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CASE FILES: BIOCHEMISTRY
randomly at various positions throughout the chain, thus yielding an
array of different length DNA fragments each terminated with the particular
dideoxyribonucleotide. The fragments of varying length are then separated
by electrophoresis in parallel lanes (each corresponding to the four deoxyri-
bonucleotides) and the positions of the fragments analyzed to determine the
sequence. The fragments are separated on the basis of size, with the shorter
fragments moving faster and appearing at the bottom of the gel. The sequence
is then read from bottom to the top, yielding the 5' to 3' sequence.
The ability to rapidly sequence DNA has become an important tool for
molecular biology. For example, the polymerase chain reaction (PCR) method
needs the information of the sequence flanking the region of interest to be able
to design specific oligonucleotides (primers) to amplify the specific DNA
region. Another important use is identifying restriction sites in plasmids,
which is useful in cloning a foreign gene into the plasmid. Before DNA
sequencing, molecular biologists had to sequence proteins directly, which was
a challenging and laborious process. Now amino acid sequences can be deter-
mined more easily by sequencing a piece of cDNA and finding an open
reading frame. In eukaryotic gene expression, sequencing made it possible to
identify conserved sequence motifs and determine important sites in the pro-
moter regions. Furthermore, sequencing can be used to identify the site of a
point mutation or other changes in the genome.
Recombinant DNA technology allows for a transfer of a DNA fragment
of interest into a self-replicating genetic element such as a bacterial plas-
mid or virus. This technology has allowed many human genes to be cloned in
Escherichia coli,
yeast, or even mammalian cells, leading to easy production
of human recombinant proteins in vitro. These include insulin for diabetics,
factor VIII for males suffering from hemophilia A, human growth hormone
(GH), erythropoietin (EPO) for treating anemia, three types of interferons,
several interleukins, adenosine deaminase (ADA) for treating some forms of
severe combined immunodeficiency (SCID), angiostatin and endostatin for tri-
als as anticancer drugs, and parathyroid hormone with many more in develop-
ment.
Efforts toward gene therapy make use of recombinant DNA technology. In
the case of CF, which is the result of a defect in a single gene, direct insertion
of the normal gene should theoretically restore the function of CFTR in the
treated cells of the CF patient. Since respiratory failure is the major cause
of deaths (95 percent) in CF, lung cells have become the primary target for
efforts at treating CF with gene therapy. Another advantage is the ability of
delivering vectors containing the functional CFTR gene directly into the
patient’s airways by aerosol, without causing trauma to any other part of the
body. To date, CF trials rely on adenovirus, adeno-associated virus (AAV),
and cationic liposomes to mediate gene transfer to nasal epithelial cells and
respiratory epithelial cells. Adenoviruses do not integrate into the host chro-
mosome; therefore, the vectors derived from these viruses have the advantage
of negligible oncogenic potential. The disadvantages are the development of
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