Académique Documents
Professionnel Documents
Culture Documents
they are responsible for transfer of DNA and RNA; genetic information
Before, they thought that proteins were responsible of transfer of genetic information James Watson and Francis Crick- theoretical helix structure; based on results of different scientists
NUCLEIC ACIDS NUCLEOTIDE A. Nitrogenous Base o o o Purine- Guanine, Adenine Pyrimidine- Cytosine, Uracil, Thymine no prime
C. Phosphate Group o o with prime outside because it is polar and the environment of cell is also polar; will interact with the environment
BONDS: glycosidic bond phoshodiester/ phosphoester bond H bond Van der Waals/ Hydrophobic sugar + base sugar + phosphate base + base base + base (up and down)
DNA double helix antiparallel orientation (5'--3') o 5' - 1 sugar only attached to phosphate Chargaff: equal conc. of A-T and C-G highly polar 2 strands are complementary more stable than RNA Negatively charged deoxyribonucleases/ DNase o hydrolyze DNA
RNA single strand viruses, etc.- double strand less stable mRNA- from DNA; making of protein/enzyme; from nucleus rRNA- found in ribosomes; protein synthesis tRNA- translation from mRNA to polypeptide to tRNA o o looks like a cross start methionine AUG
more susceptible to enzymatic attack ribonuclease; RNase- hydrolyze RNA o ubiquitous- found everywhere
EXPERIMENT
DNA- Onion White Onion was used instead of red onion red onion has more contaminants red onion is more phenolic phenolics- similar to carbohydrates when phenolic conc. is increased, DNA isolation will not be successful
- mechanical cutting for faster grinding of sample -homogenizing solution SDS o o Sodium dodecyl sulfate detergent o a detergent is a denatured agent for proteins similar to action of soap to dirt
cell membrane lipid bilayer contains lipids and membrane proteins, receptor proteins, triglycerol, phospholipids, fatty acids membrane proteins- responsible for interaction of membrane/cell with its environment; says which kind of materials can go in and out
DNA disrupts the cell membrane SDS is a detergent with a long nonpolar tail and a polar head SDS interacts with nonpolar part of lipid --> interaction will make membrane will become soluble* SDS will make proteins more soluble in the solution
EDTA o o o o o ethylenediamine tetra-acetic acid chelates metals/ chelating agents metals present in the biological system: magnesium, calcium, iron, sodium, potassium particularly chelates calcium nucleases require magnesium for their activity/ function without Magnesium, it will not work if with Magnesium, DNA and RNA will be hydrolyzed ;nucleases will be activated chelate magnesium in order to prevent nucleases (DNase; RNase) to cleave/ hydrolyze DNA/RNA o always there to remove magnesium from the solution so as to deactivate the nucleases
NaCl o o o o a salt DNA is highly negative due to phosphate backbone stabilizes DNA (since DNA is negative, sodium will react with DNA) electrostatic interaction
Sodium Citrate o o o citrate- buffers maintains pH of solution for integrity of DNA papain protease enzyme hydrolyzes protein prevents amino acid sequence we did not use papain, instead we used bromylate (similar to papain; a protease; difference: amino acid recognized for the hydrolysis) o pancreatic ______*
purpose of enzymes: hydrolyze proteins; release DNA from nucleoproteins ex. histones to get pure DNA and get rid of contaminants by uncoiling 4 histones--> coiled --> ikot ulit (packing)
incubate for an hour/30 mins- facilitate release of DNA from cell- catalysis if hot, enzymes will be excited * ??
o o
filter precipitated out proteins equal volume of ice cold ethanol layer to precipitate out DNA ice cold- for faster precipitation room temperature- more contamination since DNA is highly negative and polar, it will definitely precipitate out of the solution
Sources of error o o o o o o drying of mortar and pestle handling nucleases are not deactivated contaminated with proteins proteins and DNA won't separate
RNA added NaOH to baker's yeast o purpose: deactivation of proteases* (SHE SAID SOMETHING HINDI KO MARINIG)
RNA- ubiquitin; found anywhere hard to degrade* - even if you autoclaved it 3x already Glacial acetic acid o o acidification- adjust pH; causes* proteins to precipitate out dissociate RNA from any proteins attached to it
Store in ref o o substitute for centrifugation for RNA to settle at the bottom
A260/ 280- 1.8-2.0 (for both DNA and RNA) 260 nm- wavelength where nitrogenous bases absorb light 280- ring of Tryptophan and phenylalanine, tyrosine will be maximally absorbed o o o if large/ increased, ratio will go down signifies protein contamination proteins absorbed in aromatic amino acids
DNA RNA
larger than 230- carbohydrate contamination very pure/ slightly pure/ not so pure
acid hydrolysis most susceptible to attack: purine product after acid hydrolysis: no more glycosidic bond --> release of nitrogenous base each individual (nitrogenous base/ pentose sugar/ phosphate) -is "extracted" quali test: (+) of DNA hydrolysate: diphosphate/ deoxyribose/purine/ pyrimidine less susceptible to NaOH hydrolysis (mixture of 2' and 3' * due to presence of hydroxyl group of 2')
3/3/2012 10:26:00 PM
Qualitative Test Phosphate Test Other name Reagents Concentrated HNO3, (NH4)2 MoO4 (add phosphate group to (NH4)2 MoO4 ) Visible Result yellow ppt. (If white ppt, only (NH4)2 MoO4 is present)
Ribose Test
Bial-Orcinol
Orcinol FeCl3, HCl (OH will become =O Furfural react with orcinal ) (complexation reaction)
Deoxyribose Test
Dische
Diphenylamine, glacial HoAC, conc'd H2SO4 (ring in the sugar will become linear--> diphenylamine reacts--> sticks to Oxygen)
Purine Test
Murexide (color)
red residue
Pyrimidine Test (Thymine will be negative due to CH3, inhibits action in Br2, alcohol will react with bromide--> bridge)
purple coloration