A chromosome map describes the order of genes and other markers, and the spacing between them on the chromosome. These maps are constructed with different levels of characterization and resolution. Physical mapping of a chromosome is obtained by determining the specific physical location of its genes and markers. This kind of mapping is an important process when searching for disease genes by positional cloning methodology and for DNAsequencing. It is to be distinguished from genetic mapping, also known as linkage mapping, which shows the position of genes and markers relative to each other rather than as physical landmarks on the chromosome.
The distances between markers on a genetic map are statistical estimates made by determining how frequently the two markers are passed together from parent to child. These distances are given in centiMorgan (cM), a unit related to the frequency of meiotic recombination and the pattern of segregation and inheritance of different markers within families. A genetic distance of 1 cM corresponds to roughly 1 megabase (Mb). The markers usually followed in genetic mapping studies are either polymorphic sequences that are reflected in varying susceptibility of the studied DNA to restricted enzymes, resulting in restriction fragment length polymorphisms (RFLPs), or variation in the length and number of tandem repeats called microsatellites. Studies relying on the latter markers are usually referred to as analysis of segregation of simple sequence length polymorphism (SSLP). The most important application of genetic mapping in clinical medicine is that an inherited disease gene can be identified and mapped by following the pattern of inheritance of a DNA marker that is linked to that disease gene by being present in affected individual but absent in unaffected ones.
Cytogenetic mapping, also known as chromosomal mapping, is obtained by staining and microscopic observation of chromosomes, and is considered a low-resolution physical mapping. The unique appearance of each chromosome that has been properly stained in a cytogenetic analysis is determined by visually distinct regions, which appear as either light or dark bands. Cytogenetic mapping, which was originally used to map DNA fragments that are at distances more than 10 megabases (Mb, a unit of length for DNA fragments equal to 1 million nucleotides) apart, can now be used to produce maps with 100 Kb (kilobase) resolution. This improvement is due to the availability of novel DNA probes and the use of the powerful staining technique of fluorescence in situ hybridization (FISH).
To obtain physical maps, geneticists currently proceed by first obtaining a single chromosome separated by flow cytometry. Genomic DNA prepared from this chromosome is cut by rare-cutter restriction enzymes producing relatively large DNA fragments. This macro-restriction mapping strategy depicts the order and distances between sites at which rare-cutter enzymes cleave DNA, and is known as the top-down mapping methodology. It produces continuous maps with fewer gaps between markers, but with low resolution. To obtain high resolution physical mapping an approach called contig mapping is used. A large number of genomic DNA clones can be obtained by cloning segments of genomic DNA from a specific chromosome in cloning vectors such as yeast or bacterial artificial chromosomes. These sets of clones can be ordered into overlapping groups called contigs. As more contigs are obtained, characterized and assembled, the equivalent of a whole chromosome is obtained. Ordering and assembly of sets of clones into contig is best performed by PCR screening the clones for sequence-tagged sites (STS). These are relatively small unique sequences of DNA known to map to particular chromosomes or part of chromosomes. These genomic sequences were obtained by amassing a large number of genomic clones that cover the whole genome. Random short regions of these clones have been sequenced, thus generating sequence-tagged sites that are dispersed over the whole genome. Pairs of DNA primers were designed from these sequences that will amplify these STS if they are present in a cloned genomic insert. Clones prepared from a single chromosome and that can be shown to have specific STS in common must have overlapping inserts and will therefore be aligned in the same contig. To map the human chromosome 21, the smallest and the second human chromosome to be completely sequenced by the Human Genome Project, researchers screened a total of 134,000 clones from three separate yeast artificial chromosome (YAC) libraries. Using primer pairs from 198 STS, a total of positive 810 clones were obtained allowing the construction of the contiguous chromosome.
Sequencing, the complete determination of all base pairs of the ordered DNA clones is the ultimate physical mapping of the genome. The two main DNA sequencing approaches available, the chemical degradation method and the chain termination or dideoxy method, rely on the production of DNA fragments of varying lengths that are separated by gel electrophoresis and visualized by auto-radiography or by fluorescence tag labeling. All steps in any of these two techniques (known as the Maxam-Gilbert and Sanger methods, respectively) are now fully automated and fluorescent labeling and detection has been developed over the years. These improvements have made large-scale sequencing projects feasible. Researchers involved in major genome sequencing projects have decided to take advantage of two main sequencing methods known as the random or shotgun method an the ordered or directed sequencing method. The shotgun method involves randomly sequencing small cloned fragments of the genome, with no prior knowledge about the mapping of these fragments in a chromosome. These short sequences are subsequently re-assembled in ordered generating larger contigs of sequenced DNA, a task that requires the generation of redundant sequencing data and can be very difficult to accomplish. The directed sequencing strategies use adjacent and cloned fragments of DNA, which can be easily mapped. The sequences produced by the draft sequence of the human genome have a size of roughly 10 Kb and their locations on the chromosomes are only fairly accurate. This uncertainty about the exact physical mapping of the sequenced fragments is the reason why only a few of all the chromosomes are considered completely sequenced even though the order of base pairs in each chromosomal was determined at least 4 to 5 times. Scientists expect to perform this repeated sequencing, known as genome depth of coverage, at least 8 to 9 times in the finished version of the whole genome sequence.
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