OBJECTIVE 1. To extract and prepare Genomic DNA from blood sample; 2. To extract and prepare Genomic DNA from E.coli DH5alpha by using Alkaline method and boiling method; and 3. To compare and contrast between the two different methods of DNA extraction. INTRODUCTION There are a number of different procedures for the preparation of genomic DNA. DNA extraction or isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. Nevertheless, the DNA is present in the cell as an association with many proteins that we called as nucleoprotein complex. A nucleoprotein is any protein which is structurally associated with nucleic acid either DNA or RNA. The DNA must be separated away from these proteins prior to characterization. RNA and polysaccharides can interfere with DNA characterization methods, so the DNA must always be purified away from these macromolecules. In addition, a single cell may contain several different types of DNA molecule. The more simple approach to the purification of DNA involves purifying the total DNA present in the cell without regard the separation of different types of DNA molecules. A) PREPARATION OF GENOMIC DNA FROM EUKARYOTE CELLS (BLOOD) In preparation of genomic DNA from eukaryote cell (blood), there are three basic and one optional step in a DNA
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OBJECTIVE
1. To extract and prepare Genomic DNA from blood sample;
2. To extract and prepare Genomic DNA from E.coli DH5alpha by using Alkaline
method and boiling method; and
3. To compare and contrast between the two different methods of DNA
extraction.
INTRODUCTION
There are a number of different procedures for the preparation of genomic
DNA. DNA extraction or isolation is a routine procedure to collect DNA for
subsequent molecular or forensic analysis. Nevertheless, the DNA is present in the
cell as an association with many proteins that we called as nucleoprotein complex. A
nucleoprotein is any protein which is structurally associated with nucleic acid either
DNA or RNA. The DNA must be separated away from these proteins prior to
characterization.
RNA and polysaccharides can interfere with DNA characterization methods,
so the DNA must always be purified away from these macromolecules. In addition, a
single cell may contain several different types of DNA molecule. The more simple
approach to the purification of DNA involves purifying the total DNA present in the
cell without regard the separation of different types of DNA molecules.
A) PREPARATION OF GENOMIC DNA FROM EUKARYOTE CELLS (BLOOD)
In preparation of genomic DNA from eukaryote cell (blood), there are three
basic and one optional step in a DNA extraction. First basic is breaking the cell open,
commonly referred to as cell disruption or cell lysis. It is important to expose the DNA
within. This is commonly achieved by grinding or sonicating the sample. The lysis
solution contains several components to help stabilize the DNA during the
purification process. A TE buffer which10mM Tris-C1 &1mM EDTA is included to
maintain pH at a constant value, usually around pH 7.5 to 8.0.
Next, the second basic is removing membrane lipids by adding a detergent to
release the contents of the cells into the solution. Then, the third basic is removing
proteins by adding a protease (optional but almost always done) and extractions with
organic solvents (generally phenol/or chloroform) that denature and unfold protein
and remove them from DNA. RNA that may contaminate DNA preparations is often
removed by treatment with enzymes that degrade RNA but not DNA.
Finally, for optional step which is to precipitate the DNA with an alcohol. It is
usually ice-cold ethanol. Since DNA is insoluble in these alcohols, it will aggregate
together, giving a pellet upon centrifugation. This step also removes alcohol-soluble
salt. The precipitated DNA can then be dried and resuspended in a small volume of
buffer.
C) Preparation of plasmid DNA from bacteria cells (E.coli) – Alkaline Method
Alkaline lysis was first described by Birnboim and Doly in 1979 (Nucleic Acids
Res. 7, 1513-1523) and has, with a few modifications, been the preferred method for
plasmid DNA extraction from bacteria ever since. Alkaline method or alkaline lysis is
a technique of DNA purification that based on differential denaturation of
chromosomal and plasmid DNA in order to separate both. Alkaline lysis depends on
a unique property of plasmid DNA. It is able to rapidly anneal after being denatured.
This is what allows the plasmid DNA to be separated from the bacterial
chromosome.
There are several general steps that must been conducted to prepare the
DNA from the E. coli. First is to extract the DNA from E. coli by lysing the cell wall.
Due to this process, DNA will be denatured. The next step is followed by
neutralization that allows only the covalently closed plasmid DNA to reanneal and to
stay solubilised whereas the chromosomal DNA remains denatured. Next, the
separated chromosomal DNA fragments will aggregate with the protein isolated from
the DNA by phenol-chloroform extraction and form precipitate. This precipitate will be
removed by centrifugation. The plasmid DNA will concentrate and further purified.
Lastly, the plasmid DNA will be further analysed by electrophoresis method.
Several reagents which are mixed in three different solutions are used to
perform this method. In Solution I, Glucose is used to help in stabilizing the DNA to
minimize random breaking or shearing due to the long-rod like molecule. It also
helps in giving osmotic shock that leads to the rupture of cell wall and bacterial
membrane. The Tris-C1 buffer is used to maintain pH at constant value which is pH8
via the ability to absorb counter ions (H+ and OH-). Next is Ethylene diaminetetra
acetic acid (EDTA). It eliminates divalent cations by chelating the Mg2+ that essential
for the integrity of the bacterial outer membrane and destabilizing the membrane.It
also inhibits DNAses to prevent the degradation of DNA. RNAse is also been added
to degrade the RNA.For the solution II, it is known as lysis buffer. The lysis buffer
contains sodium hydroxide (NaOH) and the detergent Sodium Dodecyl Sulfate
(SDS). SDS is there to degrade the cell membrane. In the alkaline lysis procedure,
bacterial cells are exposed to alkaline .This causes the cell walls and membranes to
burst and the contents of the bacteria are spilled out. But more importantly it disrupts
the hydrogen bonding between the DNA bases(due to high pH), causing the DNA
double strand to denature.Addition of potassium acetate in Solution III decreases the
pH of the mixture. Under these conditions the hydrogen bonding between the bases
of the single stranded DNA can be re-established. Next, phenol-chloroform
extraction is used. Both phenol and chloroform cause proteins to become denatured
and become soluble in the organic phase or interphase, while nucleic acids remain in
the aqueous phase. The ethanol is also used to precipitate the nucleic acid in order
to further purify the solution. (Dale &Schantz, 2002)
D) Preparation of plasmid DNA from bacteria cells (E.coli) – Boiling Method
Boiling method is another method used to extract DNA. This method is
adapted from Holmes and Quigley in 1981. The principle and the reagents that been
used in this method are the same with the alkaline lysis method. However, an
enzyme known as lysozyme is used to break the bacterial cells. Lysozyme is an
enzyme that naturally present in egg white and tears to break down the bacterial cell
wall (Dale &Schantz, 2002). Besides that, Triton X-100 is added which is a non-ionic
detergent. It reduces the levels of unwanted peroxides, carbonyl compounds and
salts. After that, the lysate is boiled to denature the nucleic acids and the proteins.
When the temperature is lowered, the bases of plasmid DNA will be reanneal. The
next procedure is to remove the protein and further purification of the nucleic acid by
applying the phenol-chloroform extraction and centrifugation. Sodium acetate is also
added to recover the plasmid. This followed by concentrating the nucleic acids by
ethanol precipitation.
MATERIALS/ APPARATUS
EXPERIMENT A:
A) Blood that were collected using Heparinized/EDTA VenoJet tube