Download DNA Extraction

Extraction of Human
DNA
Experiment Goals
• Isolation of genomic DNA from human
blood
• Analysis of isolated DNA using
– Agarose gel electrophoresis
– Spectrophotometry
What is a DNA?
• DNA, also known as deoxyribonucleic acid,
• A fundamental molecule found in all living
things
• Carries the genetic information in the cell
• Contains instructions for our body cells to
perform their specific functions
• The sequence of nucleotides determines
individual hereditary characteristics
What is a DNA?
• Basic unit of information in
DNA is the gene
• Human beings have about
30,000 gene
• Size of organism’s genome
is roughly a measure of its
complexity
• Viruses
• E. coli
• Human
5-10 kb
4,640 kb
2,900,000 kb
DNA Extraction
• DNA extraction is a routine procedure to isolate
& collect DNA.
• DNA extraction is the first step for subsequent
molecular or forensic analysis.
• DNA can be extracted from almost any intact
cellular tissue
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Skin,
blood,
saliva,
semen,
mucus,
muscle tissue,
bone marrow, etc.
Nucleic Acid Preparation Applications
• Medical studies
– Understanding genetic disorders at molecular
level
– Rapid detection of genetic disorders in a
patient
• Agricultural studies
– Plant and animal breeding
• Criminology/Paternity testing
– DNA fingerprinting to identify individuals.
Basic steps in DNA extraction
• There are three basic steps in a DNA extraction,
the details of which may vary depending on the
type of sample and any substances that may
interfere with the extraction and subsequent
analysis.
– Break open cells and remove membrane lipids
– Remove cellular and histone proteins bound to the
DNA, by adding a protease, by precipitation with
sodium or ammonium acetate, or by using a
phenol/chloroform extraction step.
– Precipitate DNA in cold ethanol or isopropanol, DNA
is insoluble in alcohol and clings together, this step
also removes salts.
Procedure
Overview of Procedure
1- Lyse RBCs & WBCs
2- Lyse WBCs nuclei & Denature/digest proteins
3- Separate contaminants (e.g., proteins, heme)
4- Precipitate DNA
5- Resuspend DNA in final buffer
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
1- RBCs Lysis
• Pipette 3 mls of whole blood in a conical
centrifuge tube
• Add 9 mls of 1X erythrocyte lysing buffer
(0.155M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4)
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Leave 10 min. at RT, mix occasionally
Centrifuge at 4000 rpm for 5 min
Discard supernatent
White pellet is observed at bottom of tube
Wash pellet 3 times by adding 3 mls of buffer,
incubate 10 min at RT, & centrifuge
2- WBCs nuclei Lysis & proteins
digestion
• Add 1.5 mls of SE buffer (75mM NaCl, 25
mM Na2EDTA, pH 8.0) containing
100µg/ml of Proteinase K & 1% sodium
dodecyl sulphate (SDS) to the pellet
• Incubate at 37-55oC overnight in a water
bath or incubator
• WBCs nuclei denatured & DNA goes out
in solution
3- Separate contaminants from DNA
• After incubation add 1.5 mls of SE buffer, 750 µl
of 6M NaCl & 3.75 mls chloroform
• Mix vigorously on vortex for 20 sec
• Mix for 30 min (on rotator)
• Centrifuge for 10 min at 2000 rpm
• 2 phases are observed
• DNA is extracted in supernatant & proteins in the
lower phase
• Transfer upper aqueous phase (containing DNA)
to a clean tube
4- Precipitate DNA
• Add an equal volume of isopropanol
• DNA will be precipitated by gentle swirling &
observed as a white thread like strand
• Using a sterile spatula or loop transfer the DNA
strand into a sterile microcentrifuge tube
containing 1 ml of 75% ethanol
• Wash by inversion to remove any remaining
salts
• Centrifuge, discard supernatent
• Repeat the washing step, then centrifuge
• Remove supernatant, and dry the pellet
5- Resuspend DNA in final buffer
• Dried pellet is resuspended in TE buffer
and left overnight on a rotator
DNA Analysis
• Different methods for assessing quantity
& quality of extracted DNA
– Agarose gel electrophoresis
– UV spectrophotometry
Checking the Quality of DNA
• The product of DNA extracted will be used
in subsequent experiments
• Poor quality DNA will not perform well in
PCR
Quality from Agarose Gel
Electrophoresis
• Quality of DNA extracted is assessed
using the following simple protocol:
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Mix 5 µL of DNA with 5 µL of loading Dye
Load this mixture into a 1% agarose gel
Stain with ethidium bromide
Electrophorese at 70–80 volts, 45–90
minutes.
DNA Quality from
Agarose Gel Electrophoresis
• High molecular
weight band
• Smearing indicates
DNA degradation
Nucleic Acid Characterization
• Absorption Spectra
– Absorb light in ultraviolet range, most strongly
in the 254-260 nm range
• Useful for quantification of samples
Quantity from UV Spectrophotometry
Calculating Yield
Multiply the concentration of the
DNA sample by the
volume of hydrating solution added.
Example for DNA: 150 µg/mL X 0.1 mL = 15 µg
Concentration from UV
Spec. (µg DNA per ml
of hydrating solution)
Volume of
hydration
solution
DNA yield
Spectrophotometric analysis of DNA
Quality from UV Spectrophotometry
• DNA absorb maximally at 260 nm.
• Proteins absorb at 280 nm.
• Background scatter absorbs at 320 nm.
Quality from UV Spectrophotometry
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
Storage Conditions
• Store DNA in TE buffer at 4 °C for weeks or at –
20 °C to –80 °C for long term.
<4 Months
2–25 °C
1–3 Years
<7 Years
>7 Years
2–8 °C
–20 °C
–70 °C
Recommended
for long-term
storage in ethanol