DNA Extraction - Intro

OBJECTIVE

1. To study the total DNA extraction method.

2. To study the Quantification and Qualification of DNA extracted.



INTRODUCTION

DNA or DeoxyriboNucleic Acid is the blueprint for life, as studied by
all the scientist, each of living materials contains DNA. In order to study
more about DNA of a specific living materials, the DNA content of the
living materials need to be extracted. DNA extraction was one of the method
used these days in many diagnostic processes used to detect bacteria and
viruses in the environment as well as diagnosing disease and genetics
disorders and also the identity of the living materials by diagnosing their
DNA.

The definition of DNA extraction is the removal of DNA from the cells or
viruses in which it normally resides. It is the most crucial method used in
molecular biology(Siun and Beow). it was first isolated by Swiss physician,
Friedrich Miescher in 1869 while working in the laboratory of the
biochemist Felix Hoppe-Seyler.

The extraction of DNA comes with a lot of benefit especially in
biotechnology department. It is the starting point for numerous
applications, ranging from fundamental research to routine diagnostic and
therapeutic decision making. The extraction and purification of DNA are
also important to determine the unique shape characteristics of DNA,
including its size, shape and function.

Perfect DNA extraction depends on the instrumentation used in DNA
extraction. Breaking or grinding instrument, separation and purifying and
lastly the identification of DNA content. In breaking, the instrument used
has a purpose to break apart or lysing of cells in order to bring the DNA
of the cell accessible. Other methods of lysing cell including a french
press and sonication device. For separation and purifying instrument,
mostly lysed cell separated by using a centrifuge to precipitate the DNA.
Last but not least, the instrument used for DNA identification by using a
gel box to separate DNA in an agarose gel with an electrical charge. The
DNA migrates through the gel toward the positive charge due to the net
negative charge of the molecule. Here, different sized of DNA moved at
different rates, larger pieces of DNA moves slower through the porous
medium and vive versa for small pieces of DNA.

Nowadays, scientist prefer more safe and simple way of DNA extraction
without using any hazardous chemical but having the maximum value of DNA
extracted. In this experiment, the DNA will be extracted by using KAPA
Express Extract buffer and enzyme. KAPA Express Extract did not required
any hazardous and washing of sample which automatically reduced the risk of
sample loss and contamination but maximizing the DNA extract. To complete
the lysis step, incubating the sample with the specified temperature was
one of the requirement. Which actually in the first incubation was to
degrade the cell, nucleus and proteins to released DNA and the second
incubation was to inactivate the enzyme. The application of KAPA Express
Extract suitable for any DNA from crude sample such as human tissue, animal
tissue, fish tissue, insects, birds feathers and many more.

In this experiment, the sample used for DNA extraction was a manufacture
sausage. The DNA extraction of the sausage was done in aseptic technique to
prevent any contamination. Figure below shows the general DNA extraction
used as a guideline in this experiment.



Figure 1.0 : KAPA Express Extraction protocol



In this experiment, they were two sample of sausage prepared. Small
pieces of sausage was put into the PCR tube, grinded and mix with KAPA
Express Extract buffer and enzyme then incubated to be lyses by heat. The
separation of sausage supernatant and pellet was done by using micro
pipette into another PCR tube. For a longer usage of DNA, it must be stored
at -20 OC.

The determination of DNA concentration was done by using
spectrophotometer instrument. The step was done by transferring 3 µl of DNA
supernatant into another PCR tube to be mixed with 567 µl of double
deionised water. The DNA concentration and purity were calculated by using
tis formula shown in figure below.









Figure 1.1 : DNA concentration and DNA purity



There are four approaches that can be used to determine both quantity or
quality of DNA, while four of them permit to quantifying of DNA, not all of
them are enable a comprehensive assessment of DNA quality. These four
approaches are Ultraviolet Absorption, Fluorescence Measurement, Yield Gel
Measurements and lastly the Slot/dot Blot and Human DNA Probe.

In this experiment, the agarose gel analyzer for the DNA visualization,
an imager was used to visualize the DNA in the agarose gel. DNA can be
visualized under UV (ultraviolet) light under the right condition(Aiping).









REFERENCE

Paez. J.G. 2004 : EFGR mutations in lung cancer : correlation with
clinical response to gefitinib therapy.
(https://www.kapabiosystems.com/assets/KAPA_Express_Extract_KAPA2G_Robust
_HotStart_ReadyMix_FFPE_PCR_Note.pdf )

Siun Chee Tan and Beow Chin Yiap. November 20, 2009 : DNA, RNA and
Protein Extraction : The Past and The Present.
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2789530/ )

George Rice, Montana State University : DNA Extraction
(http://serc.carleton.edu/microbelife/research_methods/genomics/dnaext.ht
ml )

F. Samuel Baechtel : THE EXTRACTION, PURIFICATION AND QUANTIFICATION OF
DNA.
(http://projects.nfstc.org/workshops/resources/articles/The%20Extraction,
%20Purification%20and%20Quatification%20of%20DNA.pdf )

Anonymous. ; What Is Biotechnology ; DNA Extraction
(http://www.whatisbiotechnology.org/science/extraction)

Aiping ; Imager - Ultraviolet Absorption. Gabrielle Duchesne, CFRE
University of Toronto, Senior Department Officer, Psychiatry 6 Queen's
Park Crescent West, Room 12 Toronto, ON M 5S 3H2 (
http://www.tapscottchair.com/imager/ )



-----------------------
[DNA] = A260 X 50 X dilution factor (dilution factor) (ng/ µl)
DNA purity = A260 /A280