Download + + מורן גרינברג 2008

‫אנאליזה של ‪ DNA‬בעזרת ‪PCR‬‬
‫זיהוי ‪ DNA‬של הרצפטור ל‪TSH -‬‬
‫נוכחות‬
‫בעזרת ‪PCR‬‬
‫מורן גרינברג ‪2008‬‬
‫על מה נדבר?‬
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‫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
2008 ‫מורן גרינברג‬
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
2008 ‫מורן גרינברג‬
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
2008 ‫מורן גרינברג‬
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)
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2008 ‫מורן גרינברג‬
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
2008 ‫מורן גרינברג‬
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
2008 ‫מורן גרינברג‬
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
2008 ‫מורן גרינברג‬
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)
2008 ‫מורן גרינברג‬
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.
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The smaller the DNA
molecule, the less EtBr
bound, the fainter the
image seen.
2008 ‫מורן גרינברג‬
Agarose Gel Electrophoresis
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
2008 ‫מורן גרינברג‬
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
2008 ‫מורן גרינברג‬
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
2008 ‫מורן גרינברג‬
Photograph of DNA in Agarose Gel
Typical gel results
2008 ‫מורן גרינברג‬
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
2008 ‫מורן גרינברג‬
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
2008 ‫מורן גרינברג‬
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
2008 ‫מורן גרינברג‬
‫מורן גרינברג ‪2008‬‬
‫‪PCR‬‬
‫מורן גרינברג ‪2008‬‬
PCR Réaction Mixture
• DNA or cDNA
• dNTPs
• MgCl2 ; MgSO4
• Primers ; Tm= melting température
• Taq-polymerase ; PFU polymérase
2008 ‫מורן גרינברג‬
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
2008 ‫מורן גרינברג‬
•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
2008 ‫מורן גרינברג‬
PCR animation
2008 ‫מורן גרינברג‬
PCR Animation
Please click here.
Process
Denature
Anneal Primer
Replicate
DNA
1st cycle
2nd cycle
3rd cycle
2008 ‫מורן גרינברג‬
PCR methods
• Purpose of PCR: amplification
,specificity, sequencing
• Potential Problems with Taq and PFU
• PCR Applications
• RT-PCR
• Real-Time PCR
• Mutagenesis
2008 ‫מורן גרינברג‬
RT-PCR reverse transcriptase-pcr
• RNA containing virus
– PCR doesn’t work on
RNA templates
Extract RNA from virus/cells
RNA
– RT PCR
• make cDNA copy
of RNA sequence
first
• PCR the cDNA
copy of RNA
2008 ‫מורן גרינברג‬
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)
2008 ‫מורן גרינברג‬
‫מורן גרינברג ‪2008‬‬
Alkaline Lysis Method
2008 ‫מורן גרינברג‬
‫‪Lysis‬‬
‫מורן גרינברג ‪2008‬‬
‫מורן גרינברג ‪2008‬‬
‫מורן גרינברג ‪2008‬‬
‫שאלות לדוגמא לבחינה‪...‬‬
• Real time pcr
• Genomic DNA and Plasmid
‫!! ‪GO HOME‬‬
‫מורן גרינברג ‪2008‬‬