Use of partial digests, end labeling and hybridization in restriction mapping

Use of partial digests, end labeling and hybridization in restriction mapping
The above technique of individual and double digests can be supplemented with other techniques for actual construction of maps. For instance, by permitting incomplete digestion,-fragments longer than those obtained by complete digestion may be obtained. These will be called partial digests. In the example used in the last section, in partial digests with enzyme A, instead of getting A-1000, A-2100, A-1400, A-500, we may get A-3100, A-1400, and A-500, which will suggest that A-1000 and A-2100 lie in adjacent regions. In another partial digest if we get A-1000, A-3500 and A-500, this will suggest that A-2100 and A-1400 are adjoining, which will mean that A-1000 and A-1400 are found on the two sides of A-2100. This technique will thus allow the arrangement of fragment in a linear order.

Another useful technique involves labeling of the ends (end labeling) of DNA molecule before digestion, so that the fragments containing these ends can be identified due to labeling, even after digestion. In above example if A-1000 and A-500 are radioactively labeled, these will be present at the two ends of the restriction map.

Some of the features of restriction map can be confirmed by nucleic acid hybridization. For instance, if A-2100 and B-2500 have an overlapping region of 1900bp, they should hybridize with each other, which will confirm overlapping.

The above technique will help in accurate completion of a restriction map, but will require that we have a complete set of restriction fragments which make the entire DNA region being mapped. Once the restriction map is ready, it can be compared with the genetic map. Although large changes (due to deletions, duplications, inversions and interchanges) on the genetic map can be easily located on restriction map, but point mutations can not be easily mapped on the restriction map, since the restriction sites often do not change due to mutation. In such situations one may like to determine the nucleotide sequences, of individual fragments and compare them in the normal and the mutant individuals. Since sequencing of nucleotides is more laborious and can not be easily undertaken for the whole genome, another technique of molecular genetic markers in the form of restriction fragment length polymorphisms (RFLPs) has been utilized during 1980s to generate genetic linkage maps in human beings, maize, tomato, etc. This technique will be briefly discussed in the following section.