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Extracting

DNA from
Whole Blood
Group 4 - 4Bio2
Binag, Sam Dominic
Caringal, Joe Mari Isabella
Chiang, Rowena
Verin, Maryneil

Introduction
DNA EXTRACTION
process of purification of DNA from sample
using a combination of chemical and
physical methods
one of the most widely used molecular
biology techniques
usually the first step in a longer laboratory
process

Introduction
DNA EXTRACTION
important routine procedure in the fields of
biotechnology and forensics
allow scientists to detect genetic diseases,
analyze forensic evidence, produce DNA
fingerprint of individuals, and create of
genetically engineered organisms that can
produce beneficial products.

Introduction
DNA EXTRACTION OVERVIEW
1. lyse or break open the cell to expose the
DNA of interest.

2. washing with detergent

3. precipitation and resuspending of DNA

Introduction
BLOOD
excellent source of human DNA

Friedrich Miescher- isolated the first crude


extract of DNA from white blood cells in
1869
- discovered the substance nuclein now
known as DNA

Introduction
Blood
Whole blood contains three types of
cells: red blood cells (RBCs), white
blood cells (WBCs), and platelets.
RBCs do not have any DNA

WBCs contain DNA

Objectives
To prepare a flow diagram of the
experimental protocol
To extract DNA from blood

Hypothesis

Different DNA sources produce


different amounts of DNA and the
amount and freshness of the blood
affect the quality and quantity of the
DNA obtained.

Methodology

Results and
Discussion
Cell Concentration
Centrifugation
Phosphate Buffered Saline (PBS)

Cell Lysis
STE, SDS, Proteinase K

Results and
Discussion
STE
Sodium chloride: for DNA to precipitate
Tris: buffer system
EDTA: chelates cations

SDS
Detergent
Disruption of cell membranes
Emulsification of lipids and proteins

Results and
Discussion
Proteinase K
Denatures proteins
Disables nucleases

DNA Extraction
Phenol-chloroform extraction
Liquid-liquid extraction
For isolation of DNA, RNA, proteins

Results and
Discussion
Forms lower organic phase and upper
aqueous phase

Results and
Discussion
Aqueous Phase
Contains hydrophilic DNA

Organic Phase
Contains hydrophobic lipids

White interphase
Interactions of hydrophobic molecules of proteins
with phenol

Results and
Discussion
Phenol: constitutes lower organic
phase

Chloroform
Miscible with phenol
Forces a sharper separation between
organic and aqueous phase

Isoamyl alcohol
Added in small amounts
Prevents foaming

Results and
Discussion
DNA Concentration (Washing)
Sodium acetate (Salt)
Makes DNA less soluble in the aqueous solution
More efficient precipitation of DNA in alcohol

Ethanol Precipitation
Washing with 70% Ethanol
Wash away residual salts

Results and
Discussion
TE Buffer
Tris-EDTA Buffer
Solubilize DNA while preventing
degradation

Guide Questions
1. What is the role of the following in DNA
extraction?
a. Ethanol
b. NaCl
c. SDS
d. TE Buffer
e. EDTA

Guide Questions
a. Ethanol
Ethanol is used in DNA extraction to
force the DNA to precipitate in a solution.
Ethanol prevents the DNA from dissolving
into the water, instead causing it to
precipitate out so it can be separated
and extracted. Without the addition of
ethanol, DNA will just dissolve in water.

Guide Questions
b. NaCl

Salt neutralizes the charges on the


sugar
phosphate
backbone.
The
positively
charged
sodium
ions
neutralize the negative charge on the
PO3- groups on the nucleic acids, making
the molecule far less hydrophilic, and
therefore much less soluble in water.

Guide Questions
c. SDS

SDS is a strong anionic detergent that can


solubilize the proteins and lipids that form the
membranes. This will help the cell membranes and
nuclear envelopes to break down and expose the
chromosomes that contain the DNA. In addition to
removing the membrane barriers, SDS helps
release the DNA from histones and other DNA
binding proteins by denaturing them.

Guide Questions
d. TE Buffer

The purpose of TE buffer is to solubilize


DNA or RNA, while protecting it from
degradation.

Guide Questions
e. EDTA

EDTA behaves as chelating agent for


divalent ions like Ca++ and Mg++. Ca++ ions
are initiator of blood clotting and behave as
cofactor for clotting enzymes. Hence, EDTA
prevents blood coagulation by chelating these
ions.

Guide Questions
2.Give the complete chemical names of the
following:
a. SDS Sodium dodecyl sulfate

b.Tris BufferTris(Hydroxymethyl)aminomethane

c. EDTA - Ethylenediaminetetraacetic acid

Guide Questions
3. Why should the extracted DNA be
immersed in TE buffer?
TE buffer protects DNA from
degradation.
"TE"
stands
for
its
components Tris and EDTA. Tris serves as
a buffering agent while EDTA serves as a
chelating agent. TE buffer is commonly
used to protect the DNA while storing it.

Conclusion
The purpose of DNA extraction is to obtain DNA
in a relatively purified form, which can be used
for further investigations.
An efficient DNA extraction method for whole
blood was carried out using phenol-chloroform
extraction process.
The protocol designed for blood yielded DNA.
The number, age, and freshness of the source
affect the quality and quantity of the DNA
obtained.

Thank you!