The nucleus of a cell contains the chromosomal DNA for an organism. Since each cell in an organism must have a complete compliment of DNA, all of the DNA in the nucleus must be replicated before a cell divides. Because the organization of chromosomal DNA is complicated, description of the cellular replication process may be unnecessarily complicated. Fortunately, for the purposes of biotechnological applications, the process of DNA replication has has been automated and can be done on a laboratory bench top.
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The process of bench top DNA replication is referred to as “Polymerase chain reaction”, or PCR. PCR has revolutionized not only the biotechnology industry, but related industries such as forensics. A small amount of DNA, potentially less than a thousandth of a gram, can be duplicated, or amplified, into a quantity that can be used for DNA fingerprinting, or unique identification. Here I will describe some of the basic principles of bench top DNA replication, or PCR.
In broad terms, The DNA will be cycled through multiple series of steps of first breaking segments of double stranded ( helical ) DNA (dsDNA) into to single stranded DNA (ssDNA) chains. The two strands of the DNA double helix are held together by “hydrogen bonds”, or bonds that can be easily broken with heat. Hydrogen bonds are what gives motor oil its viscosity, and for example if motor oil is warmed up it becomes less viscous. Likewise, warming of DNA above a given temperature will cause the double stranded DNA to break into two strands of single stranded DNA. At this point, each single strand can be duplicated in a process mediated by an enzyme known as “DNA polymerase”, hence the name polymerase in the acronym for PCR. Once polymerization step has taken place, which takes place at low temperature, the mixture can be heated again to separate the newly created double stranded DNA. Thus the cycle of heating and cooling is repeated as many times as is necessary to create the amount of DNA desired. For now obvious reasons, the machine responsible for mediating this process in a bench top is called a “thermo cycler”, and is a common piece of equipment in every molecular biology laboratory.
Now I will talk in a little bit more detail about the actual replication step in the PCR cycle. First of all, the DNA polymerase used is from an aquatic microorganism found in hot springs. A unique characteristic of this organism’s DNA polymerase is that it has evolved the ability to withstand the high temperatures commonly found in hot springs, and also characteristic of the environment inside a thermocycler. The scientific name of the organism is thermos aquas, abbreviated taq. The taq polymerase that is added to the DNA to be expanded cannot start replicating DNA by itself. Taq polymerase must start from a double stranded DNA, and then extend the second strand using the first strand as a template. For that reason, “primers” must be designed to amplify a specific region. A primer usually consists of a strand of DNA about 25 -35 nucleotides long that is complementary to one end of the region of DNA that one wants to expand. By designing primers carefully, one can select a particular region of DNA in the template sequence to copy. Typically, computer programs are used to assist in the designing of a pair of primers in order to assure that they both have the same annealing temperature. The annealing temperature of a primer is the temperature at which a single stranded primer will bind with it’s complementary region on the DNA template.
Once the dsDNA has been heated and separated to ssDNA, the primers have annealed, or bound to their complementary regions on the DNA, and the taq polymerase has bound and is ready to extend the primer to create dsDNA, there is one more ingredient to the mixture and that is free nucleotides. The nucleotides, usually represented by the first letters of their chemical names, C,G,T,and A are added to the mixture. Once all components are in a sample to be amplified, a program will be executed automatically by the thermocycler, and the end product, a large amount of the DNA region of interest, will be produced automatically.
In the cell, the DNA replication process parallels the process described in the thermocycler. Instead of separating the strands of dsDNA using heat, a “replication fork” is initiated using a series of enzymes to separate the strands of the DNA. Likewise, the primers in the cell are RNA sequences instead of the designed primers used in PCR. In bacterial cells, known as prokaryotic cells, the DNA is organized ino rings called plasmids. Each plasmid can replicate independently. In higher plant and animal cells with well formed nuclear structures called chromosomes, DNA replication is carefully regulated by sophisticated cellular machinery so that each daughter cell of cell division gets exactly one copy of DNA.
