Abstract
The detection of genetic defects in human embryos following in vitro fertilization (IVF) or preimplantation genetic diagnosis (PGD) allows the selection and transfer of unaffected embryos in couples known to be at risk of transmitting an inherited disorder. This avoids the need to termiate an affected pregnancy, following prenatal diagnosis at later stages (1). Diagnosis of a single gene defect is usually performed on one or two single cells (blastomeres) biopsied from 8- to 10-cell embryos on the 3rd d postinsemination using nested polymerase chain reaction (PCR) to amplify informative fragments. Nested PCR allows amplification from a limited number of target sequences (2), and under carefully optimized conditions, amplification of as few as one or two target copies present in a single haploid or diploid cell is possible (3–5). PGD was first achieved for X-linked diseases by determining the sex of the embryos using a Y chromosome-specific repetitive sequence and selective transfer of only female embryos (6). More recently, specific diagnosis has been achieved for cystic fibrosis (CF), by amplifying across the cystic fibrosis transmembrane regulator (CFTR) gene †F508 locus (7) and for Lesch-Nyhan syndrome by amplifying across a familial base substitution nullifying a natural XhoI restriction site in the hypoxanthine phophoribosyl transferase (HPRT) gene (8). In both instances, nested PCR strategies were chosen to amplify the mutated sequence allowing sufficient amplification for detection on ethidium bromide-stained gels. The limited cycling with the outer primers (20 cycles) reduces nonspecific amplification, and only specific fragments that contain the complementary sequence to the internal primers are amplified to a detectable level in the second round of PCR. Although extra handling is involved, any genomic contaminant introduced after the first round of amplification would not be amplified to a detectable level by the inner primers alone. The efficiency of the second amplification is improved because the denaturation of the first amplification product (amplicon) is easier. Also, the great excess of these amplicons compared with nonspecific sequences eliminates competition, thereby enhancing specificity and yield.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Handyside, A. H (1993) Diagnosis of inherited disease before implantation Reprod. Med Rev 2, 51–61.
Mullis, K. and Faloona, F. (1987) Specific synthesis of DNA in vitro vta a polymerase-catalysed chain reaction. Methods Enzymol. 155, 335–350.
Li, A., Gyllenstein, U. B., Cut, X., Saiki, R. K., Erhch, H. A, and Arnheim, N. (1988) Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 335, 414–419.
Cui, X. F., Li, H. H., Goradia, T. M., Lange, K., Kazaztan, H.H.J, Galas, D., and Amheim, N. (1989) Single-sperm typing: determination of genetic distance between the G gamma-globm and parathyroid hormone loci by using the polymerase chain reaction and allele-specific oligomers. Proc Natl Acad Sci USA 86, 9389–9393.
Coutelle, C., Williams, C., Handyside, A., Hardy, K., Winston, R., and Williamson, R. (1989) Genetic analysis of DNA from single human oocytes a model for preimplantation diagnosis of cystic fibrosis. Br Med. J 299, 22–24.
Handyside, A. H., Kontogianni, E. H., Hardy, K, and Winston, R. M. (1990) Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 344, 768–770.
Handyside, A. H., Lesko, J. G., Tarin, J. J., Winston, R. M., and Hughes, M. R. (1992) Birth of a normal girl after in vitro fertilization and preimplantatron diagnostic testing for cystic fibrosis. N Engl J. Med. 327, 905–909.
Ray, P F, Winston, R M. L, and Handyside, A. H. (1994) Single cell analysis for diagnosis of cystic fibrosis and Lesch-Nyhan syndrome in human embryos before implantation. Miami Bio/Technol 5, 46 (abstract).
Ao, A., Ray, P., Lesko, J., Harper, J. C., Handyside, A. H., Hughes, M. R., and Winston, R. M. L (1996) Clinical experience with preimplantation diagnosis of the †F508 deletion causing cystic fibrosis. Prenat Diagn (in press).
Lesko, J., Snabes, M., Handyside, A. H., and Hughes, M. (1991) Amphficatton of the cystic fibrosis DF508 mutation from single cells: applmations toward genetic diagnosis of the preimplantation embryo. Am. J. Hum. Genet. 49(4), 223 (abstract).
Ray, P. F. (1996) Increasing the denaturation temperature during the first cycles of nested amplification reduces allele dropout from single cells for preimplantation genetic diagnosis. Mol. Hum. Reprod (in press).
Zhang, L., Cui, X., Schmitt, K., Hubert, R., Navidt, W., and Amheim, N. (1992) Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl. Acad Sci USA 89, 5847–5851.
Jeffreys, A. J., Wilson, V., and Thein, S. L. (1985) Hypervariable “mimsatellite” regions in human DNA. Nature 314, 67–73.
Nakamura, Y., Leppert, M., O’Connell, P., Wolff, R., Holm, T., Culver, M., et al. (1987) Vanable number of tandem repeat (VNTR) markers for human gene mapping. Science 235, 1616–1622.
Ray, P. F., Harper, J., Mountford, R., Elles, R., and Handyside, A. H. (1992) Analysis of simple tandem repeats following whole genome amplification from single cells for preimplantation diagnosis of Duchenne muscular dystrophy. J Reprod Fert. Abstr. Series No. 10, 52.
Knstjansson, K., Chong, S. S., Van den Veyver, I. B., Subramanian, S., Snabes, M. C., and Hughes, M. R (1994) Prelmplantation single cell analyses of dystrophin gene deletions using whole genome amphfication. Nature Genet 6, 19–23.
Fmdlay, I., Ray, P., Qmrque, P., Rutherford, A., and Lilford, R. (1995) Allelic drop-out and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and cystic fibrosis. Hum. Reprod 10, 1609–1618.
Findlay, I., Urquart, A., Quirque, P., Sullivan, K. M., Rutherford, A, and Lilford, R. (1995) Simultaneous DNA “fingerprinting”, diagnosis of sex and single-gene defect status from a single cell. Hum Reprod 10, 1005–1013.
Harper, J. C. (1996) Preimplantation diagnosis of inherited disease. An update of world figures. J Assoc Reprod Genet (in press).
Li, H., Cui, X., and Amheim, N. (1991) A Companton to Methods in Enzymology, Academic, New York.
Nakahon, Y., Hamanao, K., Iwaya, M., and Nakagome, Y. (1991) Sex identification by polymerase chain reaction using X-Y homologous primer. Am J Med Genet 39, 472,473.
Handyside, A. H., Pattinson, J. K., Penketh, R. J., Delhanty, J. D., Winston, R. M., and Tuddenham, E. G. (1989) Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet 1, 347–349.
Delhanty, J. D. A., Griffin, D. K., Handyside, A. H., Harper, J., Atkinson, G. H. G., Pieters, M. H. E. C., and Winston, R. M. L. (1993) Detection of aneuploidy and chromosomal mosaicism in human embryos during preimplantation sex determination by fluorescent in situ hybridization (FISH). Hum Mol. Genet 2(8), 1183–1185.
Chong, S. S., Kristjansson, K., Cota, J., Handyside, A. H., and Hughes, M. R. (1993) Prelmplantation diagnosis of X-linked disease: reliable and rapid sex determination of single human cells by restriction site analysis of simultaneously amphfied ZFX and ZFY sequences. Hum Mol. Genet 8, 1187–1191.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Ray, P.F., Handyside, A.H. (1996). PCR from Single Cells for Preimplantation Diagnosis. In: Elles, R. (eds) Molecular Diagnosis of Genetic Diseases. Methods in Molecular Medicine™, vol 5. Humana Press. https://doi.org/10.1385/0-89603-346-5:245
Download citation
DOI: https://doi.org/10.1385/0-89603-346-5:245
Publisher Name: Humana Press
Print ISBN: 978-0-89603-346-7
Online ISBN: 978-1-59259-589-1
eBook Packages: Springer Protocols