1. Lecturer Bahiya Osrah 2 nd Term 2012

3. DNA isolation from strawberries See the attached File on my website Why we chose strawberries: It is a good candidate for demonstrating DNA extraction since it is octoploid - with eight copies of each gene in the strawberry genome. Explanation The detergent in the shampoo helps to dissolve the phospholipid bilayers of the cell membrane and organelles. The salt helps keep the proteins in the extract layer so they aren’t precipitated with the DNA. DNA is not soluble in ethanol. When molecules are soluble, they are dispersed in the solution and are therefore not visible. When molecules are insoluble, they clump together and become visible. The colder the ethanol, the less soluble the DNA will be in it yielding more visible “clumping.” This is why it is important for the ethanol to be kept in a freezer or ice bath. DNA isolation Lab

4. Isolation of DNA from Tissues: To extract DNA from tissue/cells of interest, the cells have to be separated and the cell membranes have to be disrupted. Reagents : Lysing step 1.EDTA (Ethylenediaminetetraacetate) which removes Mg+2 and Ca+2 ions These ions are essential for preserving the overall structure of the cell membrane To inhibit Dnase ( Mg+2 and Ca+2 are cofactors) 2.SDS (Sodiumdodecylsulfate), which aids in disrupting the cell membranes by removing the lipids of the cell membranes, and solubilized the proteins both are included in the extraction buffer which lysing the cells, 3. Protein enzyme or protease can be used to break down proteins.

5. After the lysing step and centrifugation the cell extract now contain: DNA, protein and RNA. to remove these contaminants, leaving the DNA in a pure form we need to add: 1.Phenol: The standard way to de-proteinize (precipitate protein) a cell extract is to add phenol or a 111 mixture of phenol1 chloroform. These organic solvents precipitate proteins but leave the nucleic acids in aqueous solutions.The aqueous solution of nucleic acid can be removed with a pipette. 2. Ribonuclease enzyme: Ribonuclease enzyme to degrade RNA.

6.  Isolation of DNA from spleen Have a look on the protocol of the isolation of DNA from spleen  STUDY: The reagents and their purposes: Reagents: The tissue is homogenized in isotonic saline buffered with sodium citrate PH7.4: Because at this ionic strength, the deoxyribonucleoprotein is insoluble and separates well from other proteins. The sodium citrate or EDTA (Ethylenediaminetetraacetate) is used to: Inhibit deoxyribonuclease activity by binding Ca++ and Mg++, which are cofactors for this enzyme. Some DNA isolation protocols used SDS (sodiumdodecylsulfate) to: Disturb the cell membranes by removing thr lipids and solubilized the protein. Don’t forget to know

7. Proteinase is used to: Break down the yield protein during the isolation process. The extraction procedure is carried out under cold condition: so that any residual DNA'ase activity is minimal. ice-cold ethanol: The DNA is finally precipitated as a fibrous white mass by the addition of ethanol. DNA less soluble in cold ethanol. Don’t forget to know

9.  RNA exist as a single strand.  Ribose Sugar (5 carbon sugar)  Phosphate group  Adenine, Uracil, Cytosine, Guanine  For RNA, nucleosides are formed similarly to DNA.  Hairpin is a common secondary/tertiary structure.  Double stranded RNA can also exists in mammals Structure of RNA

10. RNA Types

11. snRNA - Small nuclear RNA is a class of small RNA molecules that are found within the nucleus of eukaryotic cells. eukaryotic They are involved in a variety of important processes such as RNA splicing (removal of introns), regulation of transcription factors and maintaining the telomeres.intronstranscription factorstelomeres snoRNA - Small nucleolar RNA snoRNAs are involved in rRNA modification. They are located in the nucleolus.nucleolus Other RNA’s involved in Gene Expression Regulation: siRNA- RNA interference miRNAs- Micro RNA New types of RNA

12. siRNA (Gene Silencing) Degrades mRNA then it inactivates the gene translation

13. miRNAs Tiny 21–24-nucleotide RNAs Non coding small RNAs unlike siRNAs, miRNAs downregulate expression after translation initiation without affecting mRNA stability stem-loop structure is highly Conserved Translation Inhibition

15. Tris-Borate: To allow the current conductivity EDTA (Ethylenediaminetetraacetate): Divalent cations chelating agents, which inhibits subsequent enzymatic reactions. Note: The TBE should not be very concentrated:Because that would make the current very high and a lot of heat would be generated which could cause gel melting and DNA denaturation. TBE buffer

16. The Bromophenol Blue Dye: 1.blue tracking dye that migrate usually as 300 bp DNA 2.This allows the samples to be seen when loading onto the gel 3.increases the density of the samples, causing them to sink into the gel wells DNA ladder To identify the separated or migrated DNA bands sizes in the gel Ethidium bromide Interchelates the DNA bases and glows under the UV light to give the pink color which allows the DNA visuilization DON’T forget::

17. Agarose: 1.It is a polysaccharide extracted from seaweed 2.It is typically used at concentrations of 0.5% to 3%. 3. Its preparation is easier than polyacrylamide 4.Agarose gels have a large range of separation, but relatively low resolving power. 5.DNA fragments from about 0.2 kbp to 50 kbp can be separated in agarose. The differences between Agarose and polyacryamide

18. Polyacrylamide: 1.It is a cross-linked polymer of acrylamide. 2.A wide range of conc. can be used between 3.5% to 20% (Advantage to give higher resolution to separate very small DNA fragments that are differ in a one bp and so used for DNA sequencing). 3.Polyacrylamide gels are more annoying to prepare than agarose gels and toxic (Disadvantage). Because oxygen inhibits the polymerization process, they must be poured between glass plates (or cylinders). 4.Polyacrylamide gels have a small range of separation, but very high resolving power. 5.DNA less than 0.5 kbp can be separated by polyacrylamide.

19. SDS Gel Electrophoresis SDS-PAGE For protein separation In SDS separations, migration is determined not by intrinsic electrical charge of polypeptides but by molecular weight Sodium dodecylsulfate (SDS) is an anionic detergent SDS confers a net negative charge to the polypeptide in proportion to its length denatures secondary and non–disulfide–linked tertiary structures by wrapping around the polypeptide backbone.

20. SDS Gel Electrophoresis

21. Finally Identify the separated DNA band sizes

22.  100  200  300  1,650  1,000  500  850  650  400  12,000 bp  5,000  2,000 DNA Ladder Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs. - + DNA migration bromophenol blue  Note: bromophenol blue migrates at approximately the same rate as a 300 bp DNA molecule

23. All the information about the sequence of human genome can be obtained from large databases such as NCBI (National Center for Biotechnology Information) DDBJ (DNA Data Bank of Japan) EMBL (European Molecular Biology Laboratory) Protein structure databases PDB =Protein data bank PubMed Literature references Nucleotide sequence databases NCBI (GeneBank) Bioinformatics