The Chemistry of Biotechnology

1
The Chemistry of Biotechnology

• Introduction
• Definitions
• Basic Science
• Applications
• Significance

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North Carolina State University
Goals for International Program in Poland

• Establish new interactions with researchers
• collaboration
• - joint projects
• - new funding opportunities
• exchange
• - faculty
• - students
• education

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What is Biotechnology?

• The ability to determine gene sequences and
• manipulate them
• - genes code for proteins
• - recognition and binding events important
• Application to new catalysts and drugs
• - enzymes for industry and bioremediation
• - drugs influence signaling, gene
  transcription, etc.
• Potential to influence evolution at a critical
• stage in human evolution
• - shape environment by breeding new plants
  and animals
• - develop efficient processes and clean up
  waste streams
• - treat diseases at the molecular level

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What is Chemistry?

• The understanding of molecular interactions
• that control gene transcription
• - molecular recognition involves specific
  chemical interactions
• - gene transcription requires specific
  enzymatic control
• The development of new catalysts and drugs
• - synthetic approaches are complemented by
  biology
• - the high efficiency of biological catalysis
  is goal
• A science poised to link biology to engineering
• - the genetic breakthrough requires a firm
  understanding and
• synthetic capability to make use of the new
  technology
• - biology is specific, molecular level
  understanding requires
• thinking in terms of specific molecular
  mechanisms

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The components of DNA

• The individual nucleobases are
• The nucleoside is formed by a bond with the
  ribose sugar at the N9 position of A and G and
  the N6 position of C and T.

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The stability of double-helical DNA
The double helical form of DNA is formed by
phosphodiester linkages between the 5 -OH end
of one ribose and the 3-OH of the next. The
double helix is stabilized by hydrogen bonding
in canonical base pairs and by stacking
interactions between the bases.
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DNA conformations
A form
Z form
B form
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Conformations of the ribose

• B is 2-endo anti
• A is 3-endo anti
• Z is 2-endo syn

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The first X-ray crystal structure of d(GCGCGC)
was in the Z-form

• Watson-Crick DNA was determined based on fiber
  diffraction. This was B form.
• The expectation that crystalline DNA would also
  be in the B-form was shattered by the structure
  of the hexamer d(GCGCGC)
• Raman and infrared spectroscopy have played a key
  role in comparisons of the various forms of DNA.

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The Dickerson dodecamer
The first x-ray crystal structure of DNA in the
B-form was obtained by Dickerson using the
dodecamer with the sequence d(CGCGAATTCGCG)2. T
he structure shown has the stacking pattern of
B-form DNA found in most DNA in solution. DNA
can, however, be bent or coiled. Coiling is
facilitated by regions of adenines known as
phased A tracts. Such coiling permits
the packing of DNA in structures that for
chromosomes.
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DNA sequence encodes proteins
DNA Information
transcription mRNA Message
translation Protein sequence Assembly
folding Folded protein Function
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DNA sequence encodes proteins
Ribosome
DNA Information
transcription mRNA Message
translation Protein sequence Assembly
folding Folded protein Function
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The codon table
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Translating the DNA sequence
The order of amino acids in any protein is
specificed by the order of nucleotide bases in
the DNA. Each amino acid is coded by the
particular sequence of three bases. To convert a
DNA sequence First, find the starting codon.
The starting codon is always the codon for
the amino acid methionine. This codon is AUG
in the RNA (or ATG in the DNA)
GCGCGGGUCCGGGCAUGAAGCUGGGCCGGGCCGUGC....
Met In this
particular example the next codon is AAG. The
first base (5'end) is A, so that selects the 3rd
major row of the table. The second base (middle
base) is A, so that selects the 3rd column of
the table. The last base of the codon is G,
selecting the last line in the block of four.
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Translating the DNA sequence
This entry AAG in the table is Lysine (Lys).
Therefore the second amino acid is Lysine.
The first few residues, and their DNA sequence,
are as follows (color coded to indicate the
correct location in the codon table) Met
Lys Leu Gly Arg ... AUG AAG CUG
GGC CGG GCC GUG C.. This procedure is exactly
what cells do when they synthesize proteins based
on the mRNA sequence. The process of
translation in cells occurs in a large complex
called the ribosome.
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DNA sequence encodes proteins
Folding intermediates of apomyoglobin
DNA Information
transcription mRNA Message
translation Protein sequence Assembly
folding Folded protein Function
U unfolded E early intermediate I
intermediate N native
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Levels of Protein Structure Secondary Structure
ordering along short stretches of the protein
b-sheets
a-helices
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Levels of Protein Structure Tertiary Structure
overall conformation of the protein chain
information contained in the proteins sequence
of residues is sufficient to dictate the specific
3-dimensional structure that the chain will adopt
(native state)
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Mutations

• A gene is a blueprint for construction of
  protein.
• Any change in a DNA sequence is called a
  mutation.
• The change may result in a protein that does not
  fold
• or that is not functional.
• There are controlling sequences that determine
• whether a gene will be expressed.
• Changes in these controlling sequences can
  result
• in altered levels of protein expression in a
  cell.
• Altering DNA sequences is a basic research tool
• and is an important tool in biolotechnology.
• The tool for making mutations are part of
  molecular
• biology. The job of analyzing the result of
  mutations
• is a research area for chemistry.

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DNA sequencing by the Sanger method
The standard DNA sequencing technique is the
Sanger method, named for its developer,
Frederick Sanger, who shared the 1980 Nobel
Prize in Chemistry. This method begins with the
use of special enzymes to synthesize fragments
of DNA that terminate when a selected base
appears in the stretch of DNA being sequenced.
These fragments are then sorted according to size
by placing them in a slab of polymeric gel and
applying an electric field -- a technique called
electrophoresis. Because of DNA's negative
charge, the fragments move across the gel toward
the positive electrode. The shorter the
fragment, the faster it moves. Typically, each of
the terminating bases within the collection of
fragments is tagged with a radioactive probe
for identification.
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DNA sequencing example
Problem Statement Consider the following DNA
sequence (from firefly luciferase). Draw the
sequencing gel pattern that forms as a result of
sequencing the following template DNA with ddNTP
as the capper. atgaccatgattacg... Solution
Given DNA template 5'-atgaccatgattacg..
.-3' DNA synthesized
3'-tactggtactaatgc...-5'
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DNA sequencing example
Given DNA template 5'-atgaccatgattacg...-3'
DNA synthesized 3'-tactggtactaatgc...-
5' Gel pattern -----------------------
-- lane ddATP W
lane ddTTP W
lane ddCTP W
lane ddGTP W

-------------------------
Electric Field

Decreasing size where "W" indicates the well
position, and "" denotes the DNA bands on the
sequencing gel.
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A sequencing gel
This picture is a radiograph. The dark color of
the lines is proportional to the radioactivity
from 32P labeled adenonsine in the transcribed
DNA sample.
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Reading a sequencing gel
You begin at the right, which are the smallest
DNA fragments. The sequence that you read will
be in the 5'-3' direction. This sequence will be
exactly the same as the RNA that would be
generated to encode a protein. The difference is
that the T bases in DNA will be replaced by U
residues. As an example, in the problem given,
the smallest DNA fragment on the sequencing gel
is in the C lane, so the first base is a C. The
next largest band is in the G lane, so the DNA
fragment of length 2 ends in G. Therefore the
sequence of the first two bases is CG. The
sequence of the first 30 or so bases of the DNA
are CGTAATCATGGTCATATGAAGCTGGGCCGGGCCGTGC....
When this is made as RNA, its sequence would
be CGUAAUCATGGUCAUAUGAAGCUGGGCCGGGCCGUGC....
Note that the information content is the same,
only the T's have been replaced by U's!.
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Automated procedure for DNA sequencing
A computer read-out of the gel generates a false
color image where each color corresponds to a
base. Then the intensities are translated into
peaks that represent the sequence.
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High-throughput seqeuncingCapillary
electrophoresis
The human genome project has spurred an effort
to develop faster, higher throughput, and less
expensive technologies for DNA sequencing.
Capillary electrophoresis (CE) separation has
many advantages over slab gel separations. CE
separations are faster and are capable of
producing greater resolution. CE instruments can
use tens and even hundreds of capillaries
simultaneously. The figure show a simple CE setup
where the fluorescently-labeled DNA is detected
as it exits the capillary.
Sheath flow
Laser
Focusing lens
Sheath flow cuvette
Beam block
Collection Lensc
Collection Lensc
PMT
filter
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Fluorescent end labeling of DNA
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RNA amplification and labeling by reverse
transcription

• Sample must be reverse transcribed and
  re-transcribed
• Expensive equipment
• Poor dynamic range due to self-quenching

Fluorophore
Fluorophore
mRNA
Fluorophore
Fluorophore
Labeled antisense DNA
cDNA
Requires Expertise
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RNA and DNA hybridization
DNA Array Technology
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DNA arrays
DNA Array Technology
DNA arrays are tools to determine Genomic
sequences Gene expression Levels Advantages of
DNA arrays High throughput Fast screening
compared to traditional screening
techniques Obstacles to application Poor
Selectivity (False positive responses) Low
sensitivity (Amplification of the probes is
required) Cost
cDNA Target
Probe B
Probe B
Probe B
Probe C
Probe C
Probe C
Probe D
Probe D
Probe D
DNA Array, Overall Principle
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GeneChipTM Technology
DNA Array Technology
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Polymerase chain reaction
Who would have thought a bacterium hanging out in
a hot spring in Yellowstone National Park would
spark a revolutionary new laboratory technique?
The polymerase chain reaction, now widely used
in research laboratories and doctor's offices,
relies on the ability of DNA-copying enzymes to
remain stable at high temperatures. The
polymerase from Thermus aquaticus (Taq), a
bacterium from Yellowstone can produce millions
of copies of a single DNA segment in a matter of
hours. In nature, most organisms copy their DNA
in the same way. PCR mimics the natural process,
only it does it in a test tube. When any cell
divides, enzymes called polymerases make a copy
of all the DNA in each chromosome. The first
step in this process is to "unzip" the two DNA
chains of the double helix. As the two strands
separate, DNA polymerase makes a copy using each
strand as a template.
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The role of the primer
To copy DNA, polymerase requires two other
components 1. a supply of the four nucleotide
bases 2. a primer. DNA polymerases, whether from
humans, bacteria, or viruses, cannot copy a
chain of DNA without a short sequence of
nucleotides to "prime" the process, or get it
started. So the cell has another enzyme called a
primase that actually makes the first few
nucleotides of the copy. This stretch of DNA is
called a primer. Once the primer is made, the
polymerase can take over making the rest of the
new chain.
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Step I DNA melting
The three parts of the polymerase chain reaction
are carried out in the same vial, but at
different temperatures. The first part of the
process separates the two DNA chains in the
double helix. This is done simply by heating the
vial to 95 oC for 30 seconds.
double-stranded DNA single-stranded
DNA
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Step II Primer annealing
The primers cannot bind to the DNA strands at
such a high temperature, so the vial is cooled
to 55 oC. At this temperature, the primers bind
or "anneal" to the appropriate location in the
DNA strands. This takes about 20 seconds.
single-stranded DNA primer
annealed DNA The final step of the reaction is
to make a complete copy of the templates. Since
the Taq polymerase works best at ca. 75 oC, the
temperature of the vial is raised.
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Step III Primer extension
The Taq polymerase begins adding nucleotides to
the primer and eventually makes a complementary
copy of the section of the template that lies
between the primers. This completes one PCR
cycle. primer-annealed DNA
primer-extended DNA with Taq polymerase and dATP,
dTTP, dGTP, dCTP
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Thermal cycling
At the end of a cycle, each piece of DNA in the
vial has been duplicated. Since the cycle can be
repeated 30 or more times and each newly
synthesized DNA piece can act as a new template,
one can obtain 230 or ca. 1 million copies of a
single piece of DNA.
amplified
region Note that the region of the DNA between
the two primers will be amplified. The flanking
sequences not. The entire process takes about
three hours.
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PCR amplification
The figure on the left shows the series of
steps in a single cycle. The exponential growth
of the double helical segment between the
two primers is illustrated above.
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Taq polymerase
Taq Polymerase In Complex With Tp7, An Inhibitory
Fab
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Cloning into a plasmid
The procedure begins with the insertion of a DNA
sequence of interest into a plasmid. A plasmid
is a circular piece of dsDNA (typically 3 kb).
The plasmid can be isolated from E. coli and
manipulated using cutting enzymes known as
restriction endonucleases. Once the plasmid is
cut the target gene (DNA sequency of interest)
will bind to complementary "sticky ends." The
ends of gene can be made to match the sequence
cut by a particular restriction endonuclease by
appropriate design of the primer used in PCR. The
opened plasmid and the gene are allowed mix and
are then bonded by an enzyme called DNA ligase,
which reforms the two pieces as recombinant DNA.
The plasmid carries also genes for antibiotic
resistance that will be once the plasmid is
introduced into a cell.
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Cutting Ligation Transformation Selection
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Significance
The tools described here provide the basis
for sequencing and manipulating the genome. We
can understand cell development,
biological chemistry and disease using these
tools. The development of new enzymes and
processes result from the cloning of genes in the
expression vectors of E. Coli and using the
baculovirus. We can study disease states and
target them using peptide drugs, drug design and
gene therapy.
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The Role of Spectroscopy

• Spectroscopy is a key method for detecting
• biological molecules and binding interactions.
• It is useful for DNA sequencing, detection of
  DNA and proteins on chips, assays for screening
• new drugs and many other applications. In this
• Course we will describe the applications of
• spectroscopy including
• Absorption and Fluorescence (energy transfer)
• Vibrational Raman and Infrared Spectroscopy
• Ultraviolet Circular Dichroism