Appendix A: 

 Primer on Molecular 

 Genetics 



Fig. 10. Multiple levels of mapping of human chromosomes. At the lowest level of 

 resolution, the human genome is characterized by 22 pairs of autosomal chromosomes 

 and two sex chromosomes — X and Y. Individual chromosomes can be distinguished by 

 size, position of centromeric constriction relative to telomeres (centromeric index), and 

 patterns of banding induced by staining with DNA-specific dyes after treatments that 

 partially remove chromosomal proteins (G- and Q-banding). Cloned DNA probes can be 

 used to determine by in situ hybridization the locations of specific genes or markers (A 

 and B in this diagram) along a chromosome. To establish the order of — and relative 

 distances between — genes or markers along specific human chromosomes, polymorphic 

 DNA markers or fragments of gene regions are used to follow the inheritance of specific 

 alleles in multigeneration families with large numbers of siblings. Distances on genetic 

 linkage maps are given as percent recombination between markers: a distance of 1% 

 recombination [1 centiMorgan (cM)] is generally estimated to represent approximately 

 1 Mbp of DNA. Radiation hybrid techniques can be used to obtain a higher-resolution 

 (hundreds to thousands of kilobase pairs) physical map [S. Goss and H. Harris, Nature 

 255, 1445-1458 (1975) and D, Cox et al.. Genomics^, 397-407 (1988)]. The next level 

 of resolution can be represented by the analytical physical — or macrorestriction — map 

 that is developed using rare-cutting restriction enzymes and pulsed-field gel 

 electrophoresis to separate megabase-sized DNA fragments. Markers are located on 

 macrorestriction maps relative to restriction sites, and distances can be measured in 

 kilobase pairs. 



CHROMOSOME ( Siim) 

 CYTOGENETIC MAP 



CHROMOSOMAL DNA 

 {4-8 X 10''^nl) 

 (-1.3 X lo'bp) 



LINKAGE OR 

 GENETIC MAP 



RADIATION HYBRID 

 MAP 



PHYSICAL MAPS 



MACRORESTRICTION 



MAP 



OVERLAPPING SET 

 OF CLONES 



SEQUENCE TAGGED SITES 

 (STS) 



DNA SEQUENCE 



c 



-mK 



* 



f 



^XSOOO EXPANSION MARKER* (GENES)\ 



Disease Locus 



RECOMBINATION 

 FREQUENCY 



11 It 



DIseas i Gene 



PFG MAP (20 kt)-10 Mb) 



YACs (100 - 1000 kb) 



COSMIDS (40 kb) 

 PHAGE (17 kb) 



■ m iMi m m 



^^p<Z><!^^^ ,1 BASE PAIR, 



DISEASE GENE SEQUENCE 



As an example of the value of long- 

 range restriction maps, distances 

 can be determined between markers 

 flanking a disease locus. Over- 

 lapping sets of cloned DNA from 

 phage or cosmid vectors and from 

 yeast chromosomes — all engineered 

 to accept foreign DNA — can be used 

 for constructing these macro- 

 restriction maps. A clone subset 

 provides the substrate for obtaining 

 the DNA sequence — the ultimate 

 physical map. Maps from the 

 cytological level to the DNA 

 sequence are retained in various 

 databases. The proposal regarding 

 sequence-tagged sites (STSs) has 

 offered that evenly spaced STSs 

 would be used as identifiers to 

 interrelate different levels of maps 

 and to diminish requirements for 

 storage of large numbers of 

 biological reference materials 

 [M. Olson et al., Science 245, 

 1434-1435 (1989)]. (Figure orovided 

 by C. E. Hildebrand, Los Alamos 

 National Laboratory.) 



130 



