Download Why ONION?

DNA isolation
Platon, Mabel Justine
Panuncio, Muriel Erika
Pao, Christel
Ramilo, Kelvin
Ramos, genie anne
Salalima, Bea
Salon, Mae Angellie
Sanding, Eliza Maureen
Sebastian, 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 lysis
2.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
Cut into pieces
Pour 50 mL
homogenate
into nalgene
centrifuge tubes
Place in
chilled
blender w/
300 mL
cold citrate
saline buffer
Centrifuge at
7000 rpm
for 15 mins at
4 oC
Homogenize for
30-60 secs.
Decant supernatant
and discard
PROCEDURES
Resuspend pellet
in 20 mL of
cold citrate saline
buffer
using vortex
Recentrifuge at
7000
rpm for 15
mins at 4oC
Break pellets
using vortex.
Shake vigorously
Decant
supernatant
Pour off 50 mL
suspension into
centrifuge tubes
Add 20 mL
cold 2.6 M NaCl
Recentrifuge at
13,000 rpm
for 20 mins
PROCEDURES
Pour supernatant
into a glass tube
Pour off 50 mL
suspension into
centrifuge tubes
Add 5 mL cold
95% ethanol
Recentrifuge at
13,000 rpm
for 20 mins
Collect DNA
by gently stirring
Decant alcohol
Remove DNA
from the solution
Dissolve extracted
DNA in 15 mL of cold
citrate saline buffer
in a 125 mL
Erlenmeyer flask
PROCEDURES
Add pancreatic
ribonuclease A
to a final conc.
of 100 ug/mL
& agitate slowly
for 1 hr
Add pronase to
a final conc.
of 50 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.
Place solution
into centrifuge tubes
PROCEDURES
Centrifuge at
3000 rpm for
5 mins at 4oC
Remove upper
aqueous phase
using a pipettor
Respool DNA
from the solution
onto a glass rod
Add 2 volumes
of 95% cold
ethanol
Wash the spooled
DNA twice w/
cold 70% ethanol
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 Research
Improved 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 Engineering
Recombinant 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 selfassembling 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 noncovalent 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.