Download What is a DNA?

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.
• Capable of self-replication and synthesis
of RNA.
• Contains instructions for our body cells to
perform their specific functions.
• RNA. DNA consists of two long chains of
nucleotides twisted into a double helix
• Joined by hydrogen bonds between the
complementary bases adenine and thymine or
cytosine and guanine.
• The sequence of nucleotides determines
individual hereditary characteristics.
DNA plays an important role in two processes
• The process of replication, DNA provides
information to copy itself, so genetic information
can be passed on from generation to generation
of cells.
• also provides instructions for making proteins,
which are vital to the maintenance and function
of cells.
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
5-10 kb
E. coli
4,640 kb
Human
2,900,000 kb
(about 2.9 billion base pairs)
• Only about 5% code for protein. Intervening
sequences and other noncoding sequences make
up the remainder.
• In addition to genomic DNA, mitochondria, which
are cellular organelles, contain their own DNA
(mitochondrial
DNA)
which
replicate
independently from cell chromosomal DNA.
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
• 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.
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 mL of whole blood in a conical
centrifuge tube.
• Add 9 mL of 1X erythrocyte lysing buffer.
• Leave 10 min. at RT, mix occasionally.
• Centrifuge at 4000 rpm for 5 min.
• Discard supernatant.
• White pellet is observed at bottom of tube.
• Wash pellet 3 times by adding 3 mL of buffer,
incubate 10 min at RT, & centrifuge
2- WBCs nuclei Lysis & proteins digestion
• Add 1.5 mL of SE buffer 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 mL of SE buffer, 750 µl
of 6M NaCL & 3.75 mL 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 micro centrifuge tube
containing 1 mL of 75% ethanol.
• Wash by inversion to remove any remaining salts.
• Centrifuge at 11000 g for 4 minutes and then
discard supernatant.
• 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:
•
•
•
•
Mix 5 µL of DNA with 5 µL of loading Dye
Load this mixture into a 1% agarose gel
Stain with ethidium bromide
Electrophoreses at 70–80 volts, 45–90
minutes.
• view on UV transilluminator.
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