**7. Conclusion**

*Synthetic Biology - New Interdisciplinary Science*

genome structure. It is simply to amplify nucleotide sequences that are specific to species, variety, or individual. In eukaryotes, in particular, these sequences are very numerous and offer a vast palette that allows identification in a very precise and very selective way. Indeed, the genomes of eukaryotic organisms have, unlike prokaryotes, coding sequences and noncoding sequences. The coding sequences correspond to the genes and are therefore translated into proteins. The noncoding sequences, which are therefore not translated, represent a large proportion of eukaryotic genomic DNA (up to 98%). The coding sequences are highly homologous in individuals of the same species. Indeed, the species is characterized by characters and common traits that are guaranteed by its genes. The phenotypic differences between the individuals that compose it are based on the allelic variations and the different alleles of the same gene show sequence differences that are minute (of the order of 1 base pair per 1000) [61, 62]. From one species to another, depending on the phylogenetic distance that separates them, the sequences of the genes that code for the same function have very strong homologies, all the more so that the function of the gene is essential to the embryogenesis or metabolism. As a result, coding sequences are of little relevance in terms of identification. On the other hand, the noncoding sequences are very polymorphous between species as between individuals of the same species. They thus present a large choice of genetic markers that make it possible to establish identification tests which are highly discriminating. Among these markers are minisatellites (or variable number of tandem repeats) and microsatellites (or STR, short tandem repeats) [61–63]. VNTRs and STRs are repetitive polymorphisms composed of sequences that are repeated in tandem. These repeat sequences measure from 10 to 40 base pairs for VNTRs and from 1 to 5 base pairs for STRs. From one individual to another, the repeated sequence of a VNTR or STR is identical but the number of repetitions and therefore the size of the VNTR or the STR can be very variable (we speak of alleles). On the other hand, there is a wide variety of VNTRs and STRs on eukaryotic genomes. Detection of STR or VNTR polymorphism is by PCR using primers that hybridize to nonpolymorphic flanking sequences. The amplification products are then either analyzed by electrophoresis or undergo fragment analysis using a capillary sequencer. It is now possible to simultaneously amplify several STRs or VNTRs by using several pairs of primers. The variety of amplification products obtained leads to footprints that are specific individuals. On the other hand, the power of PCR makes it possible to amplify micro- and minisatellites from very little DNA. DNA fingerprinting has become much more commonplace in recent years in the context of judicial investigations. But these techniques are equally as effective in other species as humans and allow not only identifying individuals but also varieties or species. The type of identification depends simply on the choice of markers. Similarly, for varietal identification purposes, one can commonly proceed according to protocols derived

Two techniques that are relevant are the random amplification of polymorphic DNA (RAPD) and the amplification of fragment length polymorphism (AFLP). (Random amplification of polymorphic DNA (RAPD) is a PCR for varietal identification that uses pairs of random primers of reduced size (about 10 base pairs). These primers will hybridize randomly, but PCR usually results in an electrophoresis amplification profile which is specific to the variety from which the matrix DNA is derived. Amplification of fragment length polymorphism (AFLP) is a much more efficient method. It first consists hydrolyzing the genomic DNA with one or better two restriction endonucleases. Then, we proceed with the ligation of adapters (defined sequences of DNA of about 15 nucleotides) at the level of the generated cohesive ends by restriction enzymes. Finally, the product of the ligation is amplified by PCR with a pair of primers that hybridizes at the level of the adapters. The

**158**

from the PCR [64–66].

The extension of genotyping approaches to all living organisms has made significant advances in the reconstruction of the history of life. At the population level, the distribution and frequency of known genetic polymorphisms in a species can highlight the evolving forces at play, reveal the effects of natural selection, and infer demographic change. Moreover, the comparison of the sequences of the same genes between different species and that of whole genomes is at the origin of the molecular phylogenies that currently prevail in the classification. They make it possible to trace the relationships between species on the basis of the divergence of their DNA sequences. As such, the PCR is a key stage at two levels. The first concerns the isolation of homologous genes in several species and their characterization. The second is the production of amplified total genomic DNA for genome sequencing and comparative analysis. But PCR is also used to identify the genetic heritage of missing organisms. The DNA breaks down by fragmentation after the death of the body. If we can recover these fragments and amplify them, it becomes possible, in spite of its state, to deduce all or part of the initial genome of the individual. PCR has thus become the primary tool in the field of palaeogenetics, which consists in recovering and analyzing DNA sequences of more or less old organisms, and this as well from the remains preserved in museum collections, from historical site where the skeletal or mummified remains of extinct organisms for hundreds thousands or even hundreds of thousands of years. The uses of the PCR thus quickly stopped being limited to the studies of biology, to gain other disciplines or fields of activities.
