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Molecular Genetic Pathology
DNA Structure and
Function
Prepare By
Aishan KuraciDirected By
Dr. Mehri Igci
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DNA :is the genetic material ( stores information)
DNA (deoxyribonucleic acid) is the molecule that stores
genetic information in most living systems (exception RNA
viruses).
Nucleic acids: are formed from nucleotide subunits.
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Nucleotide
Each nucleotide consists ofPhosphate ester, a pentose sugar,
and a heterocyclic nucleobase.
Nucleotide :is the building block of DNA
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Nucleotide is composed of:
1. Nitrogen base
Purine :two kind (Guanine , Adnine )
Pyrimidine(Thymine ,Cytosine )
2.Phosphate group
3.Pentose sugar
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Antiparallel : oppsite Direction
Double helix
Double strand
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DNA must be packaged
Contour length of DNA is usually much larger than the size of the
cell.
The main role of histon protein use to pachaged of genetic material
DNA
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Difference between eukaryotic and prokaryotic DNA
Eukaryotic DNA Linear DNA contains many origin of replication
Contains bound histone proteins to form chromosomes
Contain base pair more than of prokaryotic DNA
-prokaryotic DNA
Circular DNA contains single origin of replication
Not contain histone protein style in prokaryotic by condense
Region called nucleoid (partially package)
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DNA TOPOLOGY
DNA topology studies the shape and path of the DNA helix inthree dimensional space. The topology of DNA topoisomers is
important to replication, transcription and recombination,
including the recombination events important to the life cycles of
many viruses. Topoisomerasesare enzymes that change the
topology of DNA.
http://encyclopedia.thefreedictionary.com/Topologyhttp://encyclopedia.thefreedictionary.com/Topoisomerhttp://encyclopedia.thefreedictionary.com/DNA+replicationhttp://encyclopedia.thefreedictionary.com/Transcription+(genetics)http://encyclopedia.thefreedictionary.com/Recombinationhttp://encyclopedia.thefreedictionary.com/Virushttp://encyclopedia.thefreedictionary.com/Topoisomerasehttp://encyclopedia.thefreedictionary.com/Topoisomerasehttp://encyclopedia.thefreedictionary.com/Virushttp://encyclopedia.thefreedictionary.com/Recombinationhttp://encyclopedia.thefreedictionary.com/Transcription+(genetics)http://encyclopedia.thefreedictionary.com/DNA+replicationhttp://encyclopedia.thefreedictionary.com/Topoisomerhttp://encyclopedia.thefreedictionary.com/Topology7/31/2019 DNA Isolation - Copy
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Twist is the number of times one strand completelywraps around the other strand.
Writhe is the number of times that the long axis of the
double helical DNA crosses over itself in 3-D space.
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Goals:. Cell lysisremoval of proteins and fats
Destruction of DNAase andRNAaseDNA vs RNA
isolation of a specific typeof DNA (or RNA)
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Types of Methods:differential solubilityadsorption methodsdensity gradient centrifugation
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Types of DNA:genomic (chromosomal)Organellar (satellite)plasmid (extra-chromosomal)
phage/viral (ds or ss)complementary (mRNA)
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A routine procedure to collect DNA for subsequent molecular orforensic analysis.
http://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Forensicshttp://en.wikipedia.org/wiki/Forensicshttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/DNA7/31/2019 DNA Isolation - Copy
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High MW Genomic DNA Isolation
Typical Procedure
1 Cell Lysis
0.5% SDS + proteinaseK (55o several hours)
2 Phenol Extraction
gentle rocking severalhours
3 Ethanol Precipitation
4 RNAse followed byproteinase K
5 Repeat phenol extrac-tion and EtOH ppt
Phenol Extraction mix sample with equal volume
of sat. phenol soln
retain aqueous phase
optional chloroform/isoamyl
alcohol extraction(s)
aqueous phase
(nucleic acids)
phenol phase
(proteins)
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High MW Genomic DNA Isolation
Typical Procedure
1 Cell Lysis
0.5% SDS + proteinaseK (55o several hours)
2 Phenol Extraction
gentle rocking severalhours
3 Ethanol Precipitation
4 RNAse followed byproteinase K
5 Repeat Phenol Extrac-tion and EtOH ppt
EtOH Precipitation 2-2.5 volumes EtOH, -20o high salt, pH 5-5.5 centrifuge or spool out
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Isolation of RNASpecial Considerations
RNAse inhibitors! extraction in guanidine salts phenol extractions at pH 5-6
(pH 8 for DNA) treatment with RNase-free DNase selective precipitation of high MW
forms (rRNA, mRNA) with LiCl oligo-dT column
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Plasmid Miniprep Protocol
1. Solubilize bacteria in alkalisolution
2. Neutralize with Na-acetate3. Centrifuge, discard pellet
4. Mix supernatant with resin+ chaotropic agent
5. Wash resin6. Elute DNA with low salt
buffer
Adsorption Methods
nucleic acids selectively absorb to silica orresins in the presence of certain chaotropicagents or salts
applications: plasmid preps fragments after
electrophoresis PCR templates
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Density Gradient Centrifugation
rate zonal/sucrose (size fractionation) electrophoresis more common
isopycnic/CsCl (density)
DNA ~1.7 g/cm3
protein ~1.3 g/cm3 RNA > DNA ssDNA > dsDNA GC content
20 40 60 80
% GC base pairs
1.68
1.70
1.72
1.74
density(g/cm3)
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CsCl Gradients
Applications large scale preparations high purity satellite DNA RNA cushions
CsCl Gradients
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DNA absorbs UV light with a major peak at 260 nm
OpticalDensity
Wave Length
This absorption is useful
because it varies with the
structure of DNA (&RNA)
i.e. extinction coefficient
depends on the structure
dsDNA
Low extinction
coefficient
ssDNA
Higher extinction
coefficient
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Evaluation of Nucleic Acids
A260 1.0 50 g/mlDNA
A260/A280 1.6 - 1.8
A260 1.0 40 g/mlRNA
A260/A280 ~2.0
spectrophotometrically quantity quality
fluorescent dyes
gel electrophoresis
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Agarose Gel
Stained with ethidium bromide (EtBR) to Visualize the DNA
Screening PCR
products to test
for the presenceof specific DNA
sequences
500 bp
molecularweight
markers
molecularweight
markers
correctPCR
product
600 bp
700 bp
1000 bp
slots where
DNA is loaded
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Several hydrophobicmolecules containingflat aromatic and fusedheterocyclic rings can
insert between thestacked base pairsof DNA. Thesemolecules are calledintercalating agents.
Intercalating agentsare potentialCancer-inducingreagents.
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Dideoxy Chain Termination
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Thank you