DNA Synthesis

Background

Deoxyribonucleic acid (DNA) synthesis is a process by which copies of nucleic acid strands are made. In nature, DNA synthesis takes place in cells by a mechanism known as DNA replication. Using genetic engineering and enzyme chemistry, scientists have developed man-made methods for synthesizing DNA. The most important of these is poly-merase chain reaction (PCR). First developed in the early 1980s, PCR has become a multi-billion dollar industry with the original patent being sold for $300 million dollars.

History

DNA was discovered in 1951 by Francis Crick, James Watson, and Maurice Wilkins. Using x-ray crystallography data generated by Rosalind Franklin, Watson and Crick determined that the structure of DNA was that of a double helix. For this work, Watson, Crick, and Wilkins received the Nobel Prize in Physiology or Medicine in 1962. Over the years, scientists worked with DNA trying to figure out the "code of life." They found that DNA served as the instruction code for protein sequences. They also found that every organism has a unique DNA sequence and it could be used for screening, diagnostic, and identification purposes. One thing that proved limiting in these studies was the amount of DNA available from a single source.

After the nature of DNA was determined, scientists were able to examine the composition of the cellular genes. A gene is a specific sequence of DNA base pairs that provide the code for the construction of a protein. These proteins determine the traits of an organism, such as eye color or blood type. When a certain gene was isolated, it became desirable to synthesize copies of that molecule. One of the first ways in which a large amount of a specific DNA was synthesized was though genetic engineering.

Genetic engineering begins by combining a gene of interest with a bacterial plasmid. A plasmid is a small stretch of DNA that is found in many bacteria. The resulting hybrid DNA is called recombinant DNA. This new recombinant DNA plasmid is then injected into bacterial cells. The cells are then cloned by allowing it to grow and multiply in a culture. As the cells multiply so do copies of the inserted gene. When the bacteria has multiplied enough, the multiple copies of the inserted gene can then be isolated. This method of DNA synthesis can produce billions of copies of a gene in a couple of weeks.

In 1983, the time required to produce copies of DNA was significantly reduced when Kary Mullis developed a process for synthesizing DNA called polymerase chain reaction (PCR). This method is much faster than previous known methods producing billions of copies of a DNA strand in just a few hours. It begins by putting a small section of double stranded DNA in a solution containing DNA polymerase, nucleotides and primers. The solution is heated to separate the DNA strands. When it is cooled, the polymerase creates a copy of each strand. The process is repeated every five minutes until the desired amount of DNA is produced. In 1993, Mullis's development of PCR earned him the Nobel Prize in Chemistry. Today, PCR has revolutionized the fields of medical diagnostics, forensics, and microbiology. It is said to be one of the most important developments in genetic research.

Background

The key to understanding DNA synthesis is understanding its structure. DNA is a long chain polymer made up of chemical units called nucleotides. Also known as genetic material, DNA is the molecule that carries information that dictates protein synthesis in most living organisms. Typically, DNA exists as two chains of chemically linked nucleotides. These links follow specific patterns dictated by the base pairing rules. Each nucleotide is made up of a deoxyribose sugar molecule, a phosphate group, and one of four nitrogen containing bases. The bases include the pyrimidines thymine (T) and cytosine (C)and the purines adenine (A) and guanine (G). In DNA, adenine generally links with thymine and guanine with cytosine. The molecule is arranged in a structure called a double helix which can be imagined by picturing a twisted ladder or spiral staircase. The bases make up the rungs of the ladder while the sugar and phosphate portions make up the ladder sides. The order in which the nucleotides are linked, called the sequence, is determined by a process known as DNA sequencing.

In a eukaryotic cell, DNA synthesis occurs just prior to cell division through a process called replication. When replication begins the two strands of DNA are separated by a variety of enzymes. Thus opened, each strand serves as a template for producing new strands. This whole process is catalyzed by an enzyme called DNA polymerase. This molecule brings corresponding, or complementary, nucleotides in line with each of the DNA strands. The nucleotides are then chemically linked to form new DNA strands which are exact copies of the original strand. These copies, called the daughter strands, contain half of the parent DNA molecule and half of a whole new molecule. Replication by this method is known as semiconservative replication. The process of replication is important because it provides a method for cells to transfer an exact duplicate of their genetic material from one generation of cell to the next.

Raw Materials

The primary raw materials used for DNA synthesis include DNA starting materials, taq DNA polymerase, primers, nucleotides, and the buffer solution. Each of these play an important role in the production of millions of DNA molecules.

Controlled DNA synthesis begins by identifying a small segment of DNA to copy. This is typically a specific sequence of DNA that contains the code for a desired protein. Called template DNA, this material is needed in concentrations of about 0.1-1 micro-grams. It must be highly purified because even trace amounts of the compounds used in DNA purification can inhibit the PCR process. One method for purifying a DNA strand is treating it with 70% ethanol.

While the process of DNA replication was know before 1980, PCR was not possible because there were no known heat stable DNA polymerases. DNA polymerase is the enzyme that catalyzes the reactions involved in DNA synthesis. In the early 1980s, scientists found bacteria living around natural steam vents. It turned out that these organisms, called thermus aquaticus, had a DNA polymerase that was stable and functional at extreme levels of heat. This taq DNA polymerase became the cornerstone for modern DNA synthesis techniques. During a typical PCR process, 2-3 micrograms of taq DNA polymerase is needed. If too much is used however, unwanted, nonspecific DNA sequences can result.

The polymerase builds the DNA strands by combining corresponding nucleotides on each DNA strand. Chemically speaking, nucleotides are made up of three types of molecular groups including a sugar structure, a phosphate group, and a cyclic base. The sugar portion provides the primary structure for all nucleotides. In general, the sugars are composed of five carbon atoms with a number of hydroxy (-OH) groups attached. For DNA, the sugar is 2-deoxy-D-ribose. The defining part of a nucleotide is the hetero-cyclic base that is covalently bound to the sugar. These bases are either pyrimidine or purine groups, and they form the basis for the nucleic acid code. Two types of purine bases are found including adenine and guanine. In DNA, two types of pyrimidine bases are present, thymine and cytosine. A phosphate group makes up the final portion of a nucleotide. This group is derived from phosphoric acid and is covalently bonded to the sugar structure on the fifth carbon.

The first phase of polymerase chain reaction (PCR) involves the denaturation of DNA. This "opening up" of the DNA molecule provides the template for the next DNA molecule from which to be produced. With the DNA split into separate strands, the temperature is lowered—the primer annealing step. During the next phase, the DNA polymerase interacts with the strands and adds complementary nucleotides along the entire length. The time required at this phase is about one minute for every 1,000 base pairs.

To initiate DNA synthesis, short primer sections of DNA must be used. These primer sections, called oligo fragments, are about 18-25 nucleotides in length and correspond to a section on the template DNA. They typically have a C and G nucleotide concentration of about 60% with even distribution. This provides the maximum efficiency in the synthesis process.

The buffer solution provides the medium in which DNA synthesis can occur. This is an aqueous solution which contains MgCl2, HCI, EDTA, and KCI. The MgCl2 concentration is important because the Mg2+ ions interact with the DNA and the primers creating crucial complexes for DNA synthesis. The recommended concentration is one to four micromoles. The pH of this system is critical so it may also be buffered with ammonium sulfate. To energize the reaction, various energy molecules are added such as ATP, GTP, and NTP. These compounds are the same ones that living organisms use to power metabolic reactions.

Other materials that may be used in the process include mineral oil or paraffin wax. After DNA synthesis is complete, the DNA is typically isolated and purified. Some common reagents used in this process include phenol, EDTA and Proteinase K.

The Manufacturing Process

DNA synthesis is typically done on a small scale in laboratories. It involves three distinct processes including sample preparation, DNA synthesis reaction cycle and DNA isolation. These manufacturing steps are typically done in separate areas to avoid contamination. Following these procedures scientists are able to convert a few strands of DNA into millions and millions of exact copies.

Preparation of the samples

• 1 To begin DNA synthesis, the various solutions are prepared. This is typically done in a laminar flow cabinet equipped with a UV lamp to minimize contamination. Scientists use fresh gloves during each production step for similar reasons. Typically, all of the starting solutions except the primers, polymerases and the dNTPs are put in an autoclave to kill off any contaminating organism. Two separate solutions are made. One contains the buffer, primers and the polymerase. The other contains the MgCl2 and the template DNA. These solutions are all put into small tubes to begin the reaction.

Kary Banks Muilis.

Kary Banks Muilis was born in Lenoir, North Carolina, in 1944. Upon graduation from Georgia Tech in 1966 with a B.S. in chemistry, Muilis entered the biochemistry doctoral program at the University of California, Berkeley. Earning his Ph.D. in 1973, he accepted a teaching position at the University of Kansas Medical School in Kansas City. In 1977, he assumed a postdoctoral fellowship at the University of California, San Francisco.

Muilis accepted a position as a research scientist in 1979 with a growing biotech firm—Cetus Corporation, in Emeryville, California—that synthesized chemicals used by other scientists in genetic cloning. While there, he designed polymerase chain reaction (PCR), a fast and effective technique for reproducing specific genes or DNA (deoxyribonucleic acid) fragments that can create billions of copies in a few hours. The most effective way to reproduce DNA was by cloning, but it was problematic. It took time to convince Mullis's colleagues of the importance of this discovery but soon PCR became the focus of intensive research. Scientists at Cetus developed a commercial version of the process and a machine called the Thermal Cycler (with the addition of the chemical building blocks of DNA [nucleotides] and a biochemical catalyst [polymerase], the machine would perform the process automatically on a target piece of DNA).

Cetus awarded Muilis $10,000 for developing the PCR patent, then sold it for $300 million. Leaving Cetus in 1986, Muilis became a private biochemical research consultant and was awarded the Nobel Prize in 1993.

DNA synthesis cycle

• 2 After the reacting solutions are prepared, the PCR cycle is started. The first phase involves the denaturation of DNA. One of the most important initial steps is the complete denaturation of the DNA template. Denaturation of the DNA essentially means breaking apart of the double bonded strand. This "opening up" of the DNA molecule provides the template for the next DNA molecule from which to be produced. An incomplete denaturation will result in an inefficient copy in the first cycle which negatively impacts each subsequent cycle. The initial denaturation is done by heating up the DNA template solution to 203°F (95°C) over one to three minutes. The total time depends on the template composition. In repeat cycles, the denaturation step lasts about two minutes and involves heating the solution to 201PF (94°C). Additional materials may be added to the solution to facilitate DNA denaturation such as glycerol, DMSO, or formamide.
• 3 With the DNA split into separate strands, the temperature is lowered to 122-149°F (50-65°C). This is known as the primer annealing step and lasts for about two minutes. At this point, the left and right primers match up and chemically link with their complementary bases on the template DNA.
• 4 The next phase involves the extending step. This part of the reaction is when most of the DNA strand gets copied. The temperature of the system is heated to about 162°F(72°C) and held there depending on the length of DNA to copy. At this stage, the DNA polymerase interacts with the strands and adds complementary nucleotides along the entire length. The time required at this phase is about one minute for every 1,000 base pairs.
• 5 After this first cycle, the DNA synthesis cycle is repeated. The number of cycles depends of the amount of initial DNA and the amount of DNA desired. If less than 10 copies of the template DNA are available, 40 cycles are needed. With more initial DNA, 25-30 cycles is sufficient.
• 6 During the last cycle the sample is held at 162°F (72°C) for about 15 minutes. This allows the filling in (with nucleotides) of any protruding ends of a new DNA strand. At this stage, the polymerase adds extra A nucleotides on one end of the DNA strands.

DNA isolation

• 7 When the reactions are complete, the DNA is isolated from the PCR reacting materials such as the DNA polymerase, MgCl2 and the primers. This is done by adding compounds like phenol, EDTA and Proteinase K. Centrifugation is also helpful in this regard.

The Future

While scientists use PCR for DNA synthesis on a regular basis, there is still much that is not understood about DNA replication. In the future, research should elucidate the details of several important steps of the process, such as the components and intermediates involved. Additionally, improved polymerases may be developed, making it possible to create more DNA from smaller starting samples. It is hoped that one day DNA synthesis will help unlock some of the key aspects of living organisms and lead to the development medicines that will cure various cancers, viral and bacterial infections.