An enormous number of individual genes have been sequenced over the last two decades or more. Of latter years in particular, genome projects have also begun to make available the sequence of the entire complement of genes present in many species. The bulk of this sequence information has been made publicly available by its deposition in sequence databases. As a result, scientists who now wish to clone a particular gene will usually have prior access to partial/entire sequence information from the relevant organism or a closely related species. This sequence information allows them to obtain large amounts of the gene of interest by using the PCR technique (Figure 3.15). This
J.Addprimers & other reaction constituents, heat & then cool to allow primers to anneal
Repeat heating & cooling cycles
Figure 3.15 The PCR is initiated by separation of the double-stranded DNA into its two constituent strands. This is achieved by heating the sample (usually to 94 °C). Also present in the reaction mixture are: (a) two chemically synthesized oligonucleotide primers ('oligos') whose sequences are complementary to the sequences flanking the gene of interest; (b) the enzyme DNA polymerase, which can extend the primers to synthesize a new DNA strand of complementary sequence to the single stranded DNA template; (c) all the nucleoside precursors required for synthesis of the growing DNA strand (i.e. the deoxynucleoside triphosphates or dNTPs). Once strand separation has been achieved the reaction temperature is reduced, in order to allow primers to anneal to complementary sequences on each strand and allow the DNA polymerase to extend the primers. This extension phase is normally carried out at 74 °C. The DNA polymerase used is sourced from the thermophilic microorganism Thermus aquaticus; therefore, it is heat stable and not inactivated by PCR operational temperatures. This completes the first cycle of the PCR process; the result is a doubling of the amount of target DNA present in the reaction mixture. The cycle is then repeated and with each repeat comes a doubling of the amount of target DNA. After 25-30 cycle repeats, several hundred million copies of the target DNA have been generated approach to cloning has now come to the fore, as it is faster and more convenient than the more classical methods described above. The process begins by extraction of total genomic DNA from the source of interest (e.g. human cells if you wish to clone a specific human gene). Oligonucleotide primers whose sequences flank the target gene/DNA segment are synthesized and used to amplify that portion of DNA selectively. Recognition sites for REs can be incorporated into the oligonucleotides to allow cloning of the amplified gene, as outlined earlier. Because the target gene sequence is the only segment of the extracted DNA to be amplified by the prior PCR step, the vast majority of clones in the library now generated should contain the desired gene. This can be confirmed by direct sequencing of the inserted DNA fragment from several of the colonies. Sequencing is important not only to prove definitively that the cloned DNA is the target gene, but also that its sequence perfectly matches the published sequence. The PCR process is prone to the introduction of sequence errors.
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