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Molecular Biology I
Some basic concepts
Aspects to Cover
DNA: structure, replication
RNA: transcription and processing
Protein: translation
Gene Expression: levels of transcription and translation
Common Techniques:
DNA quantitation
Gel electrophoresis
Southern/ Northern blot
Gene cloning
Polymerase chain reaction (PCR)
Quantitative Real-time PCR
DNA - Molecular Structure
Deoxyribonucleic acid (DNA) is the genetic
code that exists within every cell.
Consists of two associated strands that wind
together in a helical fashion. It is often
described as a double helix.
DNA - Molecular Structure
A single nucleotide
subunit consists of a
Each strand is a deoxyribose
linear polymer
in which
sugar
the monomers (deoxynucleotides),
are
joined to a phosphate
linked together by means of
group. Attached to the
phosphodiester bonds.
other side of the sugar
molecule is one of four
nitrogen bases.
Nitrogen Base Pairing
Purines
Pyrimidines
DNA Double Helix
5`end
3`end
3`end
5`end
DNA – The Genetic Code
DNA is essential for all living
organisms.
The genetic code (genotype)
determines how an organism looks
and functions (phenotype)
The more complex the organism
the larger the genome
Genotype
wild type
Phenotype
normal
Genotype
db/db
Phenotype
obese
“Central Dogma”
Central dogma describes
information flow from
DNA→RNA→protein
Protein considered the
functional unit within the
cell
DNA Replication
Structure of Genomic DNA
Introns
Promoter
5`
1
Exon
2
3
4
5
3`
Gene
Promoter:
Gene:
Introns:
Exons:
coding
regulatory
intervening
sequences
and sequences
regulatory
within
the
elements
that,
gene
that
together
do
that
that
not
encode
combine
encode
with
transcription
the
the
toprotein
result
proteinfactors,
structure
instructure
a protein
Transcription:
process
tosequences
produce
RNA
from
the
gene
determine the amount of gene expression
RNA – From gene to protein
There is more than one type of ribonucleic acid...
Messenger RNA (mRNA): carries the genetic
information out of the nucleus for protein synthesis.
Transfer RNA (tRNA): decodes the information from
mRNA.
Ribosomal RNA (rRNA): constitutes 50% of a ribosome,
which is a molecular assembly involved in protein
synthesis.
Catalytic/ functional RNAs: involved in reactions in the
cell.
mRNA – The Cellular Messenger
RNA is similar to single
stranded DNA sense
strand
Sugar is ribose rather
than deoxyribose
Uracil (U) replaces
thymidine (T)
Carries the ‘message’
to protein construction
centers - ribosomes
DNA
sense strand
5` ATGCGTTAGACTTGACACTGACTAC 3`
3` TACGCAATCTGAACTGTGACTGATG 3`
antisense strand
RNA synthesis
mRNA
5` AUGCGUUAGACUUGACACUGACUAC 3`
RNA has same sequence as DNA sense strand
RNA is complementary to DNA antisense strand
RNA
DNA
mRNA Processing
Pre-mRNA is transcribed from DNA
in nucleus by RNA polymerase
Splicing removes introns from premRNA to create mRNA
mRNA is transported from the
nucleus to the rough
endoplasmic reticulum (rER)
Protein is translated from the RNA
at the cytoplasm at the ribosome
Protein Translation
Amino acids
polymerise
in the
order
by tRNA
the
Occurs
at ribosomes
using
mRNA
asdetermined
template and
sequence
of mRNA
for
assembly
of protein building blocks – amino acids
Polymerisation
ofinamino
acids
occurs
until
a stop codon
Ribosome
shuffles
along
mRNA
to next
codon
mRNA
arranged
codons
–
3
bases
is read
tRNA contains specific amino acids for particular anticodons
Decoding the genetic code
20 common amino acids, 1
start codon (Met - AUG), 3 stop
codons (UAA, UAG, UGA)
Each amino acid is encoded
by one or more codons –
degenerate code
RNA to protein: one possible
sequence
Protein to RNA: many possible
sequences
“Gene Expression”
Expression refers to both RNA and protein
Gene expression is regulated at both the transcriptional and
translational levels – RNA and protein expression don’t always correlate
A single gene does not always produce a single protein
Leptin Background
Hypothalamus
Ob-R
-ve
NPY
Leptin (Ob)
-ve
-ve
Adipocytes
Alternate splicing of Ob-R
Ob-RL and Ob-RS are identical in mRNA up until exon 18
Ob-RL uses exon 18a & 18b and Ob-RS uses exon 18 a
Different stop codon result in different proteins
Ob-RS
Ob-RL
Leptin Receptor – Ob-R
G
T
Extracellular
Transmembrane
Intracellular
Ob-RS
Ob-RL
Summary
Small
Translation
changes
decodes
at
theat
the
DNA
information
level
produce
from the
drastic
gene/mRNA
DNA
replicates
itself
every
cellcan
division,
placing
a copy of
changes
into ainprotein
at thecell
– protein
theinfunctional
level
endpoint of gene expression
itself
every
every
organism
Transcription and
translation
are
–
produces
a copy
of independently
the DNA calledregulated
RNA
mRNA and protein levels are not necessarily correlated
DNA/RNA Quantitation
DNA/RNA can be extracted from virtually any tissue using
special chemicals and purification procedures
Once extracted, DNA/RNA can be dissolved in water and
used for a variety of different techniques – PCR etc
Absorbance
Spectrophotometer can analyse the spectral properties of
the nucleic acid
A260/A280 = 1.8
1.60
A260 = 1.0  50 mg/ml
260
280
Similarly for RNA
0
200
350
Wavelength (nm)
A260/A280 = 2.0
A260 = 1.0  40 mg/ml
Gel Electrophoresis
DNA can originate from a variety of sources:
genomic DNA - from organisms
plasmid DNA - circular, cloned fragments
amplified DNA - specific fragments from PCR
Knowing the size of the DNA is beneficial in identifying the
fragments – distance migrated is inversely proportional to
the size of the molecule
DNA size is usually measured in numbers of base pairs:
bp (1 – 1000)
kb (thousands of bp)
Mb (millions of bp)
Gel Electrophoresis
DNA
size
moves
approximated
according
tobysize
electrophoresis
– small
through
more
Due
phosphate
DNA
has fragments
negative
charge
Loadto
DNA
onto
gelbackbone
agarose
faster electric
than
andlarge
comparison
fragments
with
fragments
of known size
Apply
current
to gel
(100
V)
Negatively charged DNA migrates to positive electrode
-
1 kb
500 bp
400 bp
300 bp
200 bp
~400 bp
~250 bp
100 bp
+
Gel Electrophoresis
Standard methodology resolves relatively small DNA
molecules (0.1-50kb)
% agarose determines range of DNA sizes resolved
0.3% w/v resolves DNA of 5-50 kb
2.0% w/v resolves DNA of 0.1-2 kb
Ethidium bromide used to stain DNA – binds and
fluoresces under UV illumination