1. Pusat Kedokteran Tropis Fakultas Kedokteran - Universitas Mulawarman
DNA ISOLATION
Sunarto Ang
Pusat Kedokteran Tropis
RSUD. A. Wahab Sjahranie
Fakultas Kedokteran - Universitas Mulawarman
Samarinda

2. DNA Isolation DNA isolation: The aim: Basic Protocol:
is an extraction process of DNA from various sources.
The aim:
is to separate DNA present in the nucleus of the cell from other cellular components.
Basic Protocol:
break the cells open to expose the DNA within.
remove the nuclear membrane lipids by adding a detergent.
precipitate the DNA with an alcohol. This step will also remove alcohol-soluble salts.

3. Application of DNA isolation
Scientific:
use DNA in number of Applications, such as introduction of DNA into cells & animals or plants for diagnostic purposes (gene cloning)
Medicine:
is the most common. To identify point sources for hospital and community-based outbreaks and to predict virulence of microorganisms
Forensic science:
needs to recover DNA for identification of individuals, (for example rapists, petty thieves, accident, or war victims), and paternity determination.

4. Overview of Procedure Lyse RBCs & WBCs
Lyse WBCs nuclei & Denature/digest proteins
Separate contaminants (e.g., proteins, heme)
Precipitate DNA
Resuspend DNA in final buffer

6. Samples for DNA Isolation
Blood
Semen
Saliva
Urine
Hair (w/Root & Shaft)
Teeth
Bone
Tissue
Cigarette Butts
Envelope & Stamps
Fingernail Clippings
Chewing Gum
Bite Marks
Feces

7. Blood Collection Blood collected in disodium EDTA tube
Samples can be stored at -20oC or -70oC
Fresh samples are kept in freezer for a few hours to facilitate RBCs hemolysis
Allow samples to thaw before starting the extraction

8. Methods of DNA Isolation
Organic extraction
Salting out
Selective DNA binding to a solid support
1. Organic extraction
DNA is polar insoluble in organic solvents.
Traditionally, phenol:chloroform is used to extract DNA.
When phenol is mixed with the cell lysate, two phases form.
DNA partitions to the (upper) aqueous phase
Denatured proteins partition to the (lower) organic phase.

9. Genomic DNA isolation: phenol extraction
1:1 phenol : chloroform or
25:24:1 phenol : chloroform : isoamyl alcohol
Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer
Chloroform: increases density of organic layer
Isoamyl alcohol: prevents foaming
Genomic DNA is isolated as pieces up to 1 Mbp!

11. Salting out Digested proteins are removed by
salting out with high concentrations of LiCl.
However, if salts are not adequately removed,
problems could occur with the RFLP procedure due to alteration of DNA mobility (band shifting)

12. Procedure of Salting out
Cell Lysis Buffer - lyse cell membrane, nuclei are intact, pellet nuclei.
Resuspend nuclei in Protein Lysis Buffer containing a high concentration of Proteinase K.
Lyse nuclear membrane and digest protein at 65°C for 2 hours.
Temperature helps denature proteins, and Proteinase K auto digests itself
To remove proteinaceous material, LiCl is added to a final concentration of 2.5 M, and incubated on ice.
Proteins precipitate out and are pelleted by centrifugation.

13. Procedure of Salting out
4. DNA remains in solution. Transfer supernatant to a new tube, care must be taken not to take any of protein pellet.
5. DNA is precipitated by the addition of room temperature isopropanol.
LiCl will not precipitate with DNA.
6. Precipitated DNA is washed with 70% ethanol, dried under vacuum and resuspended in TE buffer.

14. Binding to a support material
Support Materials
Silica
Anion-exchange resin
Advantages
Speed and convenience
No organic solvents
Disadvantage
DNA fragmentation

15. Plants Nucleus is protected within a nuclear membrane which is
surrounded by a cell membrane and a cell wall.
Four steps are used to remove and purify the DNA
Lysis
Precipitation
Wash
Resuspension

16. LYSIS In DNA extraction from plants,
this step commonly refers to the breaking of the cell wall and cellular membranes (most importantly, the plasma and nuclear membranes)
The cell wall (made of cellulose) is disrupted by
mechanical force (for example, grinding the leaves)
Then the addition of a detergent in the which breaks down the cell membranes

17. LYSIS Detergents are able to disrupt membranes due to
the amphipathic (having both hydrophilic and hydrophobic regions) nature of both cellular membranes and detergent molecules.
The detergent molecules are
able to pull apart the membranes
The end result of LYSIS is
that the contents of the plant cells are distributed in solution.

18. PRECIPITATION
A series of steps where DNA is separated from the rest of the cellular components
The first part of precipitation uses phenol/chloroform to remove the proteins from the DNA
Phenol denatures proteins and dissolves denatured proteins.
Chloroform is also a protein denaturant

19. PRECIPITATION The second is the addition of salts
The salts interrupt the hydrogen bonds between the water and DNA molecules.
The DNA is then precipitated by isopropanol or ethanol
ethanol induces a structural change in DNA molecules that causes them to aggregate and precipitate out of solution.
The DNA is pelleted by spinning with a centrifuge and the supernatant removed

20. The precipitated DNA is laden with acetate salts.
Washing:
The precipitated DNA is laden with acetate salts.
It is “washed” with a 70% ethanol solution to remove salts and other water soluble impurities but not resuspend the DNA.
Resuspension:
The clean DNA is now resuspended in a buffer to
ensure stability and long term storage.
The most commonly used buffer for resuspension is called 1xTE

21. Overview of DNA Extraction
Break down the cell wall and membranes
Centrifuge to separate the solids from the dissolved DNA
Precipitate the DNA using isopropanol
Centrifuge to separate the DNA from the dissolved salts and sugars
Flowchart of the various steps
Dissolve DNA
Wash the DNA pellet with Ethanol and dry the pellet

22. Checking the Quality of your DNA
Poor quality DNA will not perform well in PCR
Assessment of the quality of DNA extraction:
Mix 10 µL of DNA with 10 µL of loading buffer
Load this mixture into a 1% agarose gel

23. Quality from UV Spectrophotometry
DNA absorb maximally at 260 nm.
Proteins absorb at 280 nm.
Background scatter absorbs at 320 nm.
A260/A280 = measure of purity
(A260 – A320)/(A280 – A320)
1.7 – 2.0 = good DNA or RNA
<1.7 = too much protein or
other contaminant

24. Storage Conditions
Store DNA in TE buffer at 4 °C for weeks or at –20 °C to –80 °C for long term.
2–25 °C
2–8 °C
–20 °C
–70 °C
Recommended
for long-term
storage in ethanol
<4 Months
1–3 Years
<7 Years
>7 Years

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