PCR בעזרת DNA אנאליזה של TSH של הרצפטור ל - DNA זיהוי נוכחות בעזרת PCR מורן גרינברג 2008

PCR בעזרת DNA אנאליזה של

TSH של הרצפטור ל- DNA זיהוי נוכחות

PCRבעזרת

מורן גרינברג 2008

?על מה נדבר

מהו פלסמיד?• ?cDNAמהו •שיטות להפקת פלסמידים• PCR- עקרונות ריאקצית ה• PCRסוגים שונים של • DNAשיטות להפרדת מקטעי •

(Electrophoresisאלקטרופורזה )


Gel Electrophoresis of DNA


•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

What is Gel Electrophoresis?

•Electro = flow of electricity, phoresis, from the Greek = to carry across


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


• 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)

Basis of migration and separation?

+

+

-

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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


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


•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

boiling


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)


Intercalation of EtBr in DNAIntercalation of EtBr in DNA Q6: Visualization of DNA? Q6: Visualization of DNA?

EtBr stacks between the bases of the DNA helix.

The smaller the DNA molecule, the less EtBr bound, the fainter the image seen.

•Visualize DNA by use of fluorescent dye – Ethidium bromide (EtBr) intercalates in DNA.

•UV light irradiates gel (WEAR FACE SHIELD).

+ +


Agarose Gel ElectrophoresisAgarose Gel ElectrophoresisQ5: Loading dye components and purpose?Q5: 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


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


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


Photograph of DNA in Agarose GelPhotograph of DNA in Agarose Gel

Typical gel resultsמורן 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


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


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



PCR


PCR Réaction Mixture • DNA or cDNA

• dNTPs

• MgCl2 ; MgSO4

• Primers ; Tm= melting température

• Taq-polymerase ; PFU polymérase


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


•DNA synthesis requires a primer to start DNA synthesis

•separate parent strands in preparation new strand 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


PCR AnimationPlease click here.

1st cycle 2nd cycle 3rd cycle

Process

Denature

Anneal Primer

Replicate DNA


PCR methods

• Purpose of PCR: amplification ,specificity, sequencing

• Potential Problems with Taq and PFU• PCR Applications• RT-PCR• Real-Time PCR• Mutagenesis


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

RNA


Isolation of Plasmid DNACloning 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)



Alkaline Lysis Method


Lysis




......שאלות לדוגמא לבחינהשאלות לדוגמא לבחינה......שאלות לדוגמא לבחינהשאלות לדוגמא לבחינה

• Real time pcr

• Genomic DNA and Plasmid

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Documents

PCR בעזרת DNA אנאליזה של TSH של הרצפטור ל - DNA זיהוי נוכחות בעזרת PCR מורן גרינברג 2008