93

 The sizes of fragments detected in Southern blots and by ethidium

bromide-staining or end-labeling did not always correspond. The migration of

DNA molecules in agarose, and ultimately the determination of the sizes of the

DNA fragments so separated, depends on the buffer system, the voltage

gradient, and the concentration of the agarose (Southern 1979). While the

same buffer was used for all electrophoretic separations reported here, the

voltage gradient and agarose concentration were not always the same. All

Southern blots involved 2% agarose, but the fragments produced from the

digestion of 178 and smaller regions of 178 were separated in anywhere from

1% 3% agarose. Although more than 135 Southern blots were run, some of

the fragment sizes from the Mspl single and double digestions were only

determined in one or two gels. A more precise estimation of the sizes of the

Mspl and Ddel fragments within each region of 178 could be obtained by

repeating the digests, gels, and measuring the fragment migration distances,

followed by linear regression analysis. However, the purpose of the

hybridizations and digestions performed was to localize polymorphisms, which

was accomplished. Further details can be obtained from the proposed PCR

work and by sequencing.



 Discussion


 The Mspl fragment pattern resulting from the digestion of 178 with Mspl

resembled the pattern of M103, which was found at high frequency in USA