DNA extraction

1. DNA Extraction
Presented by:
Amany ELshamy

2. Outlines
• Introduction
• What is the DNA and its composition?
• Basic Extraction Steps
• Methods
• Material
• Principle
• Equipment
• Questions
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3. Introduction
• The genomes of almost organisms are DNA
(except Some Viruses e.g.; RNA viruses).
• Genome = genetic material of an organism.
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4. What is the DNA?
Each nucleotide has three components:
1. Phosphate group.
2. Sugar { Ribose (in the case of RNA) or a
deoxyribose (in the case of DNA)}.
3. Single nitrogen base {the purines (adenine [A]
and guanine [G]), each with two fused rings, and
the pyrimidines (cytosine [C], thymine [T], and
uracil [U]}.
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5. What is the DNA?
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6. What is the DNA?
• DNA is the prime genetic molecule,
carrying all the hereditary information
within chromosomes.
• Chromosomes = DNA/protein complexes.
• DNA is composed of a series of
nucleotides.
• Gene is the segment of DNA involved in
producing a polypeptide chain (The basic
biological/functional unit of heredity).
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7. Basic Concept of Extraction
• DNA can be isolated from any part of human
body (Blood, Tissue, Hair….. Etc).
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8. Basic DNA Extraction steps
1. Cell lysis (releasing the DNA)
2. Separating DNA from proteins and other
cellular debris.
3. Precipitating the DNA with an alcohol
4. Elusion (DNA is suspended in a
slightly alkaline buffer).
5. Determine the concentration and purity of DNA
in a sample.
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9. Methods
Physical Chemical/Detergents/Enzymatic
• Mechanical disruption
• Liquid homogenization
• Sonication
• Freeze/thaw cycles
• Manual grinding
• The ideal detergent will
depend on the intended
application (Organic
solvents, Salts, Proteinase
K, Anion-exchange, Silica-
based)
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10. Methods
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11. The choice of method
It depends on many factors
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Required
Quantity
of DNA
Purity and
Application
Time and
Expenses

12. Principle of DNA Extraction kit
Lysis and Extraction buffer:
Lysis of cell wall/ cell membrane, Lysis of nuclear
membrane and Removing cell debris.
Principle:
• Break the cells.
• Protect DNA from lysis.
• Separates DNA from other cell debris.
• Maintain the PH during the extraction.
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13. Silica column-based DNA extraction
Method
 Principle:
• No precipitation or extraction steps are required
therefore, It is a unique method.
• It works on the interaction between silica and DNA (
A positively charged silica particles bind with the
negatively charged DNA and hold it during
centrifugation).
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14. DNA Extraction steps
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15. Material
Sample (Blood)
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Tubes/Spin column
Reagent/kit

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17. Reagent
 Reagents contain:
• MgCl2 protects DNA by blocking the negative charge of
the lipoproteins.
• Salts such as NaCl, KCl neutralize the charges on DNA
and helps DNA to pull out of the cell.
• EDTA is Used for Removing Mg+2 ions from a cell
membrane and block DNase activity.
• Sodium Dodecyl Sulphate (SDS) is used for removing
lipids from a cell membrane and break open the
nucleus and cell.
• Proteinase K is used for digesting the protein portion.
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18. DNA
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19. Measurement
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20. Measurement
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• Equipment: Nanodrop/spectrophotometer UV
absorbance to check the concentration and
purity of a DNA preparation.
• DNA is measured at 260nm (50µg/ml=1 OD).
• Protein is measured at 280nm.
• For pure DNA sample, the Ratio (A260/A280)
is equal to 1.8

21. Measurement
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22. Video
• https://www.youtube.com/watch?v=gmNw6C
WtN5k
• https://www.youtube.com/watch?v=qIX3UEfU
vtU
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