Unit

UNIT 5.18 Human Artificial Chromosome Assembly by Transposon-Based Retrofitting of Genomic BACs with Synthetic Alpha-Satellite Arrays

  1. Joydeep Basu1,
  2. Huntington F. Willard1,
  3. Gregory Stromberg2

Published Online: 1 JAN 2007

DOI: 10.1002/0471142905.hg0518s52

Current Protocols in Human Genetics

Current Protocols in Human Genetics

How to Cite

Basu, J., Willard, H. F. and Stromberg, G. 2007. Human Artificial Chromosome Assembly by Transposon-Based Retrofitting of Genomic BACs with Synthetic Alpha-Satellite Arrays. Current Protocols in Human Genetics. 52:5.18:5.18.1–5.18.19.

Author Information

  1. 1

    Duke Institute for Genome Sciences and Policy, Durham, North Carolina

  2. 2

    Athersys, Inc., Cleveland, Ohio

Publication History

  1. Published Online: 1 JAN 2007
  2. Published Print: JAN 2007

Abstract

The development of methodologies for the rapid assembly of synthetic alpha-satellite arrays recapitulating the higher-order periodic organization of native human centromeres permits the systematic investigation of the significance of primary sequence and sequence organization in centromere function. Synthetic arrays with defined mutations affecting sequence and/or organization may be evaluated in a de novo human artificial chromosome assay. This unit describes strategies for the assembly of custom built alpha-satellite arrays containing any desired mutation as well as strategies for the construction and manipulation of alpha satellite–based transposons. Transposons permit the rapid and reliable retrofitting of any genomic bacterial artificial chromosome (BAC) with synthetic alpha-satellite arrays and other functional components, thereby facilitating conversion into BAC-based human artificial chromosome vectors. These techniques permit identification and optimization of the critical parameters underlying the unique ability of alpha-satellite DNA to facilitate de novo centromere assembly, and they will establish the foundation for the next generation of human artificial chromosome vectors.

Keywords:

  • Transposon;
  • alpha satellite;
  • centromere;
  • BAC;
  • human artificial chromosome