Exploring Genes and Genomes Aarhus Universitet

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14 0 sequence complete genomes first small genomes from viruses then larger
CHAPTER 5 Exploring Genes and genomes from bacteria and finally eukaryotic genomes including the
Genomes 3 billion base pair human genome Scientists are just beginning to exploit
the enormous information content of these genome sequences
Finally recombinant DNA technology critically depends on our ability
to deliver foreign DNA into host organisms For example DNA fragments
can be inserted into plasmids where they can be replicated within a short
period of time in their bacterial hosts In addition viruses efficiently deliver
their own DNA or RNA into hosts subverting them either to replicate the
viral genome and produce viral proteins or to incorporate viral DNA into
the host genome
These new methods have wide ranging benefits across a broad spectrum
of disciplines including biotechnology agriculture and medicine Among
these benefits is the dramatic expansion of our understanding of human
disease Throughout this chapter a specific disorder amyotrophic lateral
sclerosis ALS will be used to illustrate the effect that recombinant DNA
technology has had on our knowledge of disease mechanisms ALS was first
described clinically in 1869 by the French neurologist Jean Martin Charcot
as a fatal neurodegenerative disease of progressive weakening and atrophy
of voluntary muscles ALS is commonly referred to as Lou Gehrig s Disease
for the baseball legend whose career and life were prematurely cut short as
a result of this devastating disease For many years little progress had been
made in the study of the mechanisms underlying ALS As we shall see
significant advances have been made with the use of research tools facili
tated by recombinant DNA technology
5 1 The Exploration of Genes Relies on Key Tools
The rapid progress in biotechnology indeed its very existence is a result
of a few key techniques
1 Restriction Enzyme Analysis Restriction enzymes are precise molecular
scalpels that allow an investigator to manipulate DNA segments
2 Blotting Techniques Southern and northern blots are used to separate and
characterize DNA and RNA respectively The western blot which uses
antibodies to characterize proteins was described in Chapter 3
3 DNA Sequencing The precise nucleotide sequence of a molecule of
DNA can be determined Sequencing has yielded a wealth of information
concerning gene architecture the control of gene expression and protein
4 Solid Phase Synthesis of Nucleic Acids Precise sequences of nucleic acids
can be synthesized de novo and used to identify or amplify other nucleic
5 The Polymerase Chain Reaction PCR The polymerase chain reaction
leads to a billionfold amplification of a segment of DNA One molecule of
DNA can be amplified to quantities that permit characterization and
manipulation This powerful technique can be used to detect pathogens and
genetic diseases determine the source of a hair left at the scene of a crime
and resurrect genes from the fossils of extinct organisms
A final set of techniques relies on the computer without which it would
be impossible to catalog access and characterize the abundant information
generated by the techniques just outlined Such uses of the computer will be 141
presented in Chapter 6 5 1 Tools of Gene Exploration
Restriction enzymes split DNA into specific fragments
Restriction enzymes also called restriction endonucleases recognize specific
base sequences in double helical DNA and cleave at specific places both
strands of that duplex To biochemists these exquisitely precise scalpels are
marvelous gifts of nature They are indispensable for analyzing chromo
some structure sequencing very long DNA molecules isolating genes and
creating new DNA molecules that can be cloned Werner Arber and
Hamilton Smith discovered restriction enzymes and Daniel Nathans pio
neered their use in the late 1960s Palindrome
Restriction enzymes are found in a wide variety of prokaryotes Their A word sentence or verse that reads the
biological role is to cleave foreign DNA molecules Many restriction same from right to left as it does from left to
enzymes recognize specific sequences of four to eight base pairs and hydro right
lyze a phosphodiester bond in each strand in this region A striking charac Radar
teristic of these cleavage sites is that they almost always possess twofold Senile felines
Do geese see God
rotational symmetry In other words the recognized sequence is palindromic Roma tibi subito motibus ibit amor
or an inverted repeat and the cleavage sites are symmetrically positioned Derived from the Greek palindromos
For example the sequence recognized by a restriction enzyme from running back again
Streptomyces achromogenes is
Cleavage site
5 C C G C G G 3
3 G G C G C C 5
Cleavage site Symmetry axis
5 G G A T C C 3
In each strand the enzyme cleaves the C G phosphodiester bond on the 39 3 C C T A G G 5
side of the symmetry axis As we shall see in Chapter 9 this symmetry cor
responds to that of the structures of the restriction enzymes themselves
5 G A A T T C 3
Several hundred restriction enzymes have been purified and character EcoRI
3 C T T A A G 5
ized Their names consist of a three letter abbreviation for the host organ
ism e g Eco for Escherichia coli Hin for Haemophilus influenzae Hae for
Haemophilus aegyptius followed by a strain designation if needed and a 5 G G C C 3
roman numeral if more than one restriction enzyme from the same strain 3 C C G G 5
has been identified The specificities of several of these enzymes are shown
in Figure 5 1
Restriction enzymes are used to cleave DNA molecules into specific frag 5 G C G C 3
ments that are more readily analyzed and manipulated than the entire parent 3 C G C G 5
molecule For example the 5 1 kb circular duplex DNA of the tumor pro
ducing SV40 virus is cleaved at one site by EcoRI at four sites by HpaI and
5 C T C G A G 3
at 11 sites by HindIII A piece of DNA called a restriction fragment pro XhoI
3 G A G C T C 5
duced by the action of one restriction enzyme can be specifically cleaved into
smaller fragments by another restriction enzyme The pattern of such frag
ments can serve as a fingerprint of a DNA molecule as will be considered Figure 5 1 Specificities of some
restriction endonucleases The sequences
shortly Indeed complex chromosomes containing hundreds of millions of
that are recognized by these enzymes contain
base pairs can be mapped by using a series of restriction enzymes a twofold axis of symmetry The two strands in
these regions are related by a 180 degree
Restriction fragments can be separated by gel electrophoresis rotation about the axis marked by the green
and visualized symbol The cleavage sites are denoted by red
arrows The abbreviated name of each
Small differences between related DNA molecules can be readily detected restriction enzyme is given at the right of the
because their restriction fragments can be separated and displayed by gel sequence that it recognizes Note that the cuts
electrophoresis In Chapter 3 we considered the use of gel electrophoresis may be staggered or even
14 2 to separate protein molecules Section 3 1 Because the phosphodiester
CHAPTER 5 Exploring Genes and backbone of DNA is highly negatively charged this technique is also
Genomes suitable for the separation of nucleic acid fragments For most gels the
shorter the DNA fragment the farther the migration Polyacrylamide gels
are used to separate by size fragments containing as many as 1000 base
A B C pairs whereas more porous agarose gels are used to resolve mixtures of
larger fragments as large as 20 kb An important feature of these gels is
their high resolving power In certain kinds of gels fragments differing in
length by just one nucleotide of several hundred can be distinguished
Bands or spots of radioactive DNA in gels can be visualized by autoradiog
raphy Alternatively a gel can be stained with ethidium bromide which
fluoresces an intense orange when bound to a double helical DNA mole
cule Figure 5 2 A band containing only 50 ng of DNA can be readily
A restriction fragment containing a specific base sequence can be
identified by hybridizing it with a labeled complementary DNA strand
Figure 5 3 A mixture of restriction fragments is separated by electro
phoresis through an agarose gel denatured to form single stranded DNA and
transferred to a nitrocellulose sheet The positions of the DNA fragments
in the gel are preserved on the nitrocellulose sheet where they are
exposed to a 32P labeled single stranded DNA probe The probe hybridizes
with a restriction fragment having a complementary sequence and autora
diography then reveals the position of the restriction fragment probe
duplex A particular fragment amid a million others can be readily identi
fied in this way This powerful technique is named Southern blotting for its
inventor Edwin Southern
Similarly RNA molecules can be separated by gel electrophoresis and
specific sequences can be identified by hybridization subsequent to their
Figure 5 2 Gel electrophoresis pattern of transfer to nitrocellulose This analogous technique for the analysis of RNA
a restriction digest This gel shows the has been whimsically termed northern blotting A further play on words
fragments produced by cleaving SV40 DNA accounts for the term western blotting which refers to a technique for detect
with each of three restriction enzymes These
fragments were made fluorescent by staining
ing a particular protein by staining with specific antibody Section 3 3
the gel with ethidium bromide Courtesy of Southern northern and western blots are also known respectively as DNA
Dr Jeffrey Sklar RNA and protein blots
Transfer Add probe
of DNA P labeled revealed
by blotting DNA probe Autoradiography
Agarose Nitrocellulose Autoradiogram
gel sheet
Figure 5 3 Southern blotting A DNA fragment containing a specific sequence can be identified
by separating a mixture of fragments by electrophoresis transferring them to nitrocellulose and
hybridizing with a 32P labeled probe complementary to the sequence The fragment containing the
sequence is then visualized by autoradiography
DNA can be sequenced by controlled termination of replication DNA to be sequenced
The analysis of DNA structure and its role in gene expression also have 3 G A AT TC G C TA ATG C
been markedly facilitated by the development of powerful techniques for 5 C T TA A
the sequencing of DNA molecules The key to DNA sequencing is the gen Primer
eration of DNA fragments whose length depends on the last base in the DNA polymerase I
sequence Collections of such fragments can be generated through the con Labeled dATP TTP
trolled termination of replication Sanger dideoxy method a method devel Dideoxy analog of dATP
oped by Frederick Sanger and coworkers This technique has superseded
alternative methods because of its simplicity The same procedure is per
formed on four reaction mixtures at the same time In all these mixtures a

DNA polymerase is used to make the complement of a particular sequence 3 G A AT TC G C TA ATG C
within a single stranded DNA molecule The synthesis is primed by a 5 C T TA A G C G A
chemically synthesized fragment that is complementary to a part of the
sequence known from other studies In addition to the four deoxyribonucle New DNA strands are separated
oside triphosphates radioactively labeled each reaction mixture contains a and subjected to electrophoresis
small amount of the 29 39 dideoxy analog of one of the nucleotides a differ Figure 5 4 Strategy of the chain
ent nucleotide for each reaction mixture termination method for sequencing DNA
Fragments are produced by adding the
2 29 39 dideoxy analog of a dNTP to each of
four polymerization mixtures For example the
P P P H2 base addition of the dideoxy analog of dATP
O O O O O shown in red results in fragments ending in
A The strand cannot be extended past the
H dideoxy analog
2 3 Dideoxy analog
The incorporation of this analog blocks further growth of the new chain
because it lacks the 39 hydroxyl terminus needed to form the next phos
phodiester bond The concentration of the dideoxy analog is low enough
that chain termination will take place only occasionally The polymerase
will insert the correct nucleotide sometimes and the dideoxy analog other
times stopping the reaction For instance if the dideoxy analog of dATP
is present fragments of various lengths are produced but all will be termi
nated by the dideoxy analog Figure 5 4 Importantly this dideoxy analog
of dATP will be inserted only where a T was located in the DNA being
sequenced Thus the fragments of different length will correspond to the
positions of T Four such sets of chain terminated fragments one for each
dideoxy analog then undergo electrophoresis and the base sequence of the
new DNA is read from the autoradiogram of the four
Fluorescence detection is a highly effective alternative AT A GT G T CAC C T A A A T AG CT TG GCG T A A T C AT GG T C A T A G C T
100 110 120 130
to autoradiography because it eliminates the use of. Exploring Genes and Genomes Since its emergence in the 1970s recombinant DNA technology has revo lutionized biochemistry The genetic endowment of organisms can now be precisely changed in designed ways Recombinant DNA technology is the fruit of several decades of basic research on DNA RNA and viruses It

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