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Molecular Human Reproduction vol.2 no.3 pp. 213-218, 1996 Increasing the denaturation temperature during the first cycles of amplification reduces allele dropout from single cells for preimplantation genetic diagnosis Pierre F.Ray1 and Alan H.Handyside Human Embryology Laboratory, Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK ^o whom correspondence should be addressed Introduction DNA amplification from single cells, typically using nested polymerase chain reaction (PCR), has been applied widely for fine genetic mapping over short distances using single spermatozoa (Li et al, 1988; Boehnke et al, 1989), the detection of low levels of viral or bacterial pathogens (Bej and Mahbubani, 1992; Zimmerman et al, 1993) and for examining clonal genetic alterations in tumour progression (Trumper et al, 1993). AJI important clinical application has been for preimplantation genetic diagnosis (PGD) in which single cells removed from human preimplantation embryos following in-vitro fertilization (TVF) are analysed for a specific genetic defect causing an inherited disease (Coutelle et al, 1989; Holding and Monk, 1989). Embryos are biopsied early on the third day post-insemination (day 3) at the 6-10 cell stage and nested PCR used for genetic analysis of one or two single cells. Unaffected embryos are then selected and transferred to the uterus later the same day. This allows couples at risk of transmitting a particular gene defect to avoid the possibility of a termination following diagnosis later in pregnancy after amniocentesis in the second or chorion villus sampling (CVS) in the first trimester of pregnancy (Handyside, 1993). For PGD, single cell PCR needs to be highly efficient and © European Society for Human Reproduction and Embryology allow accurate detection of the genetic defect causing the disease. With cystic fibrosis (CF), several nested PCR protocols have been reported for amplification of regions of exon 10 of cystic fibrosis transmembrane regulatory (CFTR) gene from single cells for detection of the predominant AF508 deletion (Strom et al., 1990; Lesko et al, 1991; Liu et al, 1992). Our protocol amplifies the whole of exon 10 with high efficiency, and detection of the AF5O8 deletion by heteroduplex formation was completely accurate in a series of single cells and blastomeres disaggregated from cleavage stage embryos (Lesko et al, 1991). A singleton pregnancy was established after PGD and the baby girl confirmed at birth to be unaffected (Handyside et al, 1992). Since then four more pregnancies and singleton births, all confirmed unaffected, have been achieved in a total of 16 PGD cycles (Ao et al, 1996). To confirm the diagnoses in these later PGD cycles and to investigate their accuracy, blastomeres from human embryos which were not transferred were disaggregated and each single cell amplified (P.Ray, R.M.L.Winston and A.H.Handyside, in preparation). With presumed homozygous normal or affected embryos the accuracy of the diagnosis was high. In only four out of 366 cells would a serious misdiagnosis have occurred because of a homozygous normal or heterozygous carrier diagnosis in an otherwise uniformly homozygous affected 213 Downloaded from http://molehr.oxfordjournals.org/ at Pennsylvania State University on March 4, 2014 Single cell polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD) needs to be highly efficient and accurate. In some single cells from human embryos presumed to be heterozygous for the AF508 deletion causing cystic fibrosis (CF), we recently observed random amplification failure of one of the two parental alleles following nested PCR. To investigate allele dropout (ADO), we have examined two different lysis protocols and the effect of altering the denaturation temperature in the primary PCR using single lymphocytes heterozygous for AF508 or for two fi-thalassaemia mutations IVS 1 nt 1 (G/T) and 5 (G/C) using a nested PCR protocol to amplify the 5' region of the p*-globin gene. Amplification rates were high after lysis in either water or lysis buffer and at all denaturation temperatures studied (> 92%). With a typical denaturation temperature (93°C), ADO was detected at both loci. When the denaturation temperature was lowered to 90°C, however, ADO increased substantially and conversely by raising the denaturation temperature to 96°C during the first 10 cycles ADO was reduced but not eliminated. ADO was also reduced with cells in lysis buffer. We suggest that ADO may be caused by a combination of inefficient denaturation and degradation of one of the genomic alleles in the first cycles of PCR. For autosomal recessive conditions in which both parents are carrying the same mutation, ADO would not cause serious misdiagnosis. For compound heterozygotes or autosomal dominant conditions, however, extensive testing of the amplification protocol with single heterozygous cells and individual calibration of each thermocycler for the effect of denaturation temperature on ADO is essential before clinical application. Key words: allele dropout/polymerase chain reaction/preimplantation genetic diagnosis/single cell P.F.Ray and A.H.Handyside embryo (presumably as a result of contamination). More striking, however, was the relatively frequent failure to amplify either the normal (8%) or affected (10%) parental allele, apparently randomly, in blastomeres from heterozygous carrier embryos. In these cases, where both partners carry the AF508 mutation, this random failure of amplification of one of the alleles, allele dropout (ADO), would not result in a serious misdiagnosis since CF is autosomal recessive. For compound heterozygotes or autosomal dominant conditions, however, this could result in the transfer of an affected embryo. To investigate the phenomenon of ADO and to distinguish between these possibilities, the effects of different lysis conditions and denaturation temperatures during the first few cycles of primary amplification with the outer primers have been examined using single heterozygous lymphocytes. To eliminate locus-specific effects, both nested PCR for AF508 detection and nested PCR for amplification of part of exon 1 and the 5' end of the intervening sequence 1 (IVS 1) of the p*-globin gene were tested (P.F.Ray, J.S.Kaeda, J.Bingham, T.Vulliamy, I.Dokal, I.Roberts, R.M.L.Winston and A.H.Handyside, in preparation), in this case, with single lymphocytes from a child affected with P-thalassaemia major and heterozygous for both mutations IVS 1 nt 1 (G/T) and 5 (G/C). Materials and methods Preparation of single lymphocytes Unclotted blood (3 ml) in EDTA was gently pipetted onto a layer of Ficoll-paque (Pharmacia, St Albans, Herts., UK), lymphocytes separated by centrifugation and washed twice in phosphate-buffered saline (PBS) supplemented with 10 mg/ml bovine serum albumin (BSA; Sigma, Poole, UK). Using a mouthtube, a finely pulled glass pipette and a dissecting microscope, lymphocytes were loaded from a dilute suspension in PBS + BSA and single lymphocytes distributed to microdrops of the same medium under silicon oil. After checking that a single lymphocyte had been transferred correctly to each microdrop, each of them was transferred to 0.5 ml thin-walled microcentrifuge tubes, loaded with either 10 ul double distilled water (ddH2O) or 5 uJ of lysis buffer (200 mM KOH, 50 mM DTT) (Li et al., 1991), again using a mouth controlled pipette. Samples in ddH2O were frozen to -20°C and prior to PCR, thawed and denatured for 5 min at 95°C. Samples in lysis buffer were either 214 Nested polymerase chain reaction (PCR) Primers for the CF nested PCR were as previously published, resulting in an amplified fragment of 154 or 151 base pairs (bp) depending on the presence or absence of the AF508 deletion (Handyside et al., 1992). Conditions for the denaturation temperature with the outer primers were modified as follows: initial denaturation at 95°C for 3 min then denaturation at 90, 93 or 96 (first 10 cycles) + 94°C (12 cycles) (to avoid excessive denaturation of Taq polymerase). Primers for amplification of the (J-globin sequence were as follows: Outer primers: 5'-TAAGCCAGTGCCAGAAGAGCC 5' -GCA ATC ATTCGTCTGTTTCCC A Inner primers: 5'-GGCATATAAGTCAGGGCAGAG 5'-CCTTAAACCTGTCTTGTAACCTTGGTA IVS 1 nt 1 (G/T) nullifies a natural BsiYl restriction site. The reverse inner primer has been modified to create a Kpnl site at IVS 1 nt 5 when amplifying from normal DNA which is not present if the IVS 1 nt 5 (G/C) mutation is present. Cycling conditions were: (i) initial denaturation at 95°C for 3 min; (ii) 22 cycles with the outer primers, denaturation at 90, 93 or 96 (first 10 cycles) + 94°C (12 cycles) for 45 s, annealing at 55°C for 45 s and extension at 72°C for 90 s; and (iii) 2 \i\ of the outer reaction product amplified with the inner primers for 30 cycles after an initial denaturation at 95°C for 3 min, denaturation at 94°C for 45 s, annealing at 56°C for 45 s and extension at 72°C for 60 s. Reaction volumes were 50 u.1 overlaid with 50 u.1 of mineral oil. Outer amplification mix contained 5 |il of lysis buffer (200 mM KOH, 50 mM DTT), 5 ul neutralizing buffer (900 mM Tris HC1, pH 8.3, 300 mM KC1, 200 mM HC1) and potassium-free 10XPCR buffer (25 mM MgCl2, 1 mg/ml gelatine, 100 mM Tris HC1, pH 8.3), 0.8 U.M of each primer, 0.2 mM of each nucleotide and 1 IU Taq DNA polymerase (AmpliTaq; Perkin Elmer, Warrington, Cheshire, UK). Inner amplification mix contained 0.8 U.M of each inner primer, 0.2 mM of each nucleotide, 1 IU of AmpliTaq (Perkin Elmer), 5 ul of 1 OX PCR buffer II (Perkin Elmer) and 3 ul of 25 mM MgCl2 (Perkin Elmer). Stringent precautions were taken to minimize contamination. Gloves and dedicated gowns were worn during the preparation of single cells and setting up the primary PCR. All reagents were filtered with 0.22 u.m filters and aliquoted in small volumes. Separate sets of micropipettors were used for the two amplification steps and filtered pipette tips were used throughout. PCR mix and aliquots were prepared in a class II flow cabinet located in a first 'clean lab'. The product of the first PCR was transferred to the secondary PCR mix in a separate air flow cabinet located in a different lab. Gel electrophoresis was performed in a third area located in a different building. No amplified product was brought back to the first lab. Gel electrophoresis and mutation detection The amplification products were run on 10% polyacrylamide gels stained with ethidium bromide and viewed with a UV transilluminator. Amplification of both the normal and AF5O8 alleles from the single heterozygous lymphocytes was detected by the presence of a heteroduplex band (Figure 1). Samples with no heteroduplex band were re-analysed by adding amplified DNA of known genotype to determine which allele had been amplified (Handyside et al., 1992). Downloaded from http://molehr.oxfordjournals.org/ at Pennsylvania State University on March 4, 2014 Although preferential amplification of a particular allele sometimes occurs with conventional PCR, often because of size differences in the amplified fragments (Walsh et al., 1992), random ADO has not been reported with non-limiting target DNA. It seems likely, therefore, that ADO is specific for single cell PCR or at least very low target copy number. With only two target molecules present in a single diploid cell, ADO could arise from strand breaks or other DNA damage during preparation. Alternatively, access to the target genomic sequence by the primers and Taq polymerase may be restricted by, for example, adjacent G/C rich regions reducing denaturation efficiency or different degrees of folding perhaps related to the stage of the cell cycle. In any case, ADO is most likely to arise in the initial cycles of the primary PCR before the number of target molecules is increased by the process of amplification. stored at -20°C or used immediately by heating to 65°C for 10 min. The alkaline lysis buffer was subsequently neutralized by adding modified PCR buffer containing 5 ul of neutralizing buffer (900 mM Tris HC1, pH 8.3, 300 mM KC1, 200 mM HC1). To control for contamination, media from drops that had contained cells were also sampled. Reduced allele dropout in single cell PCR Denaturation Temperature and Allele Drop-Oui Dt° M 1 2 3 4 5 6 7 8 9 10 B B Heteroduplex Homoduplex 90°C 93°C 96°C (a) c o (b) 100 1 100' 801 SO- 0 60- SO 1 40- | "5. a I 20- 28 20- O Q < 90 93 96 90 93 96 90 Denaturation temperature (°C) Figure 2. Amplification rate ( - • - ) and frequency of allelic dropout (ADO) (-D-) at denaturation temperatures of 90, 93 and 96°C. Panel (a) with the cystic fibrosis primers and water lysis; panel (b) with CF primers and alkaline lysis; and panel (c) fi-globin primers and lysis buffer. Error bars are SEM. Heteroduplex Homoduplex Figure 3. Polyacrylamide gel electrophoresis of PCR product after amplification with the inner primers of different ratios of CF AF508 normal and mutant alleles amplified with the outer primers. M = 100 bp molecular marker, tracks 1 = 1:1 dilution of normal/mutant; tracks 2 = 1:2 dilution; tracks 3 = 1:4; tracks 4 = 1:16; track 5 = 1:256. For detection of the IVS 1 mutations in the (3-globin amplified products, 8 u.1 aliquots were digested for 16 h with either 10 IU of BsiYl (Boehringer Mannheim UK Ltd., Lewes, East Sussex, UK) at 55°C for the detection of the ntl mutation or 10 IU of Kpnl (Pharmacia) at 37°C for the detection of the nt 5 mutation. The base pair substitution at IVS I nt 1 (G/T) nullifies a natural BsiYl 215 Downloaded from http://molehr.oxfordjournals.org/ at Pennsylvania State University on March 4, 2014 Figure 1. Polyacrylamide gel electrophoresis of polymerase chain reaction (PCR) product after amplification of single blastomeres heterozygous at the AF508 locus at three different denaturation temperatures. The absence of the heteroduplex band is indicative of allele dropout (ADO). M = molecular marker (pBR322 Haelll digested); 1-10 = single lymphocyte; B = blank control. P.F.Ray and A.H.Handyside Table I. Amplification rate (Amp.) and frequency of allelic dropout (ADO) with the cystic fibrosis or P-globin primers at different denaturation temperature, using a water or alkaline lysis protocol Denaturation temperature 90 93 96 Cystic fibrosis (water protocol) 50 62 78 Cystic fibrosis (lysis buffer) Amp. (%) ADO(%) 92 97 94 70 44 14 40 40 40 fi-globin lysis buffer Amp. (%) ADO(%) 95 97 97 21 13 5 30 32 26 Amp. (%) ADO (%) 93 94 92 46 33 4 n = number of single lymphocytes analysed. Definitions Amplification rate or efficiency was calculated as the proportion of single cells from which at least one allele amplified. The rate or frequency of ADO was calculated as the number of samples in which only one allele amplified divided by the total number in which one or both allelcs amplified. Similarly, with the P-globin primers, nine cells amplified the normal allele for IVS 1 nt 1 (and therefore the affected allele for IVS 1 nt 5) against 14 which amplified the affected allele at IVS 1 nt 1 (normal at IVS 1 nt 5). To test the possibility that ADO is not the result of complete amplification failure but reduced amplification of one allele to a level below the threshold for detection, we amplified mixtures of different proportions of the outer amplification products from homozygous normal and affected cells (AF508) (Figure 3). With increasing ratios from 1:1 to 1:256 of normal:affected outer amplification product the intensity of the heteroduplex band decreased markedly. However, even at the lowest ratio tested of 1:256 a very faint heteroduplex band could still be detected by careful inspection of the gel. Discussion Results A total of 310 single lymphocytes heterozygous for AF508 (190 lysed in ddH2O and 120 in alkaline lysis buffer) and 88 single lymphocytes heterozygous for mutations at both IVS 1 nt 1 and 5 of the p-globin gene were analysed. The efficiency of amplification and frequency of ADO under the different conditions tested are shown in Table I and Figure 2. Amplification rates were high after lysis in either ddH2O or lysis buffer and at all denaturation temperatures studied (5» 92%). With a typical denaturation temperature (93°C) during the primary PCR, ADO was detected at both loci and ranged between 13-44%. When the denaturation temperature was lowered to 90°C, ADO increased substantially (21-70%). Conversely at 96°C during the first 10 cycles, ADO was reduced but not eliminated (4-14%). ADO was also reduced by use of a lysis buffer irrespective of the denaturation temperature used. Contamination occurred in eight out of 56 (14%) control blanks analysed for AF508. As all the cells amplified here are heterozygotes, contamination can only result in a small reduction in the occurrence of ADO. Therefore, we do not think it compromises the results presented here. With the P-globin primers, 0/24 blanks amplified. Typical amplifications with CF primers at the three denaturation temperatures tested are shown in Figure 1. ADO was not allele-specific at any of the denaturation temperatures tested. After amplification with the CF primers 36 cells had amplified only the normal allele and 31 only the affected allele (16716 at 90°C, 15/10 at 93°C and 5/5 at 96°C had amplified the normal/affected allele respectively). 216 This study clearly demonstrates that random failure of amplification of one parental allele, allele dropout (ADO), can occur following nested PCR with some single heterozygous lymphocytes with both sets of primers for amplification of exon 10 of the CFTR gene and detection of the AF508 deletion causing CF, and primers for amplification of part of the Pglobin gene and detection of IVS 1 nt 1 (G/T) and nt 5 (G/C) mutations causing P-thalassaemia. There was a strong correlation between the rate of ADO and the denaturation temperature used during the first few cycles of amplification although the overall amplificate rate was not affected (Figure 2). The use of more stringent lysis conditions, using a lysis buffer originally developed for amplification from single sperm (Li et al., 1991), also decreased the occurrence of ADO at all three denaturation temperatures tested. These two points stress the need for efficient separation of the native double-stranded DNA to allow adequate amplification of both alleles. ADO was first detected following PGD in a series of couples at risk of having children with CF because both partners carry the AF508 deletion (Ray et al, 1994, 1995). The proportion of heterozygous carrier embryos diagnosed in these cases, on the basis of analysis of one or two biopsied blastomeres, was lower than the expected ratio of 1:2:1 with homozygous normal and affected embryos. We therefore disaggregated the embryos which were not transferred to confirm the diagnosis and analyse multiple single cells from each embryo. This demonstrated that, although single cell analysis of homozygous normal or affected embryos was accurate and highly consistent, Downloaded from http://molehr.oxfordjournals.org/ at Pennsylvania State University on March 4, 2014 restriction site, and a mismatch in the reverse inner primer was deliberately introduced to create a new Kpnl site when amplifying the normal nt 5 allele which is abolished by the IVS 1 nt 5 (G/C) mutation. Homozygous normal cells show a single band reduced in size to 173 bp, or 184 bp when restricted with BsiYl or Kpnl, respectively. Heterozygous cells give rise to both undigested (208 bp) and digested bands (173/184 bp). Products of known genotype were digested at the same time to control for partial digestion. Analysis of both mutations in the same amplified product also acted as an internal control. Reduced allele dropout in single cell PCR amplified, the relative amounts of both products were normally distributed with a small shift towards preferential amplification of the larger, normal allele. In contrast, ADO (around 5%) appeared to be random between the two alleles as demonstrated here. If the probability of the genomic target sequence to denature is < 1, one allele could begin to amplify in an earlier cycle than the other. Assuming that short amplification products have an equal and high probability of amplification, quantitative differences in the relative amounts of amplified fragments from both alleles are likely to be maintained in subsequent cycles and could account for this distribution. However, for one allele apparently to fail to amplify after complete nested amplification because the amount of amplified product is undetectable, our mixing experiments suggest that the difference would have to be of the order of 1:250, or less, equivalent to amplification failure for the first eight cycles of the primary PCR. This seems unlikely and is not compatible with the very high efficiency of amplification of at least one allele irrespective of the lysis or cycling conditions. We therefore propose that ADO is caused by a combination of suboptimal PCR conditions and degradation of genomic target sequences during the initial cycles. In addition, to explain the high efficiency of amplification of at least one allele, we suggest that successful denaturation, primer annealing and extension may inhibit or prevent degradation of the allele being amplified, during that cycle. A correlation between complete amplification failure and ADO would still be predicted but would be much less pronounced and perhaps not detectable without analysis of a large series of single cells. In support of this hypothesis, preliminary experiments in which single cells with all PCR reagents minus dNTPs have been subjected to five cycles at a suboptimal denaturation temperature (90°C), dNTPs added and nested amplification completed with optimal denaturation temperature (96°C) in the first 10 cycles have demonstrated that the rate of ADO is equivalent to nested amplification at the suboptimal denaturation temperature. This strongly suggests that some genomic targets have been destroyed in the preliminary cycles before amplification under optimal conditions commenced. If this early degradation of genomic targets is caused by residual cellular endonucleases or degradative enzymes from other sources it could explain failure to detect ADO in previous studies because of subtle differences in the preparation of cells or purity of the reagents used. Also, differences in the temperature calibration of different thermal cyclers could have a pronounced effect on denaturation efficiency which in turn would affect the probability of degradation before amplification had commenced from each allele. Preliminary work with single heterozygous cells is therefore essential to establish the optimal denaturation temperature to minimize ADO without compromising amplification efficiency because of excessive degradation of the Taq polymerase. Further work is necesssary to explore this possibility and investigate whether inhibitors of DNases, for example, can reduce or eliminate the incidence of ADO. Recent quantitative analysis of by fluorescent PCR suggests that fication and ADO occur following heterozygous cells (Findlay et al, Aknowledgements the primary CF product both preferential ampliamplification from single 1995). When both alleles We thank Professor Robert M.L.Winston who generously supported this work and Professor Norman Arnheim for helpful comments. 217 Downloaded from http://molehr.oxfordjournals.org/ at Pennsylvania State University on March 4, 2014 in carrier embryos only one parental allele amplified in about 20% of cells, equally distributed between the parental alleles. With blastomeres, however, it is known that amplification efficiencies tend to be lower (Pickering et al, 1992) and some ADO could be related to the high levels of chromosomal mosaicism which have been detected at these stages by fluorescence in-situ hybridization (Harper et al, 1995; Munn6 et al, 1994). For an autosomal recessive disease such as CF, ADO should not cause a serious diagnosis in which an affected embryo is misdiagnosed and transferred, at least in cases in which both partners are carrying the same mutation. In this situation, unaffected heterozygous carrier embryos would be diagnosed either as homozygous normal or affected and the latter excluded from transfer. In cases in which the partners are carrying different mutations, however, it is essential to detect the heterozygous state for both mutations. Both misdiagnoses of CF to date have been in couples of this kind (Harper and Handyside, 1994) and ADO could have been a contributory cause. Similarly, for dominantly inherited conditions, it will be essential to minimize the incidence of ADO. For these even the minimal rate of ADO achieved here, around 5%, may not be an acceptable reduction in risk. The reason for ADO is not known. The occurrence of ADO with both sets of primers for different genes seems to rule out any explanation based on the quality of the primers or locusspecific effects resulting from, for example, polymorphic sequences. Amplification failure from one or both target sequences in single haploid or diploid cells has been welldocumented (Li et al, 1988; Boehnke et al, 1989) and amplification failure of a highly repeated sequence specific for the Y chromosome from a single blastomere is the most likely cause of the misdiagnosis of sex in an early case of PGD for X-linked disease (Handyside and Delhanty, 1993). Previous work with single cells, however, prior to application for PGD has failed to detect ADO with a number of different primer sets and cycling conditions including those used here for detection of the AF508 deletion in CF (Lesko et al, 1991; Strom et al, 1991). Although preferential amplification of a particular allele sometimes occurs with conventional PCR, often because of size differences in the amplified fragments (Walsh et al, 1992), ADO has not been reported with non-limiting target DNA. It seems likely, therefore, that ADO is specific for single cell PCR or at least very low target copy number. With only two target molecules present in a single diploid cell, ADO could result from strand breaks or other DNA damage during preparation for PCR. Some ADO or indeed complete amplification failure may be caused in this way and the reduction observed with lysis using an alkaline lysis buffer could in part reflect protection from, for example, endogenous nucleases. However, the strong correlation between the initial denaturation temperature and ADO indicates that most ADO originates in the initial cycles of the primary PCR. 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(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. Handyside, A.H. (1993) Diagnosis of inherited disease before implantation. Reprod. Med. Rev. 2, 51-61. Handyside, A.H., Lesko, J.G., Tan'n, J.J. et al. (1992) Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis. N. Engl. J. Med, 327, 905-909. Handyside, A.H. and Delhanty, J.D.A. (1993) Cleavage stage biopsy of human embryos and diagnosis of X chromosome-linked recessive diseases. In Edwards, R.G. (ed.). Preconception and Preimplantation Diagnosis of Human Genetic Disease. Cambridge University Press, Cambridge, pp. 239-270. Harper, J.C. and Handyside, A.H. (1994) The current status of preimplantation diagnosis. Curr. Obstet. Gynecol., 4, 143-149. Harper, J.C, Coonen, E., Handyside, A.H. et al. (1995) Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenat. Diagn., 15, 41—49 Holding, C. and Monk, M.(1989) Diagnosis of beta-thalassaemia by DNA amplification in single blastomeres from mouse preimplantation embryos. Lancet, II, 532-535. Lesko, J., Snabes, M., Handyside, A. H. and Hughes, M.(199I) Amplification of the cystic fibrosis DF5O8 mutation from single cells: applications toward genetic diagnosis of the preimplantation embryo. (Abstr.) Am. J. Hum. Genet., 49 (4), 223. Li, A., Gyllenstein, U.B., Cui, X. et al. (1988) Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature, 335, 414-419. Li, H., Cui, X. and Amheim, N. (1991) Analysis of DNA sequence variation in single cells. In Methods: A Companion to Methods in Enzymologv. Vol. 2. Academic Press, New York, USA pp. 49-59. Liu, J., Lissens, W.. Devroey, P. et al. (1992) Efficiency and accuracy of polymerase-chain-reaction assay for cystic fibrosis allele delta F5O8 in single cell. Lancet, 339, 1190-1192. Munne\ S., Weier, H.U., Grif6, J. and Cohen, J. (1994) Chromosome mosaicism in human embryos. Biol. Reprod., 51, 373-379. Pickering, SJ., McConnell, J.M., Johnson, M.H. and Braude, P.R.(I992) Reliability of detection by polymerase chain reaction of the sickle cellcontaining region of the p^-globin gene in single human blastomercs. Hum. Reprod., 7, 630-636. 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 (Abstract) In Bio/Technology, Short Reports, Advances in Gene Technology: Molecular Biology and Human Genelic Disease 55, 46. Ray, P.F., Winston, R.M.L. and Handyside, A.H. (1995) Elimination of allele drop-out (ADO) in single cell analysis for diagnosis of cystic fibrosis (CF). (Abstr.) Hum. Reprod., 10, (Abstract Book 2), 131. Strom, CM., Verlinsky, Y., Milayeva, S. et al (1990) Preconception genetic diagnosis of cystic fibrosis [letter]. Lancet, 336. 306-307 Strom, CM., Rechitsky, S. and Verlinsky. Y. (1991) Reliability of gender determination using the polymerase chain reaction (PCR) for single cells. J. In Vitro Fertil. Embryo. Transfer, 8, 225-229. Trumper, L.H., Brady, G., Bagg, A. et al. (1993) Single-cell analysis of Hodgkin and Reed-Stemberg cells: molecular heterogeneity of gene expression and p53 mutations. Blood, 81, 3097-3115. Walsh, P.S., Erlich, H.A. and Higuchi, R. (1992) Preferential PCR amplification of alleles: mechanisms and solutions. PCR Methods Appl., 1, 241-250. 218 Zimmerman, K., Pischinger, K. and Mannhalter, J. W. (1993) Rapid nonradioacuve detection of HIV-1 RNA from a single-cell equivalent by reverse transcription PCR with nested primers. Biotechniques. 15, 806-808.