Download Molecular Biology

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
STRUCTURE AND FUNCTION
OF RNA
RNA is structurally similar to DNA!
• Both nucleic acids are sugar-phosphate polymers
• Both have nitrogen bases attached to the sugars of the backbone
But there are several important differences!
They differ in composition:
The sugar in RNA is ribose, not the deoxyribose in DNA.
The base uracil is present in RNA instead of thymine.
They also differ in size and structure:
RNA molecules are smaller (shorter) than DNA molecules,
RNA is single-stranded, not double-stranded like DNA.
Another difference between RNA and DNA is in function:
1. DNA has only one function-STORING GENETIC INFORMATION
in its sequence of nucleotide bases.
2. But there are three main kinds of ribonucleic acid
(each of which has a specific job to do.)
Three stages of transcription
The RNA polymerases
The RNA polymerases are huge multi-subunit protein complexes.
Three kinds are found in eukaryotes:
• RNA polymerase I (Pol I).
It transcribes the rRNA genes for the precursor of the 28S, 18S, and 5.8S
molecules (and is the busiest of the RNA polymerases).
• RNA polymerase II (Pol II; also known as RNAP II).
It transcribes protein-encoding genes into mRNA (and also the snRNA genes).
• RNA polymerase III (Pol III).
It transcribes the 5S rRNA genes and all the tRNA genes.
Initiation of Transcription by RNA Polymerase
Legend:
In order to begin transcription, RNA polymerase requires a number
of general transcription factors (called TFIIA, TFIIB, and so on).
(A) The promoter contains a DNA sequence called the TATA box,
which is located 25 nucleotides away from the site where
transcription is initiated.
(B) The TATA box is recognized and bound by transcription factor
TFIID, which then enables the adjacent binding of TFIIB (C).
(D) The rest of the general transcription factors as well as the RNA
polymerase itself assemble at the promoter.
(E) TFIIH then uses ATP to phosphorylate RNA polymerase II,
changing its conformation so that the polymerase is released
from the complex and is able to start transcribing. As shown,
the site of phosphorylation is a long polypeptide tail that
extends from the polymerase molecule.
RNA Processing (pre-mRNA
mRNA)
The steps:
• Synthesis of the cap. This is a modified guanine (G) which is attached to the 5′ end
of the pre-mRNA as it emerges from RNA polymerase II (RNAP II). The cap protects
the RNA from being degraded by enzymes that degrade RNA from the 5′ end.
• Step-by-step removal of introns present in the pre-mRNA and splicing of the
remaining exons. This step is required because most eukaryotic genes are split.
It takes place as the pre-mRNA continues to emerge from RNAP II.
• Synthesis of the poly(A) tail. This is a stretch of adenine (A) nucleotides.
When a special poly(A) attachment site in the pre-mRNA emerges from RNAP II,
the transcript is cut there, and the poly(A) tail is attached to the exposed 3′ end.
This completes the mRNA molecule, which is now ready for export to the cytosol.
(The remainder of the transcript is degraded, and the RNA polymerase leaves the DNA.)
DNA FUNCTION
Main Kinds of RNA
1. Ribosomal RNAs-exist outside the nucleus in the cytoplasm
of a cell in structures called ribosomes. Ribosomes are small,
granular structures where protein synthesis takes place.
Each ribosome is a complex consisting of about 60% ribosomal RNA
(rRNA) and 40% protein.
2. Messenger RNAs-are the nucleic acids that "record“
information from DNA in the cell nucleus and carry it to the
ribosomes and are known as messenger RNAs (mRNA).
3. Transfer RNAs-The function of transfer RNAs (tRNA) is to
deliver amino acids one by one to protein chains growing at
ribosomes
Ribosomal RNA
The ribosome is a large machinery (~ 20 nm in diameter, 70S sedimentation rate for bacterial ribosomes)
and is made of two subunits:
1. large subunit (~50 S)
is made of two ribosomal RNA (5S and 23S) and several ~34 proteins
2. small subunit (~ 30S).
has one ribosomal RNA (16S) and ~ 21 proteins.
S = Svedberg
Transfer RNA (tRNA) molecules:
Secondary and tertiary structures:
All tRNA molecules have very similar secondary structures in which the single-stranded chain is folded in a
'clover-leaf' structure that has three hairpins and an acceptor stem where the amino-acid is covalently attached.
The acceptor stem is the 3' end of the chain and always terminates in the sequence 5'-CCA-3'.
The secondary structure folds up to form a 3-dimensional structure which looks like an inverted L.
Two-dimensional
structure
Three-dimensional
structure
Symbol
RNA in a typical eukaryotic cell:
80 – 85 % is ribosomal RNA
15 – 20 % is small RNA (tRNA, small nuclear RNAs)
About 1 – 5 % is mRNA
• variable in size
• usually containing 3’-polyadenylation (poly A tail)
RNA is chemically unstable: spontaneous cleavage of
phosphodiester backbone via intramolecular transesterification
RNA is susceptible to nearly ubiquitous RNA-degradating
enzymes (RNases):
• RNases are released upon cell lysis
• RNases are present on the skin
• Rnases are very difficult to inactivate:
• disulfide bridges conferring stability
• no requirement for divalent cations for activity
RNA extraction
RNA preparation is difficult because RNAases are extremely stable.
o
Successful RNA isolation depends on:
1)
Suppression of endogenous RNAases.
Avoid contamination with exogenous RNAases
during extraction.
2)
Suppression of endogenous
RNAases
A.
B.
Samples should be processed immediately or stored
at -70 degree until required.
Inactivation of RNAases by strong denaturing agents
like urea, guanidinium hydrochloride, guanidinium
isothiocyanate.
Avoid contamination with exogenous RNAases during
extraction
A.
B.
C.
D.
Specify glassware, solutions, equipments to be used
for RNA extraction only.
Treat water and laboratory utensils with
diethylpyrocarbonate (DEPC) which is a strong
RNAase inhibitor. DEPC is a suspected carcinogen.
Autoclave glassware, solutions and equipments if
possible.
Use disposable gloves, disposable plastic materials
that must be RNAase free.
Nucleases are enzymes which catalyse the hydrolysis of
nucleic acids. Some are active against both DNA and
RNA; others act against only DNA or only RNA.
Nucleases may be further classified as active against both
single- and double-stranded nucleic acid, or as active
against only one of these. There are also nucleases which
can digest RNA only when it is H-bonded to a DNA
strand.
Nucleases can be also classified as:
•Exonucleases, which can only remove a terminal nucleotide
•Endonucleases, which cleave both terminal and terminal
phosphodiester bonds.
There are nucleases which yield a 3`-OH- and a 5`-P-terminated
nucleotide and those producing a 3`-P and 5`-OH terminus. Some
nucleases can cleave only those bonds next to or between a
particular pair of bases; others recognize extended sequences
having particular symmetry properties. There is also ATPdependent nucleases with several activities.
1.
2.
3.
4.
In repair
In recombination
Degradation of mRNA
Others to be discovered
1. Wear gloves and use sterile glass and plastic wears
2. Use RNase inhibitors
3. Use DEPC (diethylpyrocarbonate) treated solution as much
as possible
4. Work in cold room or use ice
5. Preserve samples at -80ºC in aliquots
6. Clean surfaces regularly; never allow dust in your lab.
Inhibitors of RNases:
DEPC (diethylpyrocarbonate):
• alkylating agent: modifying proteins, destroys enzymatic activity
by modifying –NH2, -SH and groups in Rnases and other proteins.
• DEPC treated water is autoclaved both before and after
packaging to ensure sterility and complete inactivation of DEPc.
Treatment of solutions and equipments:
• DEPC treatment is a very effective way to treat solutions that will
contact RNA.
• solutions may be treated with 0.1% DEPC, then autoclaved.
•Fill glasswares with 0.1% DEPC & let stand overnight at room
temperature.
Inhibitors of RNases:
Vanadyl ribonucleoside complexes:
• competitive inhibitors of Rnases, but need to be removed
from the final preparation of RNA.
Protein inhibitors of Rnases:
• Horseshoe-shaped, leucine rich protein, found in
cytoplasm of most mammalian tissues.
• must be replenished following phenol extraction steps
1.
2.
3.
4.
5.
6.
Homogenisation in strong denaturant
Deproteinisation
DNase treatment to remove DNA contaminants
Precipitation of RNA
Dissolve RNA in sterile DEPC-treated deionised water
Estimate RNA concentration by UV spectrophotometer
according to the following equation:
40 x A260 x D.F./1000 = Concentration of RNA sample (µg/µl)
7. Check purity (A260/A280 = 2)
8. Check the integrity of rRNA by gel electrophoresis
(denaturing agarose gel)
mRNA represents from 2 to 5% of tRNA. In need to probe rare
RNAs, it is essential to isolate mRNA. Most mRNAs contain
poly A tail, therefore they can be isolated by affinity
chromatography on oligo (dT)-cellulose columns. Non-poly A
RNA and DNA (if any) will be washed through column in high
salt concentration. Then Poly (A)+ RNA will be eluted by
changing to low salt concentration.
Methods of RNA
extraction
1)
Guanidinium isothiocyanate
extraction:
Cell lysis.
Protein denaturation by guanidinium
isothiocyanate.
Cell lysate is mixed with cesium chloride.
The density of RNA in cesium chloride is much
greater than of other cellular elements.
During ultracentrifugation, RNA pellets at the
bottom of the tube and becomes separated
from other cellular components.
2)
RNA extraction by Trizol:
Tizol is a monophasic solution of phenol &
chloroform + guanidinium isothiocyanate.
The presence of phenol & chloroform will
separate cell lysate into two layers:
o
o
Upper aqueous layer containing RNA.
Organic layer containing proteins.
RNA is then precipitated from the aqueous layer
by isopropyl alcohol.
3)
RNA extraction by spin column:
These columns use RNA adsorbing silica or
glass fiber.
RNA is then eluted by elution buffer.
RNA extraction by
Trizol
4)
RNA extraction by magnetic
separation technology:
Couple magnetic beads to silica.
Magnetic silica beads binds RNA in the lysate.
The conjugated magnetic beads are then
collected by applying magnetic field.
RNA is then eluted from the beads.
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 RNA in a sample=
OD at 260 x 40 x dilution factor
DETERMINING RNA 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 RNA 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 RNA.
4. Record absorbance of RNA 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 RNA (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 RNA.
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.
Assignment:
As a part of the semester activity, a
group of students 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.