Types Of Physical Mapping Techniques

The first technology used in physical mapping took advantage of cell lines derived from animals with identified chromosomal abnormalities (monosomics, tri-somics, or chromosomal translocations). The next technology utilized the results of fusing cells from the species of interest with rodent cell lines. A small portion of the fused cells proved to be viable and retained segments of the species of interest's genome (often whole chromosomes). Individual somatic cell hybrid lines were then characterized to determine which foreign chromosomes were present in each line. A panel of somatic cell hybrid lines was then developed, and chromosomal assignments were determined based on a gene's presence or absence in each of the lines of the panel.

The resolution of a somatic cell hybrid panel is greatly enhanced by irradiating the cells from the species of interest prior to fusion. Radiation-induced fragmentation of the genome is similar to what occurs during recombination, except that the amount of fragmentation is directly proportional to the dosage of radiation and the breakages are more random. With high doses of radiation, markers within 30,000 bases can be accurately ordered.

Another commonly used technology in physical mapping relies on visualizing a labeled segment of DNA that was hybridized to metaphase chromosomes fixed to glass microscope slides (in situ hybridization). The use of highly sensitive fluorescent-labeled DNA probes allows scientists to assign the segment of DNA to specific bands on chromosomes. These techniques are refined with multicolor fluorescent probes and by using less condensed chromosomes.

Ultimately, the highest resolution physical map uses base pairs as its unit of measurement. This type of map can be obtained by two different technologies. The first utilizes a map built of contiguous overlapping clones containing inserts of hundreds of thousands of bases. These maps most often are based on bacterial artificial chromosome (BAC) clones that have been ''fingerprinted'' by digesting each clone with a restriction endonuclease, sizing each fragment produced, and then analyzing fragment sizes to develop a contiguous BAC map. With the knowledge of which BAC clones contain which genes, the distance in bases between two genes can be determined. However, once a genome has been completely

Mb

1 cccccggagg aggcaaaacc cggcctggat ctctgtacca ctgcgctccg cacggcgcac 61cccggcgcct tcagtcccaa ctcatgccca ttgcagaggg tgcgccatca ccccgctcct 121gctgcggggc cgggctccta cgggggggtt aaaagcccct tcctccccct cctgcccgcc 181ctcccagaag cccccgggcc tggatcccgc ctctgaggat cggattcccg agagccagca 241ggcctgtttc tccgcaggcg cttggatcac gggtcagggg ctaggctggg aggcgaagaa

3 01agaagaggcg tttctgagga gcgggacgca gttctctggc gcggagggcc tggccctccc 361gcaggacggc ctctactacc tctactgtca cgtcggctac cggggccggg cacctcctcc 421cggcggggac cccctggacc gctcggtcac gctgctcagc cggctgtacc gggcgggggg

4 81cgcctacgga ccggggactc ccgagctgct gctggagggc gcggagactg tgactccggt 541cttggacccc agtcggaggc acgagtacgg gcccctctgg tacacgagcg tggggttcgg 601tggcctggtg cagctccgga ggggcgagag ggtgtacgtt aatatcagtc accccgatat 661ggtggattac aggagaggaa agaccttctt cggggcggtg atggtgggct gaggactgtc 721cgcggcccga gaggaccact gcatggtggg agtgtgtcga tggatcagcc cagacacggg 781gtcccagaca ccaggccaga caccatggcc gtggggaaaa tgcaggagat cgtgtggaaa 841ctgattttga gcctgatgaa aataaagaat gtaaaagctt taatgctgcc catacagatg 901ctgagatgtt aatgtgtctt cattcgcagt gggtacacga gtgtctgttt ttccggttgc 961ttgaaacaat tcataataac acttggtatg gcgtctgttg cctacgatgg ggatcagcgg 1021gtccagccca tccctgtggc tgaattggct gcctggccat gtgccagcat tagtctcttt 1081ggcgggtaga aggaaaggga tcttaaaccc tgaagcatct gtaaacatcc cgattctcaa 1141ggccacacag tcccctcaga ctcaacactg cactcgtttc tcttggatcc cacccagcag 1201tgatagggtc attacagggg caggcaagca ccacacagaa aggcccagcc ggcagagctc 1261atccgaatag ggctgatgag gaagcactga ggggccagcc ttctccctgc cagctgcctg 1321aagcctgtta gccctccaca atcgactctc ctcccccaag ggaagagtca cagtttaggg 1381ggtatgaagg gaggaggctg aagcagagag ttggggagtg gtgatggaga gagatggggt 1441ggggacagag tctgaggaca ccagggagaa tctggggagg aggaggagga aaaactgatg 1501ccacctgacc ccatccaggg cgaggtggaa ggttctcacc ccttgttcac acttcaggcc 1561ccttttccat atgcaggcaa gttgcggggg ctccagcgta atagcagccc cctcgttcca 1621tgaccgactt tctgcccacc actgggggag gcagagaggg acctagggct ctatctaggt 1681taccccagtg ggtcacccaa cccctgcggc acaccgcacg gttttcccct gctggcaacc 1741ctggccggaa ggaaagcaca acttcagagc ccagtgggga ctcccactct ctgagtcttc 1801atttctgact ccatatttgt ctttgactcc gtgtccagaa gaaccttcct ggcagaaagg 1861aatgcttttc tctttctttc ctttcttttt ttttttgtct ttttgctatt tcttgggcca 1921ctcccgcggc ataaggaggt tcccaggcta ggggtccaat cggagctgta gccactggcc 1981gacgccagag ccacagcaac gcaggatccg agctgcgtct gcaacctaca ccacagctca 2041cggcaatgcc agatcgttaa cccattgagc aagggcaggg accgaacccg caacctcatg 2101gttcctagtc ggattcgtta actactgcgc cacgatggga actcctcttt ctttcctttc 2161tttctgcctt gcccacagct atggaacttc ctgggccagg gattgaaccc acaccacagc 2221agtgacctga gccactgcag tgacaatgct gaatccttaa cctgttgtgc catgaagaac 2281tttttctttc tttctttctt tctttctttc tttctttctt tctttctttc tttctttctt 2341tctttctttc tttctttctt tctttctttc tttctttctc tctctctctc tctctctctc 2401tctctctctt tctttctttc tttctttctt tctttctttc tttctttctt tctgtttgtt 2461ttttagggcc aaatccacaa catatggagg ttcccatgcc aggggtctaa tcagagctgt 2521tgctgccagc ctacgccaga gccacagcaa cactagacct gagctgcgtc tgtgacgtac 2581accacagctc acagcaacgc tggatcctta atccactgag taaggccagg gatggaaccc 2641gcaacctcat ggatcctagt cggatttgct tctactgcgc cataaccgga actcttcttt 2701ctttcttttt agggctacac ctgcagcata tggaagttcc caggctaggg gttgaatcgg 2761agctgcagtt gctggcctac accacagcca cagcaacggc tggatctgag ccacatctgc 2821aacctaccct gtaccttgca gcaacaacag atccttaacc caatgagtga ggccggggat 2881cgaacctgca tcctcataga gacaatgtca ggtccctaac tgctgagcca caacaggaac 2941tcaaaggggt

Fig. 2 This diagram represents a comprehensive map of pig chromosome 6. At the far left is a diagram of the banded metaphase chromosome. The lines attaching markers to the chromosome diagram indicate these markers were physically assigned to that region of chromosome 6 by in situ hybridization. The scaled vertical bar labeled cM is the genetic map for chromosome 6 in centimorgans (cM). The next scaled bar (labeled Mb) represents a physical map based on overlapping BAC clones of the genetic map spanning 42 to 52 cM. Markers were positioned based on presence or absence in each of the BAC clones. This physical map is based on millions of bases or megabases (Mb). Finally, a 3,060 base region containing the microsatellite marker SW1057 has the complete sequence displayed. (View this art in color at www.dekker.com.)

sequenced, this information can be determined by simple sequence comparisons to the genomic sequence.

COMPREHENSIVE GENOMIC MAPS

Physical and genetic maps can be combined to form comprehensive maps if enough genes or markers have been placed on both maps. Comprehensive maps would not be necessary if there was a perfect correlation between the two different units of measure for the maps. While the linear order of markers should be the same on both maps, distances between genes may not be similar. Recombination does not occur at the same frequency throughout a chromosome. In general, recombination is suppressed near centromeres and is more frequent at telo-meric ends of chromosomes. For most mammalian species, 1 centimorgan is approximately equal to 1 million base pairs. Fig. 2 presents a representation of pig chromosome 6 displaying the genetic map with some markers assigned to chromosomal bands by in situ hybridization; a segment of this chromosome has a BAC contig map developed, and a smaller segment of the chromosome is completely sequenced.

CONCLUSIONS

Due to the rapid evolution of gene mapping technologies, many of the earlier technologies that had provided valuable information to researchers in the past are now obsolete. Once a species' genome has been sequenced, the quickest and easiest method to ''map'' a gene or segment of DNA is to know the sequence of bases of the DNA segment and then use sequence comparison software to determine the gene's location in the genome. Genetic maps are still necessary to map the chromosomal location of genes that affect quantitative traits; the technique is known as quantitative trait loci mapping (QTL mapping).

There are many useful resources available on the internet for further information. A site developed by Cornell University (http://www.ansci.cornell.edu/usdagen/ usdamain.html) explains these genetic concepts and also presents some interesting examples. The most current genetic maps for cattle and pigs can be viewed at

(http://www.marc.usda.gov), and the most current physical map for the pig can be found at (http://www.toulouse. inra.fr/lgc/pig/cyto/cyto.htm). Unfortunately, livestock gene mapping has not yet reached the stage of human gene mapping. For viewing the human genome sequence data, the following two sites are suggested (http://genome. ucsc.edu/ and http://www.ncbi.nlm.nih.gov/mapview/ map search.cgi).

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