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Tujuan
Mahasiswa dapat melakukan isolasi DNA kromosom tanaman Mahasiswa dapat melakukan analisis kuantitatif hasil isolasi DNA kromosom tanaman menggunakan metode spektrofotometri

PRAKTIKUM BIOLOGI SEL, 2011

Physical Characteristics of DNA

DNA absorbs UV light at 260 nm
Allows quantitation

DNA is water soluble DNA precipitates in alcohols DNA carries a net negative charge

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DNA extraction the basic concept

Process Common procedure
Chemical
SDS, sarcosyl CTAB
Proteinase K Lysozyme Freezing Sonication Grinding Phenol chloroform

Cell lysis

Enzymatic

Mechanic
Organic solvents Protein removal Salt DNA binding DNA precipitation Alcohol

Sodium chloride Sodium acetate

Membrane Beads Ethanol Iso-propanol
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DNA Kromosom

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Resuspend & lyse cell walls.

Ice cold TES (Tris, EDTA,
NaCl).
EDTA is a chelating agent and has great affinity with matel ions and Mg-ion present in DNase as a cofactor and responsible for DNase action that degrade the DNA,here EDTA bind with Mg-ion and nullyfy the action of Dnase Tris is the main buffering component. Tris interacts with the LPS (lipopolysaccharide) in the membrane, serving to destabilize the membrane

lysozyme (degrades peptidoglycan

cell walls)
Structure of a Gram-Negative Cell Wall

Proteinase K (to degrade protein

that are bound to the DNA molecule)

Enzymes (RNAses ) are often

added to degrade the RNA in the sample for purification cells)
The DNA in the nucleus is surrounded by proteins

SDS, CTAB (detergent to lyse the

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CTAB
Cetyltrimethylammonium Bromide cationic detergent. CTAB has been used for the isolation of plant high molecular weight DNA.

PRAKTIKUM BIOLOGI SEL, 2011

Detergent:
Breaks apart membranes by attaching to the lipids (fats) & proteins in the membranes

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The cell and nuclear membranes have been broken apart, as well as all of the organelle membranes, such as those around the mitochondria and chloroplasts. So what is left?
Proteins Carbohydrates (sugars) lipid DNA
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Problems encountered in the isolation and purification of high molecular weight DNA from plant species
(1) degradation of DNA due to endonucleases (2) inhibitor compounds like polyphenols and other secondary metabolites which directly or indirectly interfere with the enzymatic reactions (Weishing et al., 1995). The presence of polyphenols, which are powerful oxidising agents present in many plant species, can reduce the yield and purity of extracted DNA (Loomis, 1974; Porebski et al., 1997).

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B-mercaptoethanol
a strong reducing agent which can remove tannins and other polyphenols often present in the crude plant extract

2-Mercaptoethanol reacts with aldehydes and ketones to give the corresponding oxathiolanes

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B-merkaptoethanol : Cleave disulfide bond of protein

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Extract and purify DNA

Separation of DNA from other molecules
Chloroform extraction

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Sentrifugasi

DNA & RNA Dilarutkan TE

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Extract and purify DNA

Alcohol precipitation of nucleic acids Need to have at least 0.2M of monovalent cation to the shield negative charges on DNA (sodium acetate pH 5.2) Ethanol. Precipitation temperature normally at 0 oC or room temperature
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Hook or Spool out Chromosomal DNA

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Khanuja et al, 1999

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