6-5.3(b)Physical Mapping
A physical map represents a partial ordering of specific DNA sequences by their position on a chromosome. It has been developed to address the limitations of the maps generated by genetic techniques listed below-

• Less accuracy of genetic maps
• Resolution of genetic maps depends upon the number of cross-overs to be scored.

There are several types of physical maps including cytological maps, restriction maps, STS content maps, radiation hybrid maps, ordered clone collections (i.e., “contig maps”), and the DNA sequence of an entire chromosome, constructed by a wide range of experimental methods without having any complete sequence information. Various techniques employed for the generation of physical maps are described below.

• Restriction Mapping
• Fluorescent in situ hybridization (FISH)
• Sequence tagged site (STS) mapping

6-5.3(b).1 Restriction Mapping

Restriction mapping helps in obtaining structural information of an unknown piece of DNA. A restriction map is generated by digesting DNA with a series of restriction enzymes followed by separation of the resultant DNA fragments by agarose gel electrophoresis. The distance between restriction enzyme sites is determined from the fragmentation pattern in the gel.

In practice a DNA molecule is first digested with a known restriction enzyme (say Eco RI ) and the sizes of the resulting fragments are measured by agarose gel electrophoresis. Then this is repeated with another enzyme (say BamHI) that identifies a different consensus sequence. From the gel patterns the numbers of restriction sites for each enzyme are calculated. To determine the relative positions of the restriction sites w.r.t each enzyme, in yet another step DNA molecule is digested with both enzymes together.

In case of problems arising due to occurrence of more than one restriction sites of the second enzyme in a fragment generated by the first enzyme the solution is to undertake partial restriction using less enzyme or short reaction time whenever required. Finally the positions of the restriction sites obtained through the above process is plotted on a bar representing a DNA segment to obtain a restriction map.