October 14, 2012

Genomes can be mapped by linkage, restriction cleavage, or DNA sequence

Defining the contents of a genome essentially means making a map. We can think about mapping genes and genomes at several levels of resolution:
  • A genetic (or linkage) map identifies the distance between mutations in terms of recombination frequencies. It is limited by its reliance on the occurrence of mutations that affect the phenotype. Because recombination frequencies can be distorted relative to the physical distance between sites, it does not accurately represent physical distances along the genetic material.
  • A linkage map can also be constructed by measuring recombination between sites in genomic DNA. These sites have sequence variations that generate differences in the susceptibility to cleavage by certain (restriction) enzymes. Because such variations are common, such a map can be prepared for any organism irrespective of the occurrence of mutants. It has the same disadvantage as any linkage map that the relative distances are based on recombination.
  • A restriction map is constructed by cleaving DNA into fragments with restriction enzymes and measuring the distances between the sites of cleavage. This represents distances in terms of the length of DNA, so it provides a physical map of the genetic material. A restriction map does not intrinsically identify sites of genetic interest. For it to be related to the genetic map, mutations have to be characterized in terms of their effects upon the restriction sites. Large changes in the genome can be recognized because they affect the sizes or numbers of restriction fragments. Point mutations are more difficult to detect.
  • The ultimate map is to determine the sequence of the DNA. From the sequence, we can identify genes and the distances between them. By analyzing the protein-coding potential of a sequence of the DNA, we can deduce whether it represents a protein. The basic assumption here is that natural selection prevents the accumulation of damaging mutations in sequences that code for proteins. Reversing the argument, we may assume that an intact coding sequence is likely to be used to generate a protein.
By comparing the sequence of a wild-type DNA with that of a mutant allele, we can determine the nature of a mutation and its exact site of occurrence. This defines the relationship between the genetic map (based entirely on sites of mutation) and the physical map (based on or even comprising the sequence of DNA).
Similar techniques are used to identify and sequence genes and to map the genome, although there is of course a difference of scale. In each case, the principle is to obtain a series of overlapping fragments of DNA, which can be connected into a continuous map. The crucial feature is that each segment is related to the next segment on the map by characterizing the overlap between them, so that we can be sure no segments are missing. This principle is applied both at the level of ordering large fragments into a map, and in connecting the sequences that make up the fragments.

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