Unit

UNIT 5.7 Purification and Characterization of YACs Containing Large Inserts

  1. Robert M. Gemmill (restriction mapping)1,
  2. Richard Bolin (restriction mapping)2,
  3. William M. Strauss (PFGE purification)3,
  4. William Pavan (fragmentation)4

Published Online: 1 MAY 2001

DOI: 10.1002/0471142905.hg0507s00

Current Protocols in Human Genetics

Current Protocols in Human Genetics

How to Cite

Gemmill, R. M., Bolin, R., Strauss, W. M. and Pavan, W. 2001. Purification and Characterization of YACs Containing Large Inserts. Current Protocols in Human Genetics. 00:5.7:5.7.1–5.7.20.

Author Information

  1. 1

    Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado

  2. 2

    Nexagen, Boulder, Colorado

  3. 3

    Whitehead Institute, Cambridge, Massachusetts

  4. 4

    Princeton University, Princeton, New Jersey

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: FEB 1994

Abstract

This unit provides protocols for characterizing DNA segments cloned in YACs and for purifying YACs from yeast chromosomes. The first basic protocol describes Southern blotting and partial-digest restriction analysis of YACs. These methods are useful for determining the size and complexity of the cloned insert DNA, the presence and location of particular restriction sites or sequences, and even the species of origin of the insert DNA (indicated by hybridization to species-specific repetitive elements such as Alu repeats). The second basic protocol describes gel purification of YACs for use in procedures requiring pure YAC DNA, such as mammalian-cell transformation and subcloning into smaller insert vectors. The third basic protocol details characterizing and analyzing YACs: in vivo fragmentation via homologous recombination with specialized fragmentation vectors containing specific probe sequences or repetitive elements, followed by Southern blotting with YAC- and human-derived probes.