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Molecular Biology Primer – Part 2
Angela Brooks, Raymond Brown, Calvin Chen, Mike Daly,
Hoa Dinh, Erinn Hama, Robert Hinman, Julio Ng, Michael
Sneddon, Hoa Troung, Jerry Wang, Che Fung Yung
Adapted from the above authors’ slides my Mark Fienup
2 types of cells: Prokaryotes
v.s. Eukaryotes
Prokaryotes v.s. Eukaryotes
Prokaryotes
Eukaryotes
Single cell
Single or multi cell
No nucleus
Nucleus
No organelles
Organelles
One piece of circular DNA Chromosomes
No mRNA post
Exons/Introns splicing
transcriptional modification
DNA, RNA, and the Flow of Information
Replication
Transcription
Translation
Overview of DNA to RNA to Protein
•
A gene is expressed in two steps
1) Transcription: RNA synthesis
2) Translation: Protein synthesis
Central Dogma Revisited
Transcription
Splicing
Nucleus
hnRNA
mRNA
Spliceosome
DNA
protein
Translation
Ribosome in Cytoplasm
• Base Pairing Rule: A and T or U is held together by
2 hydrogen bonds and G and C is held together by 3
hydrogen bonds.
• Note: Some mRNA stays as RNA (ie tRNA,rRNA).
Splicing
Alternative splicing example: tissue specific gene
expression of a-tropomyosin
Translation
• The process of going
from RNA to
polypeptide/protein.
• Three base pairs of
RNA (called a codon)
correspond to one
amino acid based on a
fixed table.
• Always starts with
Methionine and ends
with a stop codon
Purpose of tRNA
• The proper tRNA is chosen
by having the
corresponding anticodon for
the mRNA’s codon.
• The tRNA then transfers its
aminoacyl group to the
growing peptide chain.
• For example, the tRNA with
the anticodon UAC
corresponds with the codon
AUG and attaches
methionine amino acid onto
the peptide chain.
Translation, continued
• Catalyzed by Ribosome
• Using two different
sites, the Ribosome
continually binds tRNA,
joins the amino acids
together and moves to
the next location along
the mRNA
• ~10 codons/second,
but multiple translations
can occur
simultaneously
http://wong.scripps.edu/PIX/ribosome.jpg
Protein Structure and Function
A proteins fold into their native structure due to the chemical
interactions of the various amino acids. The folded structure
of proteins bring together linearly remote sections of the
protein to form chemically interesting sites on the protein.
• Primary–sequence of amino acids constituting the protein
chain
• Secondary–local organization into secondary structures
such as a helices and b sheets
• Tertiary –three dimensional arrangements of the amino
acids as they react to one another due to the polarity and
resulting interactions between their side chains
• Quaternary–number and relative positions of the protein
subunits
Protein Structure
• The structure that a
protein adopts is vital to
it’s chemistry
• Its structure determines
which of its amino acids
are exposed carry out
the protein’s function
• Its structure also
determines what
substrates it can react
with
Chemical Bonds Review
Elements - things that cannot be further reduced by chemical reactions
Atom - individual “unit” of an element
Atoms are made up of three stable subatomic particles:
Subatomic
Particle
neutron
protron
electron
Weight
Charge
“Location”
1.7 x 10-24 grams
1.7 x 10-24 grams
about 1/1,840 as much as a protron
none
+1
-1
nucleus
nucleus
move in orbits around the nucleus at the
speed of light
Electrons and protons are electrically attracted to each other.
Almost all of the mass of an atom is in its nucleus; almost all of the volume of an atom is occupied by electrons.
The number of protons (also known as its atomic number) determines the element. Varying the number of neutrons
results in isotopes. Varying the number of electrons results in ions.
The particles within an atom are bound together by powerful forces.
Electrons are easier to add or remove from an atom than a proton or neutron. Chemical reactions largely involve atoms
or groups of atoms and the interactions between their electrons.
Chemical Reactivity
Chemical reactivity – is the degree to which an atom attracts
electrons of other atoms.
An atom's attraction for such electrons is determined by the
number of electrons in its outer energy level, or outer shell.
Atoms with completely filled outer shells do not attract
electrons and are relatively unreactive or inert.
Atoms with incomplete outer shells tend to "share" electrons
with other atoms to achieve the electron configurations of the
six inert or nobles gases-Helium, Neon, Argon, Krypton,
Xenon, and Radon- with 2, 8, 8, 8, 8 and 8 outer shell
electrons, respectively.
This sharing of electrons between atoms is called covalent
bonding
Periodic Table
Electronegativity
• Electronegativity is an atoms affinity (“desire”) for electrons.
• An atoms electronegativity is determined by how many
electrons it needs to acquire or donate to completely fill or
empty its outermost shell of orbitals.
• Hydrogen (1H) and carbon (6C) have outermost shells that are
half full so their electronegativity is about equal, so they share
electrons equally.
• Oxygen (8O) is more electronegative since it only need to gain
2 (or lose 6) electrons to complete its outermost shell.
Therefore, when oxygen bonds with hydrogen (H2O), the
electrons spend more time in the vicinity of the oxygen atom
causing a slight separation of charge, called a polar bond.
H
+
O
H
+
Amino Acid Properties
Each amino acid has the same fundamental structure, differing only
in the side-chain, designated the R-group. The carbon atom to
which the amino group, carboxyl group, and side chain (R-group)
are attached is the alpha carbon (C).
Glycine has just a hydrogen atom in place of an R-group.
Nonpolar, hydrophobic R-groups
Polar, hydrophilic R-groups
At physiological pH, some amino acid R-groups are charged, because of
dissociation or association of a proton by a carboxyl or an amino group.
Protein Folding
• The amino acids have very different chemical
properties; they interact with each other after
the protein is built
• This causes the protein to start folding and
adopting it’s functional structure
• Proteins may fold in reaction to some ions, and
several separate chains of peptides may join
together through their hydrophobic and hydrophilic
amino acids to form a polymer
Protein Folding
• Proteins tend to fold into the lowest
free energy conformation.
• Proteins begin to fold while the
peptide is still being translated.
• Proteins bury most of its hydrophobic
residues in an interior core to form an
α helix.
• Most proteins take the form of
secondary structures α helices and β
sheets.
• Molecular chaperones, hsp60 and hsp
70, work with other proteins to help
fold newly synthesized proteins.
• Much of the protein modifications and
folding occurs in the endoplasmic
reticulum and mitochondria.
Protein – Open Problems
• A protein is a polypeptide, however to
understand the function of a protein given
only the polypeptide sequence is a very
difficult problem.
• Protein folding is an open problem. The 3D
structure depends on many variables.
• Current approaches often work by looking at the
structure of homologous (similar) proteins.
• Improper folding of a protein is believed to be the
cause of mad cow disease.
http://www.sanger.ac.uk/Users/sgj/thesis/node2.html for more information on folding
Molecular Biology Tools:
• Copying DNA
• Polymerase Chain Reaction
• Cloning
• Cutting and Pasting DNA
• Restriction Enzymes
• Measuring DNA Length
• Electrophoresis
• DNA sequencing
• Probing DNA
• DNA probes
• DNA arrays
Analyzing a Genome
• How to analyze a genome in four “easy” steps.
• Cut it
• Use enzymes to cut the DNA in to small fragments.
• Copy it
• Copy it many times to make it easier to see and detect.
• Read it
• Use special chemical techniques to read the small fragments.
• Assemble it
• Take all the fragments and put them back together. This is
hard!!!
• Bioinformatics takes over
• What can we learn from the sequenced DNA.
• Compare interspecies and intraspecies.
Restriction Enzymes: Cutting DNA
• Restriction Enzymes - proteins that cut double-stranded
DNA whenever they encounter a specific sequence of
nucleotides, called its restriction site.
• Over 300 types of restriction enzymes known
Restriction Enzyme
EcoRI
HinfI
NotI
Restriction Site
5’ - GAATTC - 3’
5’ - GATC - 3’
5’ - GCGGCCGC - 3’
How large of DNA fragments would you expect with
EcoRI vs. HinfI?
Restriction Enzymes: Cutting DNA
• Restriction enzymes can cut the two DNA strands
“straight” to leave blunt ends, or “crooked” to leave
sticky ends.
5'- ... G A A T T C ... - 3'
3'- ... C T T A A G ... - 5'
Digestion with EcoRI to
get sticky ends
5'- ... G
A A T T C ... - 3'
3'- ... C T T A A
G ... - 5'
Why we need so many copies
• Biologists needed to find a way to read DNA codes.
• How do you read base pairs that are angstroms in
size?
• It is not possible to directly look at it due to DNA’s
small size.
• Need to use chemical techniques to detect what you
are looking for.
• To read something so small, you need a lot of it, so
that you can actually detect the chemistry.
• Need a way to make many copies of the base pairs,
and a method for reading the pairs.
Two Ways to Copy/Clone DNA
• DNA Cloning
• Insert the fragment into the genome of
a living organism and watch it multiply.
• Once you have enough, remove the
organism, keep the DNA.
• Use Polymerase Chain Reaction
(PCR)
Vector DNA
Polymerase Chain Reaction
• Problem: Modern
instrumentation cannot
easily detect single
molecules of DNA, making
amplification a prerequisite
for further analysis
• Solution: PCR doubles
the number of DNA
fragments at every
iteration
1…
2…
4…
8…
Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR)
• Used to massively replicate DNA sequences.
• How it works:
• Separate the two strands with low heat
• Add some base pairs, primer sequences, and
DNA Polymerase
• Creates double stranded DNA from a single
strand.
• Primer sequences create a seed from which
double stranded DNA grows.
• Now you have two copies.
• Repeat. Amount of DNA grows exponentially.
• 1→2→4→8→16→32→64→128→256…
Denaturation
Raise temperature to 94oC
to separate the duplex form
of DNA into single strands
Design primers
• To perform PCR, a 10-20bp sequence on either
side of the sequence to be amplified must be
known because DNA pol requires a primer to
synthesize a new strand of DNA
Annealing
• Anneal primers at 50-65oC
Annealing
• Anneal primers at 50-65oC
Extension
• Extend primers: raise temp to 72oC, allowing Taq
pol to attach at each priming site and extend a
new DNA strand
Extension
• Extend primers: raise temp to 72oC, allowing Taq
pol to attach at each priming site and extend a
new DNA strand
Repeat
• Repeat the Denature, Anneal, Extension
steps at their respective temperatures…
Polymerase Chain Reaction
Electrophoresis
• A copolymer of mannose and galactose,
agaraose, when melted and recooled,
forms a gel with pores sizes dependent
upon the concentration of agarose
• The phosphate backbone of DNA is
highly negatively charged, therefore
DNA will migrate in an electric field
• The size of DNA fragments can then
be determined by comparing their
migration in the gel to known size
standards.
Pasting DNA
• Two pieces of DNA can
be fused together by
adding chemical bonds
• Hybridization –
complementary basepairing
• Ligation – fixing bonds
with single strands
Probes
A probe is single-stranded DNA of 20+
nucleotide which is chosen on the basis of it
consisting of the reverse complementary
base pairs of the DNA fragment of interest.
Probes can be labeled before hybridization
either radioactively or enzymatically (e.g.
alkaline phosphatase or horseradish
peroxidase), or fluorescently.
Probes are detected by directly exposing the
membrane to X-ray film or chemiluminescent
methods.
Reading DNA, i.e., DNA Sequencing
Determining the sequence of nucleotides of the DNA
strand.
All sequencing methods employ the same basic strategy:
 generated a complete set of subfragments for a region
being studied whose lengths differ from each other by
one nucleotide
 label the subfragments ends with different nucleotide
specific labels
 separate the labeled fragments by size (e.g., using gel
eletrophoresis)
 used the different labels on the ends of the nucleotide to
read the sequence
Automated DNA sequencing using
fluorescent primers: (A)
•
•
•
•
Labeled DNA fragments are loaded into single lanes
of the electrophoresis gel.
During the electrophoresis run, a laser beam is
focused at a specific constant position on the gel.
As the individual DNA fragments migrate past this
position, the laser causes the dyes to fluoresce.
Maximum fluorescence occurs at different
wavelengths for the four dyes, and the information is
recorded electronically and the interpreted sequence
is stored in a computer database.
Automated DNA sequencing using
fluorescent primers:
(B) Shows a typical output of an automated DNA
sequencer as a succession of dye-specific (and
therefore base-specific) intensity profiles.
Automated DNA Sequencing
Technology:
 DNA fragments of about 1,000 nucleotides can be
sequenced at one time
 Each DNA fragment makes up only a small part of
an organism’s genome (eukaryotes have billions of
base pairs in their DNA)
 Each DNA fragment makes up only a small part of
an organism’s gene (eukaryotes genes can be
100,000s base pairs)
 After sequencing the DNA fragments, the fragment
sequences must be reassembled to form the
genome.
Assembling Genomes
• Must take the fragments
and put them back
together
• Not as easy as it sounds.
• SCS Problem (Shortest
Common Superstring)
• Some of the fragments will
overlap
• Fit overlapping sequences
together to get the
shortest possible
sequence that includes all
fragment sequences
Assembling Genomes
• DNA fragments contain sequencing errors
• Two complements of DNA
• Need to take into account both directions of DNA
• Repeat problem
• 50% of human DNA is just repeats
• If you have repeating DNA, how do you know where it
goes?
• Assembly is a difficult problem!!!
Microarray (DNA chip)
1,000 to 10,000s nucleotide sequences are affixed to individual
positions on the surface of a small glass chip.
Fluorescently labeled copies of the RNA transcripts (cDNA) from
an organism are washed over the chip allowing them to hybridize
to complementary nucleotides on the chip.
Unbound RNA is washing away.
A laser is used to excite the fluorescent tags and photodetectors
quantify the amount of signal associated with each spot of a
known sequence.
Application: Determination of relative RNA levels associated with
huge numbers of known and predicted genes in a single
experiment. DNA chips exist commerically for a variety of
organisms.
Labeling technique for DNA arrays
RNA samples are labeled using fluorescent nucleotides (left) or
radioactive nucleotides (right), and hybridized to arrays. For fluorescent
labeling, two or more samples labeled with differently colored fluorescent
markers are hybridized to an array. Level of RNA for each gene in the
sample is measured as intensity of fluorescence or radioactivity binding
to the specific spot. With fluorescence labeling, relative levels of expressed
genes in two samples can be directly compared with a single array.
DNA Arrays--Technical Foundations
• An array works by exploiting the ability of a given mRNA molecule
to hybridize to the DNA template.
• Using an array containing many DNA samples in an experiment, the
expression levels of hundreds or thousands genes within a cell by
measuring the amount of mRNA bound to each site on the array.
• With the aid of a computer, the amount of mRNA bound to the spots
on the microarray is precisely measured, generating a profile of
gene expression in the cell.
An experiment on a microarray
In this schematic:
GREEN represents Control DNA
RED represents Sample DNA
YELLOW represents a combination of Control and Sample DNA
BLACK represents areas where neither the Control nor Sample DNA
Each color in an array represents either healthy (control) or diseased (sample) tissue.
The location and intensity of a color tell us whether the gene, or mutation, is present in
the control and/or sample DNA.
DNA Microarray
Millions of DNA strands
build up on each location.
Tagged probes become hybridized
to the DNA chip’s microarray.
DNA Microarray
Affymetrix
Microarray is a tool for
analyzing gene expression
that consists of a glass slide.
Each blue spot indicates the location of a PCR
product. On a real microarray, each spot is
about 100um in diameter.
Affymetrix GeneChip® Arrays
A combination of photolithography and combinatorial chemistry to manufacture
GeneChip® Arrays. With a minimum number of steps, Affymetrix produces
arrays with thousands of different probes packed at extremely high density.
Enable to obtain high quality, genome-wide data using small sample volumes.
May 11,2004
http://www.affymetrix.com/technology/manufacturing/index.affx
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Affymetrix GeneChip® Arrays
Data from an experiment showing the
expression of thousands of genes on
a single GeneChip® probe array.
May 11,2004
http://www.affymetrix.com/corporate/media/image_library/image_library_1.affx
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