Download DNA

Molecular Biology
(MLMB-201)
Department of Medical Laboratory Technology
Faculty of Allied Medical Science
Lecturer:
Dr. Mohamed Salah El-Din
Intended Learning Outcomes (ILO’s):
Molecular biology course provides an overview of the molecular basis to cell structure and function.
This course focuses on the structure, biosynthesis and function of DNA and RNA on the molecular level and
how these interact among themselves and with proteins. Molecular biology techniques are essential for
modern biological and medical research. This course will give you an introduction to DNA and RNA standard
techniques.
Student will have basic knowledge of:
• Cell organization.
• DNA structure and function.
• DNA Extraction.
• RNA structure and function.
• RNA Extraction.
• Gene expression and protein biosynthesis.
• Agarose gel electrophoresis for DNA/RNA; and SDS-PAGE for protein.
• Polymerase Chain Reaction (PCR) – Theory, Types, Application.
• Gene library and screening
• DNA sequencing
Central Dogma Of Molecular Biology
Chromosomes
Thread-like structures present within the nuclei of cells. Chemically, a chromosome
consists of an extremely long chain of DNA along with a coating protein. The
combination of DNA and protein is called chromatin. The complete array of
chromosomes in an organism is termed the karyotype.
Prokaryotic cell Versus Eukaryotic cell
What is a “gene”?
A bit of DNA which acts as a code
to control part of a cell's
chemistry. Every human cell
holds, within its nucleus, more
than 50,000 different genes. Each
gene is located at a particular
position along the chromosome,
termed locus. The particular form
of the gene is termed the allele.
DNA STRUCTURE
Structure
of
Nucleotide
RNA
DNA
Cytosine (C)
Adenine (A)
Thymine (T)
Guanine (G)
Guanine (G)
Thymine (T)
Adenine (A)
Cytosine (C)
Deoxyribose
(Sugar molecule)
Phosphoric Acid
(Phosphate molecule)
Pyrimidine (T & C)
Purine (A & G)
nucleoside
nucleotide
Watson-Crick model of a linear, double-stranded helix

A single polynucleotide strand of DNA is a copolymer in which
deoxyribose and phosphate alternate; the side chains are single
organic bases, one attached to each deoxyribose.
DNA FUNCTION
DNA is the repository of genetic information in all organisms:
(except for a relatively small number of viruses)

It must transmit its encoded information to the cell which
contains it “Transcription”

It must replicate in order that a progeny cell has the same
properties as its parent “Replication”

Evolutionary pressures have produced a mechanism for
creating new information “Mutation” and for exchanging
genes “Recombination”, so that new and better organisms
can develop.
DNA Replication
Mutation
The copying process is not perfect, and very occasionally a fault occurs
leading to a mutation (change) in the nucleic acid sequence; this, in turn,
alters the structure of the DNA in one of the daughter cells-and thus leads
to a change in one of its genes.
Mutations may potentially occur at any site within the genome and may
have several effects upon the structure and function of the genome.
Recombination
DNA
extraction
Relevance of DNA isolation
Isolation of DNA is often the first step before further analysis:
• DNA profiling
• cloning
• disease diagnosis
• DNA sequencing
• genetically modified organisms (GMO) -agriculture, pharmaceutical
• Environmental testing, bioterrorism
Protocol Highlights: DNA Extraction
• Collect cells from whole blood, tissues…etc.
(NB. Ample cell collection is critical for success)
• Add Lysis buffer to cells to break open cell and
nuclear membranes and release nuclear contents
• Digest sample with protease to degrade proteins
• Precipitate DNA with cold alcohol in high salt
Lysis Buffer:
What is Lysis Buffer?
CH3
CH2
• 50 mM Tris-HCI, pH 8.0
• 1% SDS
CH2
CH2
CH2
Tris buffer to maintain the
pH of the solution at a
level where DNA is stable
CH2
CH2
CH2
CH2
CH2
CH2
O
S
-
O
O
O
1% SDS to break open
the cell and nuclear
membranes, allowing the
DNA to be released into
the solution (SDS also
denatures and unfolds
proteins, making them
more susceptible to
protease cleavage).
CH2
SDS
Why Add Protease?
• Protease is added to destroy nuclear proteins that bind DNA and
cytoplasmic enzymes that breakdown and destroy DNA.
• Protease treatment increases the amount of intact DNA that is
extracted.
Adding Salt
• The protease solution already contains salt
• Na+ ions of NaCI bind to the phosphate groups of DNA
molecules, neutralizing the electric charge of the DNA molecules.
• The addition of NaCI allows the DNA molecules to come together
instead of repelling each other, thus making it easier for DNA to
precipitate out of solution when alcohol is added.
• Salts and buffers deactivate the enzymes that degrade DNA
when released and stabilize the DNA (acid vs. base).
Adding Ice Cold Alcohol?
• DNA does not dissolve in alcohol.
• The addition of cold alcohol makes the DNA clump together and
precipitate out of solution.
• Precipitated DNA molecules appear as long pieces of fluffy,
stringy, web-like strands.
• Microscopic oxygen bubbles “aggregate” , or “fuse” together,
as the DNA precipitates.
• The larger, visible air bubbles “lift” the DNA out of solution,
from the aqueous into the organic phase.
DNA extraction
DNA is easy to prepare and store because DNAases
are easy destroyed by heating.
Phenol/chloroform/isoamyl alcohol
extraction:
1)
Phenol is organic solvent.
Nucleic acids prefer aqueous solutions.
But, proteins, lipids and carbohydrates prefer
organic environment.
So, after addition of phenol to the cell lysate, the
reaction tube will have two phases:
o
o
Upper aqueous layer containing DNA.
Lower organic layer containing phenol + proteins.
Phase separation by phenol
extraction
2)
Salting out method:
Cell lysis.
Protein digestion by proteinase enzyme.
Protein precipitation by high salt concentration.
Salt also reduces the repulsion of the negatively
charged DNA molecules.
Centrifugation will remove the precipitated
proteins.
The supernatant contains the DNA.
DNA is then precipitated by adding ethanol.
The precipitated DNA is resuspended in the
desired buffer.
3)
Spin column:
Commercial DNA extraction kits are based on
selective adsorption of DNA to silica gel
membrane columns or glass fiber matrix
columns.
Binding of DNA to silica or glass fiber is
enhanced by high salt concentration.
The bound DNA will then be eluted by elution
buffer with low salt concentration.
Extraction by spin
column
Plasmid
extraction

Alkaline lysis method:
Cell lysis.
Then, we add sodium hydroxide.
The alkaline pH will denaturate chromosomal DNA
but not the plasmid DNA.
Then we add sodium or potassium acetate that will
neutralize pH.
So, chromosomal DNA will renature and aggregate
with proteins.
By centrifugation, proteins + chromosomal DNA will
precipitate while plasmid DNA will remain in the
supernatant.
Quantification
Quantification of extracted nucleic acids is
done by using spectrophotometer as follows:
At wave length 260: optical density (OD) of 1
means that:
o
o
The concentration of DNA= 50 µg/ml
The concentration of RNA= 40 µg/ml
So, the concentration of extracted DNA in a sample=
OD at 260 x 50 x dilution factor
DETERMINING DNA CONCENTRATION AND PURITY BY SPECTROPHOTOMETRY
PROCEDURE:
1. Fill two cuvettes with TE buffer. Read and record the A260 of the sample cuvette
against the blank. Repeat at 280 nm.
2. Dilute the DNA in 400 µl of TE such that the A260 is ideally between 0.1 to 1.0. Mix
well.
3. Empty and clean the sample cuvette and add the diluted DNA.
4. Record absorbance of DNA sample at both 260 and 280 nm. Correct the readings as
necessary using the blank values you determined in step 1.
5. The absorbance at 260 nm allows calculation of the concentration of DNA or RNA in
the sample. An OD of 1 corresponds to approximately 50 ng/µl for double-stranded
DNA, 40 ng/µl for RNA, and 32 to 34 ng/µl for single-stranded DNA and typical
oligonucleotides. The A260/A280 ratio can provide a very rough estimate of the
purity of the nucleic acid. Relatively pure preparations of DNA and RNA have
A260/A280 values of 1.8 and 2.0, respectively. Phenol contamination will result in
significantly lower A260/A280 ratios. Such contamination makes accurate
quantitation of DNA or RNA impossible. Note however that the A260/A280 ratio can
not be used to determine whether there is significant protein contamination in a
nucleic acid preparation (Glasel, J. Biotechniques 18:62 (1995).
Spectrophotometer
It is very important to assess purity of the
extracted DNA (the degree of protein
contamination).
Purity is assessed by:
Determine the ratio between the OD of the
sample at 260 nm and 280 nm. (OD 260/OD
280).
If the ratio is 2: this means that protein
contamination is zero.
If the ratio is < 2: this means protein
contamination of the extracted DNA.
N.B. Notice that the concentration of the extracted nucleic acid in a given
sample can be roughly estimated by observing the fluorescence intensity of
the band obtained on agarose gel after electrophoresis.
Now You Can
Capture Your Unique Essence!
&
Prepare DNA Necklaces
The DNA in the glass
vial can last
for years. Add more
alcohol into the vial if
some evaporation
occurs.
Congratulations!
You have just created your
very own DNA Necklace!
Assignment:
As a part of the semester activity, one
student is selected every week to
prepare a short seminar about his/her
point of interest in one of the lecture
topics. That to be discussed and
evaluated during the next lecture.