DNA Isolation - Copy | Dna | Nucleotides

Molecular Genetic Pathology DNA Structure and Function

Prepare By Aishan Kuraci Directed By Dr. Mehri Igci

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Dr.Saba Abdi
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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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and a heterocyclic nucleobase.Nucleotide Each nucleotide consists of Phosphate ester. Nucleotide :is the building block of DNA Dr. a pentose sugar.Saba Abdi 3 .

Cytosine ) 2.Nucleotide is composed of : 1.Saba Abdi 4 .Pentose sugar Dr. Adnine ) • Pyrimidine(Thymine . Nitrogen base • Purine :two kind (Guanine .Phosphate group 3.

Saba Abdi 5 .Antiparallel : oppsite Direction Double helix Double strand Dr.

Saba Abdi 6 .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 Dr.

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) 7 .

DNA TOPOLOGY DNA topology studies the shape and path of the DNA helix in three dimensional space. The topology of DNA topoisomers is important to replication. transcription and recombination. Topoisomerasesare enzymes that change the topology of DNA. Dr. including the recombination events important to the life cycles of many viruses.Saba Abdi 8 .

Twist is the number of times one strand completely wraps around the other strand. Dr.Saba Abdi 9 . Writhe is the number of times that the long axis of the double helical DNA crosses over itself in 3-D space.

Saba Abdi 10 .Dr.

Saba Abdi 11 .Goals: . Cell lysis •removal of proteins and fats •Destruction of DNAase and RNAase •DNA vs RNA •isolation of a specific type of DNA (or RNA) Dr.

Saba Abdi 12 .Types of Methods: •differential solubility •‘adsorption’ methods •density gradient centrifugation Dr.

Types of DNA: •genomic (chromosomal) •Organellar (satellite) •plasmid (extra-chromosomal) •phage/viral (ds or ss) •complementary (mRNA) i 13 .

14 .A routine procedure to collect DNA for subsequent molecular or forensic analysis.

General Features: •denaturing cell lysis (SDS. boiling. chaotropic) • enzyme treatments -Protease (proteinase K) -RNase (DNase-free) -DNase (RNase-free) Dr. alkali.Saba Abdi 15 .

High MW Genomic DNA Isolation Typical Procedure 1 Cell Lysis – 0.5% SDS + proteinase K (55o several hours) Phenol Extraction • mix sample with equal volume of sat.Saba Abdi  aqueous phase (nucleic acids)  phenol phase (proteins) 16 . phenol soln • retain aqueous phase • optional chloroform/isoamyl alcohol extraction(s) 2 Phenol Extraction – gentle rocking several hours 3 Ethanol Precipitation 4 RNAse followed by proteinase K 5 Repeat phenol extraction and EtOH ppt Dr.

pH 5-5.5% SDS + proteinase K (55o several hours) EtOH Precipitation • 2-2.5 volumes EtOH.High MW Genomic DNA Isolation Typical Procedure 1 Cell Lysis – 0. -20o • high salt.5 • centrifuge or ‘spool’ out 2 Phenol Extraction – gentle rocking several hours 3 Ethanol Precipitation 4 RNAse followed by proteinase K 5 Repeat Phenol Extraction and EtOH ppt Dr.Saba Abdi 17 .

Isolation of RNA Special 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.Saba Abdi 18 . mRNA) with LiCl • oligo-dT column Dr.

Centrifuge.Saba Abdi buffer . Mix supernatant with resin + chaotropic agent 5. Solubilize bacteria in alkali solution 2. Neutralize with Na-acetate 3. Wash resin 6. Elute DNA with low salt 19 Dr. discard pellet 4.Adsorption Methods • nucleic acids selectively absorb to silica or resins in the presence of certain chaotropic agents or salts • applications: • plasmid preps • fragments after electrophoresis • PCR templates Plasmid Miniprep Protocol 1.

70 1.3 g/cm3 RNA > DNA ssDNA > dsDNA GC content 1.72 1.Saba Abdi 40 60 80 20 % GC base pairs .7 g/cm3 protein ~1.Density Gradient Centrifugation • rate zonal/sucrose (size fractionation) • electrophoresis more common • isopycnic/CsCl (density) density (g/cm3) • • • • • DNA ~1.68 20 Dr.74 1.

CsCl Gradients Applications • large scale preparations • high purity • ‘satellite’ DNA • RNA ‘cushions’ CsCl Gradients Dr.Saba Abdi 21 .

DNA absorbs UV light with a major peak at 260 nm Optical Density This absorption is useful because it varies with the structure of DNA (&RNA) i. extinction coefficient depends on the structure Wave Length dsDNA Low extinction coefficient i ssDNA Higher extinction coefficient 22 .e.

6 .Evaluation of Nucleic Acids • spectrophotometrically • quantity • quality • fluorescent dyes • gel electrophoresis A260 DNA A260/A280 A260 RNA A260/A280 1.8 1.0  40 g/ml ~2.0  50 g/ml 1.0 Dr.Saba Abdi 23 .1.

Saba product Abdi molecular weight markers 24 .Agarose Gel Stained with ethidium bromide (EtBR) to Visualize the DNA slots where DNA is loaded 1000 bp 700 bp 600 bp 500 bp Screening PCR products to test for the presence of specific DNA sequences molecular weight markers correct PCR Dr.

Several hydrophobic molecules containing flat aromatic and fused heterocyclic rings can insert between the stacked base pairs of DNA. These molecules are called intercalating agents. Intercalating agents are potential Cancer-inducing reagents. 25 .

Dr.Saba Abdi 26 .

Dideoxy Chain Termination Dr.Saba Abdi 27 .

Saba Abdi 28 .Thank you Dr.

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