features, such as restriction map data, that have two. and then multiple, clones in 

 common. TTiey then form contiguous blocks of DNA (contigs). The resulting ordered 

 library of DNA pieces is 10.000 to 100.000 base pairs in size. 



By using chromosomes purified by flow-sorters (a technique pioneered by the national 

 laboratories) or in hybrid cell lines, one can concentrate on mapping a single 

 chromosome at a time. 



Sequencing 



The DNA sequence is the ultimate physical map. Sequencing is also done by two basic 

 approaches. Both of these methods work because very high resolution separations of 

 DNA molecules are achievable with gel electrophoresis. In Maxam-Gilbert sequencing 

 (Fig. 13A). DNA is cleaved at individual specific bases, and the lengths of the resulting 

 fragments are determined. In Sanger sequencing (Fig. 13B), DNA replication is stopped 

 at one of the four types of bases, and the lengths of the resulting DNA fragments are 

 determined. Virtually all the steps in these sequencing methods are now automated. 



Double Stranded 

 DNA ot Unknown 



Chemical Trealmml 



loCul Chain al One or 

 Two Specific 

 NocleoUdes 



Reaction Mixtures 



Get Electrophoresis 



Detection of 



Radioactive Bands 



Only (Autoradiography! 



G + A T - C 



DNA Polymerase I ^-^ .,, .~. 



dCTP ; . >■ V 



dCTP 



dTTP ; KH Hy 



H H 

 Dldroxynucleoilde (ddNTP any one ol Ihe 4 bases) 



dJTTI- .JdCTT 



■■'"'^•te.,. 



RcacUon Mixtures 



Ge] EtectrophoTCsIs 



Auloradiof^phy lo 



Dclecl Radioartlvr 



Etands 



I I ' I ' i I ' Ij 



Fig. 13A. DNA sequencing by the Maxam-Gilbert method 



{Office of Technology Assessment, 1988). 



Fig. 13B. DNA sequencing by the Sanger method. (Office of 

 Technology Assessment, 1988). 



133 



