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260 Molecular genetics
DNA sequencing B. Sequencing of DNA
Thenucleotidesequenceof DNA is nowadays
A. Gene libraries usually determined using the so-called chain
termination method.In single-strand se-
It is often necessary in genetic engineering to
isolate a DNA segment when its details are not quencing, the DNA fragment (a)is cloned
into the DNA of phage M13 (see p. 404),
fully known—e. g., in order to determine its from which the coded single strand can be
nucleotide sequence. In this case, one can use
what are known as DNA libraries.A DNAli- easily isolated. This is hybridized with a pri-
mer—a short, synthetically produced DNA
brary consists of a large number of vector DNA
molecules containing different fragments of fragment that binds to 3 end of the intro-
duced DNA segment (b).
foreign DNA. For example, it is possible to take Based on this hybrid, the missing second
all of the mRNA molecules present in a cell and strand can now be generated in the test tube
transcribe them into DNA. These DNA frag- by adding the four deoxyribonucleoside tri-
ments(known ascopyDNA or cDNA)are then
randomly introduced into vector molecules. phosphates (dNTP)and a suitable DNA poly-
merase (c). The trick lies in also adding small
Alibrary of genomic DNA canbe estab-
lished by cleaving the total DNA from a cell amounts of dideoxynucleoside triphosphates
into small fragments using restriction endo- (ddNTP). Incorporating a ddNTP leads to the
termination of second-strand synthesis. This
nucleases (see p. 258), and then incorporating
these into vector DNA. Suitable vectors for can occur whenever the corresponding dNTP
oughtto beincorporated. Theillustration
gene libraries include bacteriophages,for ex- shows this in detail using the example of
ample (“phages” for short). Phages are viruses ddGTP. In this case, fragments are obtained
that only infect bacteria and are replicated by that each include the primer plus three, six,
them (see p. 404). Gene libraries have the
advantage that they can be searched for spe- eight, 13, or 14 additional nucleotides. Four
separate reactions, each with a different
cific DNA segments, using hybridization with
oligonucleotides. ddNTP, are carried out (c), and the products
areplaced sideby sideon a supporting mate-
The first step is to strongly dilute a small
5
6
part of the library (10 –10 phages in a small rial. The fragments are then separated by gel
volume), mix it with host bacteria, and plate electrophoresis (see p. 76), in which they
move in relation to their length.
out the mixture onto nutrient medium. The Following visualization (d), thesequenceof
bacteria grow and form a continuous cloudy the fragments in the individual lanes is simply
layer of cells. Bacteria infected by phages read frombottomto top (e)to directly obtain
grow more slowly. In their surroundings, the
bacterial “lawn” is less dense, and a clearer thenucleotidesequence. A detailfrom sucha
sequencing gel and the corresponding protein
circular zone known as a plaque forms. The
bacteria in this type of plaque exclusively sequence are shown in Fig. 2.
In a more modern procedure, the four
contain the offspring of a single phage from ddNTPs are covalently marked with fluores-
the library.
The next step is to make an impression of cent dyes, which produce a different color for
the plate on a plastic foil, which is then each ddNTP on laser illumination. This allows
heated. This causes the phage DNA to adhere the sequence in which the individual frag-
ments appear at the lower end of the gel to
to the foil. When the foil is incubated with a be continuously recorded and directly stored
DNA fragment that hybridizes to the DNA seg-
ment of interest (a gene probe), the probe in digital form.
binds to the sites on the imprint at which
the desired DNA is attached. Binding of the
gene probe can be detected by prior radio-
active or other labeling of the probe. Phages
from the positive plaques in the original plate
are then isolated and replicated. Restriction
cleavage finally provides large amounts of the
desired DNA.
Koolman, Color Atlas of Biochemistry, 2nd edition © 2005 Thieme
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