Genetic engineering requires the combination of a number of different molecular genetics techniques. In order to work with genes, it is useful to know their DNA sequences. We've had the ability to sequence DNA for many years. In fact, the first whole genome was sequenced in 1977. However, it has taken some improvements to the process, and automation (developed in the late 80s - early 90s) to make it fast and inexpensive enough for everyday use. Modern DNA sequencing methods depend upon a number of individual steps, made possible by the following molecular "tools":
1. PCR
Prior to the discovery of the tools that make PCR possible,short DNA sequences could be determined using the Maxam-Gilbert chemical method. However, PCR enables faster DNA sequencing of longer nucleotide strands, by the Sanger method.
2. Thermophilic Organisms
The process of PCR involves repeated heating and cooling of the PCR mixture, during which time double-stranded DNA melts and rehybridizes. The process of elongation (building new chains of DNA from templates and primers requires polymerase enzymes that can withstand the heating and cooling cycles. The original thermostable polymerase was called Taq, isolated from a thermophilic bacterium called Thermophilus aquaticus,but now there are others, like Pfu (from Pyrococcus furiosus) which has proof-reading capabilities, Pwo, Tfl, Tli,and Tma, each from a different microorganism, and with it's own unique properties.
3. Electrophoresis
Electrophoresis was made possible by the discovery that nucleotide fragments can be separated by moving them through a porous material (agarose) within an electric field and then visualized using fluorescent dyes or radionucleotides. It is used with the Sanger method of DNA sequencing, to separate the different lengths of "truncated" DNA.
4. Nucleotide Synthesis
The synthesis of short pieces of DNA, or nucleotides, is essential for generating short fragments of DNA to make primers for PCR. Thus, this is also one of the most essential tools for PCR. Another important requirement for using the Sanger gene sequencing method, is the ability to make synthetic nucleotides. The Sanger method depends upon there being nucleotides in the PCR mixture that lack the hydroxy group (-OH) at the 3' carbon atom. Incorporation of one of these synthetic nucleotides into the PCR product prevents further elongation of the DNA strand.
5. Restriction Enzymes
Since the advent of the Sanger DNA sequencing method, numerous variations of the technique have been developed. One of these, the Shotgun method of gene sequencing, makes use of restriction enzymes to cut long DNA strands into shorter pieces.