Molecular Human Reproduction, Vol. 9, No. 7, 411-420,
July 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Detailed investigation of factors influencing amplification efficiency and allele drop-out in single cell PCR: implications for preimplantation genetic diagnosis
Submitted on February 4, 2003; accepted on March 19, 2003
1 Department of Obstetrics and Gynaecology, University College London, 86–96 Chenies Mews, London WC1E 6HX, UK, 2 Department of Obstetrics and Gynaecology, Faculty of Medicine, Chiang Mai University, 110 Intawaroros Road, Sriphum, Mueang, Chiang Mai, 50200, Thailand and 3 The Institute for Reproductive Medicine and Science, Saint Barnabas Medical Center, 101 Old Short Hills Road, West Orange, NJ 07052, USA
4 To whom correspondence should be addressed. e-mail: dagan.wells{at}embryos.net
Preimplantation genetic diagnosis (PGD) of single gene disorders relies on PCR-based tests performed on single cells (polar bodies or blastomeres). Despite the use of increasingly robust protocols, allele drop-out (ADO; the failure to amplify one of the two alleles in a heterozygous cell) remains a significant problem for diagnosis using single cell PCR. In extreme cases ADO can affect >40% of amplifications and has already caused several PGD misdiagnoses. We suggest that an improved understanding of the origins of ADO will allow development of more reliable PCR assays. In this study we carefully varied reaction conditions in >3000 single cell amplifications, allowing factors influencing ADO rates to be identified. ADO was found to be affected by amplicon size, amount of DNA degradation, freezing and thawing, the PCR programme, and the number of cells simultaneously amplified. Factors found to have little or no affect on ADO were local DNA sequence, denaturing temperature (94 or 96°C) and cell type. Consideration of the causal factors identified during this study should permit the design of PGD protocols that experience little ADO, thus improving the accuracy of PGD for single gene disorders.
Key words: allele drop-out/preferential amplification/preimplantation genetic diagnosis/single cell PCR/prenatal diagnosis
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Ling, G. Zhuang, B. Tazon-Vega, C. Zhang, B. Cao, Z. Rosenwaks, and K. Xu Evaluation of genome coverage and fidelity of multiple displacement amplification from single cells by SNP array Mol. Hum. Reprod., November 1, 2009; 15(11): 739 - 747. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Lin, M. Takata, H. Murata, Y. Goto, K. Kido, S. Ferrone, and T. Saida Polyclonality of BRAF Mutations in Acquired Melanocytic Nevi J Natl Cancer Inst, October 21, 2009; 101(20): 1423 - 1427. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Curtis, B. S. Stewart, and S. A. Karl Sexing Pinnipeds with ZFX and ZFY Loci J. Hered., June 4, 2007; (2007) esm023v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Gardner, E. Oitmaa, A. Messner, L. Hoefsloot, A. Metspalu, and I. Schrijver Simultaneous Multigene Mutation Detection in Patients With Sensorineural Hearing Loss Through a Novel Diagnostic Microarray: A New Approach for Newborn Screening Follow-up Pediatrics, September 1, 2006; 118(3): 985 - 994. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Altarescu, B. Brooks, Y. Kaplan, T. Eldar-Geva, E.J. Margalioth, E. Levy-Lahad, and P. Renbaum Single-sperm analysis for haplotype construction of de-novo paternal mutations: application to PGD for neurofibromatosis type 1 Hum. Reprod., August 1, 2006; 21(8): 2047 - 2051. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Rund and E. Rachmilewitz {beta}-Thalassemia N. Engl. J. Med., September 15, 2005; 353(11): 1135 - 1146. [Full Text] [PDF]
|
|
|
|
|
|
A.R. Thornhill, C.E. deDie-Smulders, J.P. Geraedts, J.C. Harper, G.L. Harton, S.A. Lavery, C. Moutou, M.D. Robinson, A.G. Schmutzler, P.N. Scriven, et al. ESHRE PGD Consortium 'Best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS)' Hum. Reprod., January 1, 2005; 20(1): 35 - 48. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. De Rycke, I. Georgiou, K. Sermon, W. Lissens, P. Henderix, H. Joris, P. Platteau, A. Van Steirteghem, and I. Liebaers PGD for autosomal dominant polycystic kidney disease type 1 Mol. Hum. Reprod., January 1, 2005; 11(1): 65 - 71. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. E. Pierce, J. E. Rice, J.A. Sanchez, and L. J. Wangh Detection of cystic fibrosis alleles from single cells using molecular beacons and a novel method of asymmetric real-time PCR Mol. Hum. Reprod., December 1, 2003; 9(12): 815 - 820. [Abstract] [Full Text] [PDF]
|
|