dards). Measurements should be performed with a computer-assisted or computer-automated system, in which the operator identifies the positions of the bands with a digitizing pen or similar device that directly records them, visually inspects them, or both. Computer-based procedures ensure appropriate documentation of the measurement and promote objectivity.
External molecular-weight standards alone, however, are not sufficient, because anomalies in electrophoresis can lead to errors in RFLP typing caused by band shifting.5 Such anomalies can be due to differences in salt or DNA concentrations among samples (which could be corrected by repeated extraction) or to covalent or noncovalent modifications of the DNA (which might be irreversible). Band shifting could cause two DNA samples from one person to show different patterns or DNA samples from two different persons to show the same pattern. Band shifting also makes it impossible to measure fragment sizes relative to external molecular-weight standards, because the standards have migrated at a different speed.
Band shifting is easy to detect by hybridizing the Southern blot with monomorphic probes—that is, probes that detect constant-length fragments that are always in the same position in all people. If several monomorphic fragments are in the same position in both lanes, it is safe to assume that no band shifting has occurred. If the monomorphic fragments are in different positions, band shifting is present. The committee considers it desirable for all samples to be tested for band shifting by hybridization with monomorphic probes that cover a wide range of fragment sizes in the gel. That approach will eliminate the rare production of a match by shifting of bands in an evidence sample to the same positions as in a suspect sample. Testing laboratories now investigate the possibility of band shifting only when they find two samples with patterns that appear to be similar but shifted relative to one another. (Multiple monomorphic probes might not be available for some systems and might need to be developed.)
Testing for band shifting is easy, but correcting it is harder. The best approach is to clean the samples (by re-extraction, dialysis, or other measures) and repeat the experiment in the hope of avoiding band shifting. When that is impossible because too little sample is available or it fails (perhaps because of covalent modification of the DNA), it is possible in principle to determine the molecular weights of polymorphic fragments in a sample by comparing them with monomorphic human bands in the same lane—so-called internal molecular-weight standards. These monomorphic fragments are expected to have undergone the same band shift, so they should provide an accurate internal ruler for measurement. (Note that the polymorphic fragments and the internal molecular-weight standards are visualized on separate hybridizations, but can be superimposed on one another, if the external molecular-weight standards are used to align the gels.)
In practice, however, the use of internal standards presents serious dif-