Molecular Genetics

The inserts of clones of a genomic library can be arranged into a map of the molecular chromosome

Facts

• An indexed cosmid library of genomic DNA was prepared. An indexed library is a multidimensional array of numbered clones, the location of each in the array being known, or "indexed".
• The DNA of each cosmid clone was digested with HindIII, a restriction enzyme with a 6 bp recognition site, end labeled, and then digested with MboI, which recognizes a 4 bp sequence.
• The fragments were separated by polyacrylamide gel electrophoresis. Labeled ones were identified by autoradiography. Patterns from selected cosmids are shown.

Interpretations

• The pattern of labeled fragments serves as a "profile" of the cosmid.
• Cosmids with fragments from overlapping parts of the genome share fragments in their profile. In the above, the top four cosmids derive from the same region of the genome, while no. 1010 came from an unrelated region.
• Computer analysis of shared bands can order the cloned segments relative to one another. These segments form a contig since they represent DNA segments that are contiguous in the genome.
• A diagram showing the genome order of cosmid clones is a contig map.
• In this example, inserts 2001 and 4690 might be part of a minimum tiling path, the collection of cloned inserts that covers the entire genome with a minimum of overlaps. Inserts 1012 and 1352 would probably not belong since they overlap substantially with 2001 and 1012. A minimum tiling path is important for the efficiency of map-based genome sequencing projects.

Further information

• Phage, BAC, PAC and YAC libraries can also be ordered into contigs by the same strategy.
• Numerous other strategies also yield "profiles" or "fingerprints" that can be used.
• A contig can be related to the genetic chromosome by hybridization of DNA marker probes to the array of library members.
• Assembly of contig maps is often a prelude to nucleotide sequencing.
• 
  Another approach, particularly useful with a shotgun sequencing approach, is the sequencing of the ends of all inserts in a large insert (BAC or PAC) library. As sequencing contigs accumulate in a sequencing project, they can be ordered and mapped (distance between them specified) relative to one another when the sequence of one end is in one contig and that of the other end is in another. [Note: lengths of sequences are exaggerated relative to the length of the BAC insert.]

This is page 11232 of Molecular Genetics by Ulrich Melcher, © 1997, 1998, 2000