DNA isolationPlaton, Mabel Justine

Panuncio, Muriel ErikaPao, Christel

Ramilo, KelvinRamos, genie anne

Salalima, BeaSalon, Mae Angellie

Sanding, Eliza MaureenSebastian, Jamie Emmanuelle

DNA

Deoxyribonucleic Acid• is a nucleic acid that contains the

genetic instructions used in the development and functioning of all known living organisms and some viruses.

• carries the information needed to direct replication and protein synthesis.

JAMES WATSON AND FRANCIS CRICK

• Published the first description of the structure of DNA

• Double- stranded helix DNA

EUKARYOTIC VS. PROKARYOTIC DNA

EUKARYOTIC

• LINEAR• COMPLEXED with

"histones“

• ORGANIZED into CHROMOSOMES

• With varied number of chromosomes (DNA)

• Stored in the NUCLEUS

PROKARYOTIC

• CIRCULAR with no ends• NAKED “without histones”• UNORGANIZED but forms in circles called PLASMIDS• One DNA

• Stored in the CYTOPLASM or area called Nucleiod.

DNA ISOLATION

• a routine procedure to collect DNA for subsequent molecular or forensic analysis.

DNA ISOLATION

GENERAL STEPS:

1.Cell disruption or cell lysis2.Removal of membrane lipids by a

detergent.3.Removal of proteins by a protease.4.Precipitation of DNA with alcohol.

Why ONION?• It is used because it has a low starch content

which allows the DNA to be seen more clearly.• The salt shields the negative phosphate end of

DNA which allows these ends to come closer so they can precipitate out of a cold alcohol solution.

• The detergent causes the cell membrane to breakdown by emulsifying the lipids and proteins of the cell and disrupting the polar interactions that hold the cell membrane together.

• The detergent then forms complexes with these lipids and proteins, causing them to precipitate out of solution. Collectively, the salt solution and detergent are referred to as a lysing buffer.

PROCEDURES

Weigh 100 g of onion

Homogenize for 30-60 secs.

Decant supernatantand discard

Pour 50 mL homogenate into nalgene

centrifuge tubes

Centrifuge at 7000 rpm

for 15 mins at 4oC

Cut into pieces

Place in chilled

blender w/ 300 mL

cold citrate saline buffer

Resuspend pellet in 20 mL of

cold citrate saline buffer

using vortex

Add 20 mL cold 2.6 M NaCl

Recentrifuge at 13,000 rpm for 20 mins

Break pellets using vortex.

Shake vigorously

Pour off 50 mL suspension into centrifuge tubes

Decant supernatant

Recentrifuge at 7000

rpm for 15 mins at 4oC

PROCEDURES

Pour supernatant into a glass tube

Remove DNA from the solution

Dissolve extracted DNA in 15 mL of coldcitrate saline buffer

in a 125 mL Erlenmeyer flask

Pour off 50 mL suspension into centrifuge tubes

Decant alcohol

Collect DNA by gently stirring

Add 5 mL cold 95% ethanol

Recentrifuge at 13,000 rpm for 20 mins

PROCEDURES

Place solution into centrifuge tubes

Add pancreatic ribonuclease A to a final conc. of 100 ug/mL

& agitate slowly for 1 hr

Extract solution w/ chloroform:isoamyl alcohol

(24:1). Shake vigorously

Add sodium lauryl sulfate to make 1%

conc. & sodium perchlorate to a

final conc. of 1 M. Agitate for 30 mins.

Add pronase to a final conc. of 50 ug/mL

& agitate slowly for 1 hr

PROCEDURES

Wash the spooled DNA twice w/

cold 70% ethanol

Centrifuge at 3000 rpm for 5 mins at 4oC

Respool DNA from the solution onto a glass rod

Add 2 volumes of 95% cold

ethanol

Remove upper aqueous phase

using a pipettor

PROCEDURES

1. What is the rationale of homogenizing the samples using Saline Citrate buffer?

The homogenization step involves the heating and blending of the onion tissue in order to break down the cells. The heat treatment softens the phospholipid in the cell membrane and denatures the deoxyribonuclease enzymes, which if present will cut the DNA into small fragments that will not spool. The onion tissue is mixed in a blender with the homogenization medium which is the Saline Citrate buffer , which breaks down the cell wall, cell membrane, and nuclear membrane.

2. What are the substances in the supernatant liquid that must be discarded?

The substances in the supernatant liquid that

must be discarded are soluble proteins and other membrane bound organelles of the cell.

3. What is the importance of salt in the set-up? Why do you have to suspend the cells in cold salt solution? The importance of salt in the set-up is to make proteins and carbohydrates precipitate while making the DNA remained in solution. Suspending cells in cold salt solution will provide the DNA with favorable environment since salt contributes atoms that neutralize the normal negative charge of DNA. Negatively charged phosphates on DNA causes the cells to repel each other.  The NaCl Solution provides Na ions that will block charge from phosphates on DNA. The Na ions will form an ionic bond with negatively charges and allow DNA molecules to come together.

4. Describe the isolated DNA.

The DNA appears to be whitish and it looks like a twisted ladder when subjected to the UV viz. The outside of the ladder is made of deoxyribose sugars all attached to each other. The rungs of the ladder are four different bases: adenine, cytosine, guanine and thymine

5. Importance of the discovery of DNA to science

1.Modern Medicine and Genetic ResearchImproved ability to diagnosis disease, detect

genetic predisposition to disease, create new drugs to treat disease, use gene therapy as treatment, and design "custom drugs" based on individual genetic profiles.

2.Genetic EngineeringRecombinant DNA , a man-made DNA sequence that

has been assembled from other DNA sequences. They can be transformed into organisms (GMO) of desired and appropriate format.

3. Forensics DNA is used in blood, semen, skin, saliva or hair found

at a crime scene to identify a matching DNA of an individual called DNA profiling and DNA Fingerprinting.

4. Bioinformatics manipulation, searching, and data mining of DNA

sequence data. 5. DNA Nanotechnology

uses the unique molecular recognition properties of DNA and other nucleic acids to create self-assembling branched DNA complexes with useful properties.

6. What is the rationale of using the following reagents?a.Ribonuclease A (RNase A).•it specifically degrades single stranded RNA at C and U residues. It cleaves the phoshodiester bond between the S ribose of a nucleotide and the phosphate group attached to the 8’ ribose of an adjacent pyrimidine nucleotide

b. Pronase

It was used in order to breakdown proteins removing the from the DNA . It was also done in order to purify the isolated DNA

C. Addition of Sodium Lauryl Sulfate• Sodium lauryl sulfate is an anionic surfactant used in many

cleaning and hygiene products. The product can also be used to aid in lysing cells during DNA extraction and for unraveling proteins. It is commonly used in preparing proteins for electrophoresis. This compound works by disrupting non-covalent bonds in the proteins, denaturing them, and causing the molecules to lose their native shape (conformation).

D. Addition of chloroform: isoamyl alcohol• Proteins and restriction enzymes are removed by chloroform in

disrupting protein secondary structure. Chloroform is an organic solvent that very efficiently denature and cause the precipitation of proteins. Isoamyl alcohol reduces the foaming of proteins that would normally be generated by the mechanics of the extraction procedure. In the presence of these solvents RNase activity is inhibited.

Conclusion:

DNA can be isolated from its surrounding matter through a process involving homogenization, deproteinization, and the eventual separation of DNA. "Hydrion" molecules have a negative charge, as accidentally demonstrated by their attraction to the positive electrode in an electrical field. Each species of onion plant has its own DNA pattern, and members within each species share a pattern.