DNA Isolation - Copy

7/31/2019 DNA Isolation - Copy

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Dr.Saba Abdi 1

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/Topology


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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/DNA


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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

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DNA Isolation - Copy