Recombinant DnA Technology
The term recombinant DNA refers
to a combination of DNA molecules that are not found together in nature.
Recombinant DNA technology makes it possible to identify the DNA sequence in a
gene and produce the protein product encoded by a gene. The specific nucleotide
sequence of a DNA fragment can often be identified by analyzing the amino acid
sequence and mRNA codon of its protein product. Short sequences of base pairs
can be synthesized, radioactively labeled, and sub-sequently used to identify
their complementary sequence. In this way, identifying normal and abnormal gene
structures is possible.
Tests of DNA sequences are
particularly useful in identifying polymorphisms, including the previously
discussed SNPs, that are associated with
various diseases. Because genetic variations are
so distinctive, DNA
fingerprinting (analysis of
DNA sequence differences) can be used to determine family relationships or help
identify persons involved in criminal acts. The methods of recombinant DNA technology
can also be used in the treatment of disease. For example, recombinant DNA
technology is used in the manufacture of human insulin that is used to treat
diabetes mellitus.
Gene Isolation and Cloning
The gene isolation and cloning
methods used in recombinant DNA technology rely on the fact that the genes of
all organisms, from bacteria through mammals, are based on a similar
molecular organization. Gene cloning requires cutting a DNA molecule apart,
modifying and reassembling its fragments, and producing copies of the modified
DNA, its mRNA, and its gene product. The DNA molecule is cut apart by using a bacterial
enzyme, called a restriction enzyme, that binds to DNA wherever a
particular short sequence of base pairs is found and cleaves the molecule at a
specific nucleotide site. In this way, a long DNA molecule can be broken down
into smaller, discrete fragments, one of which presumably contains the gene of
interest. Many restriction enzymes are commercially available that cut DNA at
different recognition sites.
The restrictive fragments of DNA
can often be replicated through insertion into a unicellular organism, such as
a bacterium. To do this, a cloning vector such as a bacterial virus or a small
DNA circle that is found in most bacteria, called a plasmid, is used.
Viral and plasmid vectors replicate autonomously in the host bacterial cell.
During gene cloning, a bacterial vector and the DNA fragment are mixed and
joined by a special enzyme called a DNA ligase. The recombinant vectors
formed are then introduced into a suitable culture of bacteria, and the
bacteria are allowed to replicate and express the recombinant vector gene.
Sometimes, mRNA taken from a tissue that expresses a high level of the gene is
used to pro- duce a complementary DNA molecule that can be used in the cloning
process. Because the fragments of the entire DNA molecule are used in the
cloning process, additional steps are taken to identify and separate the clone
that contains the gene of interest.
Pharmaceutical Applications
Recombinant DNA technology has also
made it possible to produce proteins that have therapeutic properties. One of
the first products to be produced was human insulin. Recombinant DNA
corresponding to the A chain of human insulin was isolated and inserted into
plasmids that were in turn used to transform Escherichia coli. The bacteria
then synthesized the insulin chain. A
similar method was used to obtain the B chains. The A and B chains were then
mixed and allowed to fold and form disulfide bonds, producing active insulin
molecules. Human growth hormone has also been produced in E. coli. More
complex proteins are produced in mammalian cell culture using recombinant DNA
techniques. These include erythropoietin, which is used to stimulate red blood
cell production; factor VIII, which is
used to treat hemophilia; and
tissue plasminogen activator (tPA), which is frequently administered after a heart attack to dissolve
thrombi.
DNA Fingerprinting
The technique of DNA fingerprinting
is based in part on those techniques used in recombinant DNA technology and on
those originally used in medical genetics to detect slight variations in the
genomes of different individuals. Using restriction enzymes, DNA is cleaved at
specific regions (Fig. 6.12). The DNA fragments are separated according to size
by electrophoresis and denatured (by heating or treating chemically) so that
all the DNA is single stranded. The single-stranded DNA is then transferred to
nitrocellulose paper, baked to attach the DNA to the paper, and treated with
series of radioactive probes. After the radioactive probes have been allowed to
bond with the denatured DNA, radiography is used to reveal the labeled DNA
fragments.
When used in forensic pathology,
this procedure is applied to specimens from the suspect and the forensic specimen.
Banding patterns are then analyzed to see if they match. With conventional
methods of analysis of blood and serum enzymes, a 1 in 100 to 1000 chance
exists that the two specimens match because of chance. With DNA fingerprinting,
these odds are 1 in 100,000 to 1 million.
When necessary, the polymerase
chain reaction (PCR) can be used to amplify specific segments of DNA. It is
particularly suited for amplifying regions of DNA for clinical and forensic
testing procedures because only a small sample of DNA is required as the
starting material. Regions of DNA can be amplified from a single hair or drop
of blood or saliva.
Gene Therapy
Although quite different from
inserting genetic material into a unicellular organism such as bacteria,
techniques are avail- able for inserting genes into the genome of intact
multicellular plants and animals. Promising delivery vehicles for these genes
are the adenoviruses. These viruses are ideal vehicles because their DNA does
not become integrated into the host genome. However, repeated inoculations are
often needed because the body’s immune system usually targets cells expressing adenovirus
proteins. Sterically stable liposomes also show promise as DNA delivery
mechanisms. This type of therapy is one of the more promising methods for the
treatment of genetic disorders such as cystic fibrosis, certain cancers, and
many infectious diseases.
Two main approaches are used in
gene therapy: transferred genes can replace defective genes or they can selectively
inhibit deleterious genes. Cloned DNA sequences are usually the compounds used
in gene therapy. However, the introduction of the cloned gene into the
multicellular organism can influence only the few cells that get the gene. An
answer to this problem would be the insertion of the gene into a sperm or ovum;
after fertilization, the gene would be replicated in all of the differentiating
cell types. Even so, techniques for cell insertion are limited. Not only are
moral and ethical issues involved,
but these techniques cannot direct the inserted DNA to
attach to a particular chromosome or supplant an existing gene by knocking it out of its place.
To date, gene therapy has been used
successfully to treat children with severe combined immunodeficiency disease,
and in a suicide gene transfer to facilitate treatment of graft-versus-host disease after
donor lymphocyte infusion.
