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 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 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.
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
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.
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.
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.