Abstract
Purpose: Single-cell polymerase chain reaction (PCR) requires efficient amplification and accurate detection. We compare the accuracy of heteroduplex, fluorescent-fragment, and fluorescent single-strand conformation polymorphism (F-SSCP) analysis as detection systems for analysis of a PCR assay developed for preimplantation genetic diagnosis.
Methods: A single-cell, fluorescent multiplex PCR assay was developed for the cystic fibrosis ΔF508 mutation and the short tandem repeat, D21S11. Detection systems were compared by analyzing blinded PCR products.
Results: Amplification rates for cystic fibrosis were 89% by heteroduplex and 91% by fragment analysis, while it was 72% for D21S11 by fragment analysis. No difference in allele dropout was detected for cystic fibrosis by any method (2%). Overall accuracy was high, >97%, although SSCP was the least accurate.
Conclusions: Heteroduplex and fragment analysis proved equal in the diagnosis of a single amplified locus. We determined that fragment analysis allows maximal accuracy of detection and permits analysis of a second loci, controlling for DNA contamination and allelic dropout.
Keywords: allele dropout, fluorescence multiplex PCR, preimplantation genetic diagnosis, short tandem repeats
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REFERENCES
- 1.Verlinsky Y, Rechitsky S, Cieslak J, Ivakhnenko V, Wolf G, Lifchez A, Kaplan B, Moise J, Walle J, White M, Ginsberg N, Strom C, Kuliev A. Preimplantation diagnosis of single gene disorders by two-step oocyte genetic analysis using first and second polar body. Biochem Mol Med. 1997;62(2):182–187. doi: 10.1006/bmme.1997.2635. [DOI] [PubMed] [Google Scholar]
- 2.Lavery SA, Aurell R, Turner C, Taylor DM, Winston RM. An analysis of the demand for and cost of preimplantation genetic diagnosis in the United Kingdom. Prenat Diagn. 1999;19(13):1205–1208. [PubMed] [Google Scholar]
- 3.Kuliev A, Rechitsky S, Verlinsky O, Ivanhnenko V, Cieslak J, Evsikov S, Wolf G, Angastiniotis M, Kalakoutis G, Strom C, Verlinsky Y. Birth of healthy children after preimplantation diagnosis of thalassemias. J Assist Reprod Genet. 1999;16(4):207–211. doi: 10.1023/A:1020316924064. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Sermon K, Goosens V, Seneca S, Lissens W, De Vos A, Vandervorst M, Van Steirteghem A, Liebaers I. Preimplantation diagnosis for Huntington's disease (HD): Clinical application and analysis of the HD expansion in affected embryos. Prenat Diagn. 1998;18(13):1427–1436. doi: 10.1002/(sici)1097-0223(199812)18:13<1427::aid-pd493>3.0.co;2-3. [DOI] [PubMed] [Google Scholar]
- 5.Sermon K, De Vos A, Van de Velde H, Seneca S, Lissens W, Joris H, Vandervorst M, Van Steirteghem A, Liebaers I. Fluorescent PCR and automated fragment analysis for the clinical application of preimplantation genetic diagnosis of myotonic dystrophy (Steinhert's disease) Mol Hum Reprod. 1998;4(8):791–796. doi: 10.1093/molehr/4.8.791. [DOI] [PubMed] [Google Scholar]
- 6.el-Hashemite N, Delhanty JD. A technique for eliminating allele specific amplification failure during DNA amplification of heterozygous cells for preimplantation diagnosis. Mol Hum Reprod. 1997;3(11):975–978. doi: 10.1093/molehr/3.11.975. [DOI] [PubMed] [Google Scholar]
- 7.Ao A, Wells D, Handyside AH, Winston RM, Delhanty JD. Preimplantation genetic diagnosis of inherited cancer: Familial adenomatous polyposis coli. J Assist Reprod Genet. 1998;15(3):140–144. doi: 10.1023/A:1023008921386. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Findlay I, Ray P, Quirke P, Rutherford A, Lilford R. Allelic drop-out and preferential amplification in single cells and human blastomeres: Implications for preimplantation diagnosis of sex and cystic fibrosis. Hum Reprod. 1995;10:1609–1618. doi: 10.1093/humrep/10.6.1609. [DOI] [PubMed] [Google Scholar]
- 9.Hattori M, Yoshioka K, Sakaki Y. High-sensitive fluorescent DNA sequencing and its application for detection and massscreening of point mutations. Electrophoresis. 1992;13:560–565. doi: 10.1002/elps.11501301114. [DOI] [PubMed] [Google Scholar]
- 10.Blake D, Tan SL, Ao A. Assessment of multiplex fluorescent PCR for screening single cells for trisomy 21 and single gene defects. Mol Hum Reprod. 1999;5(12):1166–1175. doi: 10.1093/molehr/5.12.1166. [DOI] [PubMed] [Google Scholar]
- 11.Cui XF, Li HH, Goradia TM, Lange K, Kazazian HHJ, Galas D, Arnheim N. Single-sperm typing: Determination of genetic distance between the G gamma-globin and parathyroid hormone loci by using the polymerase chain reaction and allele-specific oligomers. Proc Natl Acad Sci USA. 1989;86:9389–9393. doi: 10.1073/pnas.86.23.9389. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Holding C, Bentley D, Roberts R, Bobrow M, Mathew C. Development and validation of laboratory procedures for preimplantation diagnosis of Duchenne muscular dystrophy. J Med Genet. 1993;30(11):903–909. doi: 10.1136/jmg.30.11.903. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Handyside AH, John LG, Tarin JJ, Winston RML, Hughes MR. Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis. New Engl J Med. 1992;327:905–909. doi: 10.1056/NEJM199209243271301. [DOI] [PubMed] [Google Scholar]
- 14.Wu R, Cuppens H, Buyse I, Decorte R, Marynen P, Gordts S, Cassiman JJ. Co-amplification of the cystic fibrosis delta F508 mutation with the HLA DQA1 sequence in single cell PCR: Implications for improved assessment of polar bodies and blastomeres in preimplantation diagnosis. Prenat Diagn. 1993;13:1111–1122. doi: 10.1002/pd.1970131206. [DOI] [PubMed] [Google Scholar]
- 15.Rechitsky S, Strom C, Verlinsky O, Amet T, Ivakhnenko V, Kukharenko V, Kuliev A, Verlinsky Y. Allele dropout in polar bodies and blastomeres. J Assist Reprod Genet. 1998;15(5):253–257. doi: 10.1023/A:1022532108472. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Dreesen JC, Jacobs LJ, Bras M, Herbergs J, Dumoulin JC, Geraedts JP, Evers JL, Smeets HJ. Multiplex PCR of polymorphic markers flanking the CFTR gene: A general approach for preimplantation genetic diagnosis of cystic fibrosis. Mol Hum Reprod. 2000;6(5):391–396. doi: 10.1093/molehr/6.5.391. [DOI] [PubMed] [Google Scholar]
- 17.Lewis CM, Pinel T, Whittaker JC, Handyside AH. Controlling misdiagnosis errors in preimplantation genetic diagnosis: A comprehensive model encompassing extrinsic and intrinsic sources of error. Hum Reprod. 2001;16:43–50. doi: 10.1093/humrep/16.1.43. [DOI] [PubMed] [Google Scholar]
- 18.Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA. 1989;86:2766–2770. doi: 10.1073/pnas.86.8.2766. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Grifo JA, Giatras K, Tang YX, Krey LC. Successful outcome with day 4 embryo transfer after preimplantation genetic diagnosis for genetically transmitted diseases. Hum Reprod. 1998;13(6):1656–1659. doi: 10.1093/humrep/13.6.1656. [DOI] [PubMed] [Google Scholar]
- 20.Iwarsson E, Ahrlund-Richter L, Inzunza J, Fridstrom M, Rosenlund B, Hillensjo T, Sjoblom P, Nordenskjold M, Blennow E. Preimplantation genetic diagnosis of DiGeorge syndrome. Mol Hum Reprod. 1998;4(9):871–875. doi: 10.1093/molehr/4.9.871. [DOI] [PubMed] [Google Scholar]
- 21.Gianaroli L, Magli MC, Munné S, Fortini D, Ferraretti AP. Advantages of day 4 embryo transfer in patients undergoing preimplantation genetic diagnosis of aneuploidy. J Assist Reprod Genet. 1999;16(4):170–175. doi: 10.1023/A:1020356605408. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Verlinsky Y, Kuliev A. Preimplantation genetics. J Assist Reprod Genet. 1998;15(5):215–218. doi: 10.1023/A:1022536005746. [DOI] [PMC free article] [PubMed] [Google Scholar]