Charles R. Cantor

Content and organization of the human Ig VH locus: definition of three new VH families and linkage to the Ig CH locus.

We present a detailed analysis of the content and organization of the human immunoglobulin VH locus. Human VH genes representing five distinct families were isolated, including novel members belonging to two out of three of the known VH gene families (VH1 and VH3) as well as members of three new families (VH4, VH5, and VH6). We report the nucleotide sequence of 21 novel human VH genes, many of which belong to the three new VH gene families. In addition, we provide a preliminary analysis of the organization of these gene segments over the full extent of the locus. We find that the five multi‐segment families (VH1‐5) have members interspersed over nearly the full 1500‐2000 kb of the VH locus, and estimate that the entire heavy chain locus covers 2500 kb or less. Finally, we provide the first report of the physical linkage of the variable and constant loci of a human Ig gene family by demonstrating that the most proximal known human VH segments lie within 100 kb of the constant region locus.

The Future of DNA Sequencing: After the Human Genome Project

The notion of determining the DNA sequence of the entire human genome was conceived in the mid 1980’s. The initial impulse came from a consideration of how improved DNA sequencing methodology might enhance, dramatically, our ability to find mutations and be able to quantitate the effect of radiation and other environmental insults on human inherited mutations. However, a stronger and more constant driving force has been the usefulness of DNA sequence information in finding genes and studying their activity.

6 – Purification, Specific Fragmentation, and Separation of Large DNA Molecules

Publisher Summary nThis chapter discusses the purification, specific fragmentation, and separation of large DNA molecules. Three new techniques have been recently developed that allow the fractionation and analysis of DNA molecules on a size scale much larger than previously possible. Electrophoresis techniques developed in the past 50 years have allowed effective separation of proteins and nucleic acids on the basis of molecular weight. DNA molecules above 20 kb long usually cannot be separated by gel electrophoresis because they are larger than the pore size of the matrix. In standard gel electrophoresis, the electrical field is applied constantly in one direction. Pulse times used with PFG electrophoresis can be tuned such that maximal separation of particular sized molecules is obtained.