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Mol. Hum. Reprod. Advance Access published online on August 20, 2004
Molecular Human Reproduction, doi:10.1093/molehr/gah101
© 2004 by Oxford University Press
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Received June 12, 2004
Revised July 15, 2004
Accepted July 24, 2004

Article

Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease

Alan H. Handyside 1* *, Mark D. Robinson 2 *, Robert J. Simpson 3, Mark B. Omar 3, Marie-Anne Shaw 3, J.Gedis Grudzinskas 4, Anthony Rutherford 2

1 Leeds PGD Centre, Assisted Conception Unit, Clarendon Wing, Leeds General Infirmary, Leeds, UK; The London Bridge Fertility, Gynaecology and Genetics Centre, London, UK; School of Biology, University of Leeds, Leeds, UK
2 Leeds PGD Centre, Assisted Conception Unit, Clarendon Wing, Leeds General Infirmary, Leeds, UK
3 School of Biology, University of Leeds, Leeds, UK
4 The London Bridge Fertility, Gynaecology and Genetics Centre, London, UK

* To whom correspondence should be addressed. E-mail: a.h.handyside{at}leeds.ac.uk.


  Abstract

Preimplantation genetic diagnosis (PGD) of single gene defects following assisted conception typically involves removal of single cells from preimplantation embryos and analysis using highly sensitive PCR amplification methods taking stringent precautions to prevent contamination from foreign or previously amplified DNA. Recently, whole genome amplification has been achieved from small quantities of genomic DNA by isothermal amplification with bacteriophage {Phi}29 DNA polymerase- and exonuclease-resistant random hexamer primers. Here we report that isothermal whole genome amplification from single and small numbers of lymphocytes and blastomeres isolated from cleavage stage embryos yielded microgram quantities of amplified DNA, and allowed analysis of 20 different loci, including the {Delta}F508 deletion causing cystic fibrosis and polymorphic repeat sequences used in DNA fingerprinting. As with analysis by PCR-based methods, some preferential amplification or allele drop-out at heterozygous loci was detected with single cells. With 2-5 cells, amplification was more consistent and with 10 or 20 cells results were indistinguishable from genomic DNA. The use of isothermal whole genome amplification as a universal first step marks a new era for PGD since, unlike previous PCR-based methods, sufficient DNA is amplified for diagnosis of any known single gene defect by standard methods and conditions.

Keywords: preimplantation genetic diagnosis; single gene defects; whole genome amplification.

*Joint first authors


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