dna isolation (dr sunarto ang)

8/11/2019 DNA Isolation (Dr Sunarto Ang)

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DNA Isolation

DNA isolation: is an extraction process of DNA from various

sources.

The aim:

is to separate DNA present in the nucleus of thecell from other cellular components.

Basic Protocol: break the cells open to expose the DNA within.

remove the nuclear membrane lipidsby adding a

detergent.

precipitatethe DNA with an alcohol. This step will also

remove alcohol-soluble salts.


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Application of DNA isolation

1. Scientific: use DNA in number of Applications, such as introduction

of DNA into cells & animals or plants for diagnosticpurposes (gene cloning)

2. Medicine: is the most common. To identify point sources for

hospital and community-based outbreaks and to predictvirulence of microorganisms

3. Forensic science: needs to recover DNA for identification of individuals,

(for example rapists, petty thieves, accident, or warvictims), and paternity determination.


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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


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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


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Blood Collection

1. Blood collected in disodium EDTA tube

2. Samples can be stored at -20oC or -70oC

3. Fresh samples are kept in freezer for a

few hours to facilitate RBCs hemolysis

4. Allow samples to thaw before starting the

extraction


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Methods of DNA Isolation

1. Organic extraction2. Salting out

3. 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) organicphase.


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Genomic DNA isolation: phenol extraction

1:1 phenol: chloroform or

25:24:1 phenol: chloroform: isoamylalcohol

Phenol: denatures proteins, precipitates format interface between aqueous and organiclayer Chloroform:increases

density of organic layer

Isoamyl alcohol: preventsfoaming

Genomic DNA is isolated as

pieces up to 1 Mbp!


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Salting out

Digested proteins are removed by

salting out with high concentrations of LiCl.

However, if salts are not adequatelyremoved,

problems could occur with the RFLP

procedure due to alteration of DNA mobility(band shifting)


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Procedure of Salting out1. Cell Lysis Buffer - lyse cell membrane, nuclei are

intact, pellet nuclei.

2. Resuspend nuclei in Protein Lysis Buffer containinga high concentration of Proteinase K.

Lyse nuclear membrane and digest protein at 65C for 2hours.

Temperature helps denature proteins, and Proteinase Kauto digests itself

3. To remove proteinaceous material, LiCl is added toa final concentration of 2.5 M, and incubated on ice.

Proteins precipitate out and are pelleted by centrifugation.


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Procedure of Salting out

4. DNA remains in solution. Transfersupernatant to a new tube, care must betaken not to take any of protein pellet.

5. DNA is precipitated by the addition of roomtemperature isopropanol.

LiCl will not precipitate with DNA.

6. Precipitated DNA is washed with 70%ethanol, dried under vacuum andresuspended in TE buffer.


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Plants

Nucleus is protected within a nuclearmembrane which is

surrounded by a cell membrane and a cell wall.

Four steps are used to remove and purifythe DNA

1. Lysis

2. Precipitation

3. Wash4. Resuspension


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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 whichbreaks down the cell membranes


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LYSIS

Detergents are able to disrupt membranesdue 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.


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PRECIPITATION

A series of steps where DNA is separatedfrom the rest of the cellular components

The first part of precipitation usesphenol/chloroform to remove the proteinsfrom the DNA

Phenol denatures proteins and dissolvesdenatured proteins.

Chloroform is also a protein denaturant


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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 byisopropanol or ethanol ethanol induces a structural change in DNA

molecules that causes them to aggregate andprecipitate out of solution.

The DNA is pelleted by spinning with acentrifuge and the supernatant removed


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Washing:

The precipitated DNA is laden with acetatesalts. It is washed with a 70% ethanol solution to remove

salts and other water soluble impurities but notresuspend the DNA.

Resuspension: The clean DNA is now resuspended in a buffer to

ensure stability and long term storage.

The most commonly used buffer forresuspension is called 1xTE


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Break down

the cell wall

andmembranes

Centrifuge to

separate the

solids fromthe dissolved

DNA

Precipitate

the DNA

usingisopropanol

Centrifuge to

separate the

DNA fromthe dissolved

salts and

sugarsWash the

DNA pellet

with Ethanol

and dry thepellet

Dissolve

DNA

Overview of DNA Extraction


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Checking the Quality of your DNA

Poor quality DNA will not perform well inPCR

Assessment of the quality of DNAextraction:

Mix 10 L of DNA with 10 L of loading buffer

Load this mixture into a 1% agarose gel


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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

(A260A320)/(A280 A320)

1.7

2.0 = good DNA or RNA


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Storage Conditions

Store DNA in TE buffer at 4 C forweeks or at20 C to80 C for long

term.

225 C 28 C 20 C 70 C

Recommendedfor long-term

storage in ethanol


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dna isolation (dr sunarto ang)

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