bottom of well • Dye -- to mark progress of electrophoresis • bromophenol blue -- runs same as 300 bp dsDNA • xylene cyanol -- runs same as 4 kb dsDNA) • EDTA, SDS -- to inactivate restriction enzymes מורן גרינברג 2008
Steps in running a gel •DNA is prepared by digestion with restriction enzymes •Agarose is made to an appropriate thickness (the higher the % agarose, the slower the big fragments run) and ‘melted’ in the microwave •The agarose may have a DNA ‘dye’ added (or it may be stained later). The agarose is poured onto the gel block and cooled מורן גרינברג 2008
Next? •The power source is turned on and the gel is run. The time of the run depends upon the amount of current and % gel, and requires experimentation •At the end of the run the gel is removed (it is actually quite stiff) •The gel is then visualized -UV light causes the bands of DNA to fluoresce מורן גרינברג 2008
Photograph of DNA in Agarose Gel Typical gel results מורן גרינברג 2008
Still more…. •The DNA band of interest can be cut out of the gel and the DNA extracted •Or DNA can be removed from the gel by Southern Blotting מורן גרינברג 2008
Blotting Techniques • Southern Blot • Procedure for identifying DNA fragments with DNA probe; developed by E. M. Southern, 1975 • Northern Blot • Procedure for identifying RNA fragments with DNA probe • Western Blot • Procedure for identifying proteins using antibodies as probes מורן גרינברג 2008
Blotting Techniques • Southern Blot • Procedure for identifying DNA fragments with DNA probe; developed by E. M. Southern, 1975 • Northern Blot • Procedure for identifying RNA fragments with DNA probe • Western Blot • Procedure for identifying proteins using antibodies as probes מורן גרינברג 2008
PCR מורן גרינברג 2008
PCR Réaction Mixture • DNA or cDNA • dNTPs • MgCl2; MgSO4 • Primers ; Tm= melting température • Taq-polymerase ; PFU polymérase מורן גרינברג 2008
DNA Polymerase • DNA Polymerase is the enzyme responsible for copying the sequence starting at the primer from the single DNA strand • Commonly use Taq, an enzyme from the hyperthermophilic organisms Thermus aquaticus, isolated first at a thermal spring in Yellowstone National Park • This enzyme is heat-tolerant useful both because it is thermally tolerant (survives the melting T of DNA denaturation) which also means the process is more specific, higher temps result in less mismatch – more specific replication מורן גרינברג 2008
separate parent strands in preparation new strand synthesis • DNA synthesis requires a primer to start DNA synthesis • addition of nucleotides, one at a time, to the growing end of the DNA strand (3’ end) using the parent strand as the template מורן גרינברג 2008
PCR animation מורן גרינברג 2008
PCR Animation Please click here. Process Denature Anneal Primer Replicate DNA 1st cycle 2nd cycle 3rd cycle מורן גרינברג 2008
PCR methods • Purpose of PCR: amplification ,specificity, sequencing • Potential Problems with Taq and PFU • PCR Applications • RT-PCR • Real-Time PCR • Mutagenesis מורן גרינברג 2008
RNA RT-PCR reverse transcriptase-pcr • RNA containing virus • PCR doesn’t work on RNA templates • RT PCR • make cDNA copy of RNA sequence first • PCR the cDNA copy of RNA Extract RNA from virus/cells מורן גרינברג 2008
Isolation of Plasmid DNA Cloning vehicles • Plasmids are the most extensively used tools for: • gene cloning - making many copies of particular gene. • gene expression - converting the information stored in the DNA gene into a functional molecule for the cell (RNA or Protein) מורן גרינברג 2008
Alkaline Lysis Method מורן גרינברג 2008
Lysis מורן גרינברג 2008
Real time pcr • Genomic DNA and Plasmid
GO HOME !! מורן גרינברג 2008
