New opportunities
Despite all the changes, pharmaceutical companies are maintaining a strong internal development program in areas with large markets such as oncology, neuroscience, and diabetes/obesity; and they are hiring people whose skills fit with their drug-development programs. Furthermore, the shifts in the industry may herald a new, more fluid division of labor where nontraditional partnerships take on the earliest stages of drug development. "I think it may be up to smaller biotech companies and academia to come up with new drugs," Littman says. "And smaller biotech companies are very interested in generating biomarkers, working on proof of concept, and testing in smaller patient populations."
It's a modular approach to drug discovery and translational research that both FitzGerald and others believe will become prevalent. They also think this will necessitate changes in the way industry and academia handle things such as intellectual property. People choosing industry careers should be prepared and adaptable.
"I think there is a lot of uncertainty out there in the world right now," says Will West, chief executive at CellCentric, a biotechnology company in Cambridge, United Kingdom. "If you are a young graduate, I can see how basing a career in industry may seem like a difficult choice to make. I see the opposite. I think in a changing model, there is opportunity for bright people to take advantage of that change and be drivers for the solutions.
Saturday, March 27, 2010
Sunday, March 7, 2010
Recombinant DNA Technology Has Revolutionized All Aspects of Biology
recombinant DNA technology, which has permitted biology to move from an exclusively analytical science to a
synthetic one. New combinations of unrelated genes can be constructed in the laboratory by applying recombinant DNA
techniques. These novel combinations can be cloned amplified manyfold by introducing them into suitable cells,
where they are replicated by the DNA-synthesizing machinery of the host. The inserted genes are often transcribed and
translated in their new setting. What is most striking is that the genetic endowment of the host can be permanently
altered in a designed way.
Restriction Enzymes and DNA Ligase Are Key Tools in Forming Recombinant
DNA Molecules
Let us begin by seeing how novel DNA molecules can be constructed in the laboratory. A DNA fragment of interest is
covalently joined to a DNA vector. The essential feature of a vector is that it can replicate autonomously in an
appropriate host. Plasmids (naturally occurring circles of DNA that act as accessory chromosomes in bacteria) and
bacteriophage l , a virus, are choice vectors for cloning in E. coli. The vector can be prepared for accepting a new DNA
fragment by cleaving it at a single specific site with a restriction enzyme. For example, the plasmid pSC101, a 9.9-kb
double-helical circular DNA molecule, is split at a unique site by the EcoRI restriction enzyme. The staggered cuts made
by this enzyme produce complementary single-stranded ends, which have specific affinity for each other and hence are
known as cohesive or sticky ends. Any DNA fragment can be inserted into this plasmid if it has the same cohesive ends.
Such a fragment can be prepared from a larger piece of DNA by using the same restriction enzyme as was used to open
the plasmid DNA
The single-stranded ends of the fragment are then complementary to those of the cut plasmid. The DNA fragment and
the cut plasmid can be annealed and then joined by DNA ligase, which catalyzes the formation of a phosphodiester bond
at a break in a DNA chain. DNA ligase requires a free 3 -hydroxyl group and a 5 -phosphoryl group. Furthermore, the
chains joined by ligase must be in a double helix. An energy source such as ATP or NAD+ is required for the joining
reaction,
This cohesive-end method for joining DNA molecules can be made general by using a short, chemically synthesized
DNA linker that can be cleaved by restriction enzymes. First, the linker is covalently joined to the ends of a DNA
fragment or vector. For example, the 5 ends of a decameric linker and a DNA molecule are phosphorylated by
polynucleotide kinase and then joined by the ligase from T4 phage . This ligase can form a covalent bond
between blunt-ended (flush-ended) double-helical DNA molecules. Cohesive ends are produced when these terminal
extensions are cut by an appropriate restriction enzyme. Thus, cohesive ends corresponding to a particular restriction
enzyme can be added to virtually any DNA molecule. We see here the fruits of combining enzymatic and synthetic chemical approaches in crafting new DNA molecules.
synthetic one. New combinations of unrelated genes can be constructed in the laboratory by applying recombinant DNA
techniques. These novel combinations can be cloned amplified manyfold by introducing them into suitable cells,
where they are replicated by the DNA-synthesizing machinery of the host. The inserted genes are often transcribed and
translated in their new setting. What is most striking is that the genetic endowment of the host can be permanently
altered in a designed way.
Restriction Enzymes and DNA Ligase Are Key Tools in Forming Recombinant
DNA Molecules
Let us begin by seeing how novel DNA molecules can be constructed in the laboratory. A DNA fragment of interest is
covalently joined to a DNA vector. The essential feature of a vector is that it can replicate autonomously in an
appropriate host. Plasmids (naturally occurring circles of DNA that act as accessory chromosomes in bacteria) and
bacteriophage l , a virus, are choice vectors for cloning in E. coli. The vector can be prepared for accepting a new DNA
fragment by cleaving it at a single specific site with a restriction enzyme. For example, the plasmid pSC101, a 9.9-kb
double-helical circular DNA molecule, is split at a unique site by the EcoRI restriction enzyme. The staggered cuts made
by this enzyme produce complementary single-stranded ends, which have specific affinity for each other and hence are
known as cohesive or sticky ends. Any DNA fragment can be inserted into this plasmid if it has the same cohesive ends.
Such a fragment can be prepared from a larger piece of DNA by using the same restriction enzyme as was used to open
the plasmid DNA
The single-stranded ends of the fragment are then complementary to those of the cut plasmid. The DNA fragment and
the cut plasmid can be annealed and then joined by DNA ligase, which catalyzes the formation of a phosphodiester bond
at a break in a DNA chain. DNA ligase requires a free 3 -hydroxyl group and a 5 -phosphoryl group. Furthermore, the
chains joined by ligase must be in a double helix. An energy source such as ATP or NAD+ is required for the joining
reaction,
This cohesive-end method for joining DNA molecules can be made general by using a short, chemically synthesized
DNA linker that can be cleaved by restriction enzymes. First, the linker is covalently joined to the ends of a DNA
fragment or vector. For example, the 5 ends of a decameric linker and a DNA molecule are phosphorylated by
polynucleotide kinase and then joined by the ligase from T4 phage . This ligase can form a covalent bond
between blunt-ended (flush-ended) double-helical DNA molecules. Cohesive ends are produced when these terminal
extensions are cut by an appropriate restriction enzyme. Thus, cohesive ends corresponding to a particular restriction
enzyme can be added to virtually any DNA molecule. We see here the fruits of combining enzymatic and synthetic chemical approaches in crafting new DNA molecules.
PCR Is a Powerful Technique in Medical Diagnostics, Forensics, and Molecular
In 1984, Kary Mullis devised an ingenious method called the polymerase chain reaction (PCR) for amplifying specific
DNA sequences. Consider a DNA duplex consisting of a target sequence surrounded by nontarget DNA. Millions of the
target sequences can be readily obtained by PCR if the flanking sequences of the target are known. PCR is carried out by
adding the following components to a solution containing the target sequence: (1) a pair of primers that hybridize with
the flanking sequences of the target, (2) all four deoxyribonucleoside triphosphates (dNTPs), and (3) a heat-stable DNA
polymerase. A PCR cycle consists of three steps (Figure 6.8).
1. Strand separation. The two strands of the parent DNA molecule are separated by heating the solution to 95°C for 15 s.
2. Hybridization of primers. The solution is then abruptly cooled to 54°C to allow each primer to hybridize to a DNA
strand. One primer hybridizes to the 3 -end of the target on one strand, and the other primer hybridizes to the 3 end on
the complementary target strand. Parent DNA duplexes do not form, because the primers are present in large excess.
Primers are typically from 20 to 30 nucleotides long.
3. DNA synthesis. The solution is then heated to 72°C, the optimal temperature for Taq DNA polymerase. This heatstable
polymerase comes from T hermus aq uaticus, a thermophilic bacterium that lives in hot springs. The polymerase
elongates both primers in the direction of the target sequence because DNA synthesis is in the 5 -to-3 direction. DNA
synthesis takes place on both strands but extends beyond the target sequence.
These three steps strand separation, hybridization of primers, and DNA synthesis constitute one cycle of the PCR
amplification and can be carried out repetitively just by changing the temperature of the reaction mixture. The
thermostability of the polymerase makes it feasible to carry out PCR in a closed container; no reagents are added after
the first cycle. The duplexes are heated to begin the second cycle, which produces four duplexes, and then the third cycle
is initiated . At the end of the third cycle, two short strands appear that constitute only the target
sequence the sequence including and bounded by the primers. Subsequent cycles will amplify the target sequence
exponentially. The larger strands increase in number arithmetically and serve as a source for the synthesis of more short
strands. Ideally, after n cycles, this sequence is amplified 2 n -fold. The amplification is a millionfold after 20 cycles and
a billionfold after 30 cycles, which can be carried out in less than an hour.
Several features of this remarkable method for amplifying DNA are noteworthy. First, the sequence of the target need
not be known. All that is required is knowledge of the flanking sequences. Second, the target can be much larger than the
primers. Targets larger than 10 kb have been amplified by PCR. Third, primers do not have to be perfectly matched to
flanking sequences to amplify targets. With the use of primers derived from a gene of known sequence, it is possible to
search for variations on the theme. In this way, families of genes are being discovered by PCR. Fourth, PCR is highly
specific because of the stringency of hybridization at high temperature (54°C). Stringency is the required closeness of the
match between primer and target, which can be controlled by temperature and salt. At high temperatures, the only DNA
that is amplified is that situated between primers that have hybridized. A gene constituting less than a millionth of the
total DNA of a higher organism is accessible by PCR. Fifth, PCR is exquisitely sensitive. A single DNA molecule can be
amplified and detected.
PCR can provide valuable diagnostic information in medicine. Bacteria and viruses can be readily detected with the use
of specific primers. For example, PCR can reveal the presence of human immunodeficiency virus in people who have
not mounted an immune response to this pathogen and would therefore be missed with an antibody assay. Finding
Mycobacterium tuberculosis bacilli in tissue specimens is slow and laborious. With PCR, as few as 10 tubercle bacilli
per million human cells can be readily detected. PCR is a promising method for the early detection of certain cancers.
This technique can identify mutations of certain growth-control genes, such as the ras genes (Section 15.4.2). The
capacity to greatly amplify selected regions of DNA can also be highly informative in monitoring cancer chemotherapy.
Tests using PCR can detect when cancerous cells have been eliminated and treatment can be stopped; they can also
detect a relapse and the need to immediately resume treatment. PCR is ideal for detecting leukemias caused by
chromosomal rearrangements.
PCR is also having an effect in forensics and legal medicine. An individual DNA profile is highly distinctive because
many genetic loci are highly variable within a population. For example, variations at a specific one of these locations
determines a person's HLA type (human leukocyte antigen type); organ transplants are rejected when the HLA types of
the donor and recipient are not sufficiently matched. PCR amplification of multiple genes is being used to establish
biological parentage in disputed paternity and immigration cases. Analyses of blood stains and semen samples by PCR
have implicated guilt or innocence in numerous assault and rape cases. The root of a single shed hair found at a crime
scene contains enough DNA for typing by PCR
DNA is a remarkably stable molecule, particularly when relatively shielded from air, light, and water. Under such
circumstances, large fragments of DNA can remain intact for thousands of years or longer. PCR provides an ideal
method for amplifying such ancient DNA molecules so that they can be detected and characterized (Section 7.5.1). PCR
can also be used to amplify DNA from microorganisms that have not yet been isolated and cultured. As will be discussed
in the next chapter, sequences from these PCR products can be sources of considerable insight into evolutionary
relationships between organisms.
DNA sequences. Consider a DNA duplex consisting of a target sequence surrounded by nontarget DNA. Millions of the
target sequences can be readily obtained by PCR if the flanking sequences of the target are known. PCR is carried out by
adding the following components to a solution containing the target sequence: (1) a pair of primers that hybridize with
the flanking sequences of the target, (2) all four deoxyribonucleoside triphosphates (dNTPs), and (3) a heat-stable DNA
polymerase. A PCR cycle consists of three steps (Figure 6.8).
1. Strand separation. The two strands of the parent DNA molecule are separated by heating the solution to 95°C for 15 s.
2. Hybridization of primers. The solution is then abruptly cooled to 54°C to allow each primer to hybridize to a DNA
strand. One primer hybridizes to the 3 -end of the target on one strand, and the other primer hybridizes to the 3 end on
the complementary target strand. Parent DNA duplexes do not form, because the primers are present in large excess.
Primers are typically from 20 to 30 nucleotides long.
3. DNA synthesis. The solution is then heated to 72°C, the optimal temperature for Taq DNA polymerase. This heatstable
polymerase comes from T hermus aq uaticus, a thermophilic bacterium that lives in hot springs. The polymerase
elongates both primers in the direction of the target sequence because DNA synthesis is in the 5 -to-3 direction. DNA
synthesis takes place on both strands but extends beyond the target sequence.
These three steps strand separation, hybridization of primers, and DNA synthesis constitute one cycle of the PCR
amplification and can be carried out repetitively just by changing the temperature of the reaction mixture. The
thermostability of the polymerase makes it feasible to carry out PCR in a closed container; no reagents are added after
the first cycle. The duplexes are heated to begin the second cycle, which produces four duplexes, and then the third cycle
is initiated . At the end of the third cycle, two short strands appear that constitute only the target
sequence the sequence including and bounded by the primers. Subsequent cycles will amplify the target sequence
exponentially. The larger strands increase in number arithmetically and serve as a source for the synthesis of more short
strands. Ideally, after n cycles, this sequence is amplified 2 n -fold. The amplification is a millionfold after 20 cycles and
a billionfold after 30 cycles, which can be carried out in less than an hour.
Several features of this remarkable method for amplifying DNA are noteworthy. First, the sequence of the target need
not be known. All that is required is knowledge of the flanking sequences. Second, the target can be much larger than the
primers. Targets larger than 10 kb have been amplified by PCR. Third, primers do not have to be perfectly matched to
flanking sequences to amplify targets. With the use of primers derived from a gene of known sequence, it is possible to
search for variations on the theme. In this way, families of genes are being discovered by PCR. Fourth, PCR is highly
specific because of the stringency of hybridization at high temperature (54°C). Stringency is the required closeness of the
match between primer and target, which can be controlled by temperature and salt. At high temperatures, the only DNA
that is amplified is that situated between primers that have hybridized. A gene constituting less than a millionth of the
total DNA of a higher organism is accessible by PCR. Fifth, PCR is exquisitely sensitive. A single DNA molecule can be
amplified and detected.
PCR can provide valuable diagnostic information in medicine. Bacteria and viruses can be readily detected with the use
of specific primers. For example, PCR can reveal the presence of human immunodeficiency virus in people who have
not mounted an immune response to this pathogen and would therefore be missed with an antibody assay. Finding
Mycobacterium tuberculosis bacilli in tissue specimens is slow and laborious. With PCR, as few as 10 tubercle bacilli
per million human cells can be readily detected. PCR is a promising method for the early detection of certain cancers.
This technique can identify mutations of certain growth-control genes, such as the ras genes (Section 15.4.2). The
capacity to greatly amplify selected regions of DNA can also be highly informative in monitoring cancer chemotherapy.
Tests using PCR can detect when cancerous cells have been eliminated and treatment can be stopped; they can also
detect a relapse and the need to immediately resume treatment. PCR is ideal for detecting leukemias caused by
chromosomal rearrangements.
PCR is also having an effect in forensics and legal medicine. An individual DNA profile is highly distinctive because
many genetic loci are highly variable within a population. For example, variations at a specific one of these locations
determines a person's HLA type (human leukocyte antigen type); organ transplants are rejected when the HLA types of
the donor and recipient are not sufficiently matched. PCR amplification of multiple genes is being used to establish
biological parentage in disputed paternity and immigration cases. Analyses of blood stains and semen samples by PCR
have implicated guilt or innocence in numerous assault and rape cases. The root of a single shed hair found at a crime
scene contains enough DNA for typing by PCR
DNA is a remarkably stable molecule, particularly when relatively shielded from air, light, and water. Under such
circumstances, large fragments of DNA can remain intact for thousands of years or longer. PCR provides an ideal
method for amplifying such ancient DNA molecules so that they can be detected and characterized (Section 7.5.1). PCR
can also be used to amplify DNA from microorganisms that have not yet been isolated and cultured. As will be discussed
in the next chapter, sequences from these PCR products can be sources of considerable insight into evolutionary
relationships between organisms.
Exploring Genes
Recombinant DNA technology has revolutionized biochemistry since it came into being in the 1970s. The genetic
endowment of organisms can now be precisely changed in designed ways. Recombinant DNA technology is a fruit of
several decades of basic research on DNA, RNA, and viruses. It depends, first, on having enzymes that can cut, join, and
replicate DNA and reverse transcribe RNA. Restriction enzymes cut very long DNA molecules into specific fragments
that can be manipulated; DNA ligases join the fragments together. The availability of many kinds of restriction enzymes
and DNA ligases makes it feasible to treat DNA sequences as modules that can be moved at will from one DNA
molecule to another. Thus, recombinant DNA technology is based on nucleic acid enzymology.
A second foundation is the base-pairing language that allows complementary sequences to recognize and bind to each
other. Hybridization with complementary DNA or RNA probes is a sensitive and powerful means of detecting specific
nucleotide sequences. In recombinant DNA technology, base-pairing is used to construct new combinations of DNA as
well as to detect and amplify particular sequences. This revolutionary technology is also critically dependent on our
understanding of viruses, the ultimate parasites. 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. Likewise, plasmids, which are accessory chromosomes found in bacteria, have been indispensable in
recombinant DNA technology.
These new methods have wide-ranging benefits. Entire genomes, including the human genome, are being deciphered.
New insights are emerging, for example, into the regulation of gene expression in cancer and development and the
evolutionary history of proteins as well as organisms. New proteins can be created by altering genes in specific ways to
provide detailed views into protein function. Clinically useful proteins, such as hormones, are now synthesized by
recombinant DNA techniques. Crops are being generated to resist pests and harsh conditions. The new opportunities
opened by recombinant DNA technology promise to have broad effects.
The Basic Tools of Gene Exploration
The rapid progress in biotechnology indeed its very existence is a result of a relatively few techniques.
1. Restriction-enzyme analysis. Restriction enzymes are precise, molecular scalpels that allow the investigator to
manipulate DNA segments.
2. Blotting techniques. The 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 Section 4.3.4.
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 structure.
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 acids.
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 is being used to detect pathogens and genetic diseases, to determine the source of a hair left at the
scene of a crime, and to resurrect genes from fossils.
A final tool, the use of which will be highlighted in the next chapter, is the computer. Without the computer, it would be
impossible to catalog, access, and characterize the abundant information, especially DNA sequence information, that the techniques just outlined are rapidly generating.
endowment of organisms can now be precisely changed in designed ways. Recombinant DNA technology is a fruit of
several decades of basic research on DNA, RNA, and viruses. It depends, first, on having enzymes that can cut, join, and
replicate DNA and reverse transcribe RNA. Restriction enzymes cut very long DNA molecules into specific fragments
that can be manipulated; DNA ligases join the fragments together. The availability of many kinds of restriction enzymes
and DNA ligases makes it feasible to treat DNA sequences as modules that can be moved at will from one DNA
molecule to another. Thus, recombinant DNA technology is based on nucleic acid enzymology.
A second foundation is the base-pairing language that allows complementary sequences to recognize and bind to each
other. Hybridization with complementary DNA or RNA probes is a sensitive and powerful means of detecting specific
nucleotide sequences. In recombinant DNA technology, base-pairing is used to construct new combinations of DNA as
well as to detect and amplify particular sequences. This revolutionary technology is also critically dependent on our
understanding of viruses, the ultimate parasites. 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. Likewise, plasmids, which are accessory chromosomes found in bacteria, have been indispensable in
recombinant DNA technology.
These new methods have wide-ranging benefits. Entire genomes, including the human genome, are being deciphered.
New insights are emerging, for example, into the regulation of gene expression in cancer and development and the
evolutionary history of proteins as well as organisms. New proteins can be created by altering genes in specific ways to
provide detailed views into protein function. Clinically useful proteins, such as hormones, are now synthesized by
recombinant DNA techniques. Crops are being generated to resist pests and harsh conditions. The new opportunities
opened by recombinant DNA technology promise to have broad effects.
The Basic Tools of Gene Exploration
The rapid progress in biotechnology indeed its very existence is a result of a relatively few techniques.
1. Restriction-enzyme analysis. Restriction enzymes are precise, molecular scalpels that allow the investigator to
manipulate DNA segments.
2. Blotting techniques. The 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 Section 4.3.4.
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 structure.
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 acids.
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 is being used to detect pathogens and genetic diseases, to determine the source of a hair left at the
scene of a crime, and to resurrect genes from fossils.
A final tool, the use of which will be highlighted in the next chapter, is the computer. Without the computer, it would be
impossible to catalog, access, and characterize the abundant information, especially DNA sequence information, that the techniques just outlined are rapidly generating.
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