DNA fingerprinting of sister blastomeres from human IVF embryos

M.G. Katz1,2,3, A.O. Trounson1 and D.S. Cram1,2

1 Centre for Early Human Development, Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria and 2 Monash IVF, Melbourne, Australia


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Previously published single cell DNA fingerprinting systems have been plagued by high rates of allele drop-out (ADO) and preferential amplification (PA) preventing clinical application in preimplantation genetic diagnosis. METHODS: Tetranucleotide microsatellite markers with high heterozygosity, known allelic size ranges and minimal PCR stutter artefacts were selected for chromosomes X, 13, 18 and 21 and optimized in a multiplex fluorescent (FL)-PCR format. FL-PCR products were analysed using the ABI Prism 377 DNA sequenator and Genescan software. Validation of the DNA fingerprinting system was performed on single diploid (n = 50) and aneuploid (n = 25) buccal cells and embryonic blastomeres (n = 21). RESULTS: The optimized pentaplex PCR DNA fingerprinting system displayed a high proportion of successful amplifications (>91%) and low ADO and PA (<6%) when assessed on 50 human buccal cells. DNA fingerprints of single cells from a subject with Down's syndrome detected the expected tri-allelic pattern for the chromosome 21 marker, confirming trisomy 21. In a blind study on 21 single blastomeres, all embryos were identifiable by their unique DNA fingerprints and shared parental alleles. CONCLUSIONS: A highly specific multiplex FL-PCR based on the amplification of five highly polymorphic microsatellite markers was developed for single cells. This finding paves the way for the development of a more complex PCR DNA fingerprinting system to assess aneuploidy and single gene mutations in IVF embryos from couples at genetic risk.

Key words: aneuploidy/DNA fingerprinting/embryonic blastomeres/single cell PCR


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chromosomal abnormalities such as aneuploidies are associated with human reproductive failure and early embryonic loss (Munné et al., 1993Go, 1995Go). Aneuploidies originate from meiotic non-disjunction, predominantly observed in the first meiotic division of the oocyte or sperm (Antonarakis et al., 1992Go) and increase with advanced maternal age (Verlinsky et al., 1998Go). Aneuploidies can also arise post-fertilization from mitotic non-disjunction in the second and third cleavage divisions that culminate in chromosomal mosaicism and a mixture of normal, monosomic and trisomic cells (Delhanty and Handyside, 1995Go). Various aneuploidies have been observed in cleavage stage embryos as well as the inner cell mass cells of IVF blastocysts (Evsikov and Verlinsky, 1998Go; Magli et al., 2000Go). The most common aneuploidies seen in spontaneous abortions are trisomies involving chromosomes 13, 16, 18, 21 and 22, and monosomy X (Boué and Boué, 1976Go; Eiben et al., 1990Go; Vidal et al., 1998Go). Although these chromosomal abnormalities are generally associated with implantation failure or miscarriage in the first trimester, a very small proportion can develop to term. The remaining possible trisomies or monosomies occurring in the preimplantation stage almost always result in embyronic lethality (Boué et al., 1985Go; Sandalinas et al., 2000Go).

To improve outcomes of IVF patients with a poor prognosis for pregnancy due to advanced maternal age (>35 years), a history of unexplained recurrent miscarriages and repeated failed IVF (more than three cycles), fluorescent in-situ hybridization (FISH) has been performed to identify euploidy in oocyte polar bodies or embryonic blastomeres using up to nine different chromosomal probes on one fixed nucleus (Gianaroli et al., 1997aGo,bGo; Magli et al., 1998Go; Munné et al., 1998Go; Verlinsky et al., 1998Go). FISH analysis of embryos from these patient groups consistently reveals aneuploidy and mosaicism rates of up to 50% (Munné et al., 1994Go; Harper et al., 1995Go; Kuo et al., 1998Go; Magli et al., 1998Go; Verlinsky et al., 1998Go; Gianaroli et al., 1999Go; Bielanska et al., 2000Go). In most clinical preimplantation genetic diagnosis (PGD) programmes, the selection of euploid embryos for transfer has resulted in higher implantation and ongoing pregnancy rates for poor prognosis patients, in particular for women of advanced maternal age (Magli et al., 1998Go; Damario et al., 1999Go; Giarnaroli et al., 1999; Rubio et al., 2000Go). More recently, efforts have focused on development of new methods to determine the numeracy of all 23 pairs of human chromosomes, including comparative genomic hybridization (CGH) (Wells and Delhanty, 2000Go; Vouillaire et al., 2000Go), spectral karyotyping (Marquez et al., 1998Go) and nuclear conversion (Verlinsky and Evsikov, 1999Go). However, any added clinical benefit of these methods over FISH has yet to be assessed in a large randomized trial.

Conventional and fluorescent polymerase chain reaction (FL-PCR) incorporating various microsatellite markers (Mansfield, 1993Go; Muggleton-Harris et al., 1993Go; Pickering et al., 1994Go; Pickering and Muggleton-Harris, 1995Go; Pertl et al., 1994Go) has been investigated for potential application in PGD. It has been shown that single cell multiplex FL-PCR can simultaneously provide information on multiple loci including identity of sex, an individual DNA fingerprint and genetic status for inherited conditions such as cystic fibrosis (Findlay et al., 1995Go; Findlay and Quirke, 1996Go). FL-PCR DNA fingerprinting using chromosome-specific microsatellite markers has also been used in prenatal diagnosis for the detection of chromosomal aneuploidies (Mansfield, 1993Go; Pertl et al., 1994Go, 1996Go) and on single cells (Sherlock et al., 1998Go; Findlay et al., 1999Go). Theoretically, the amount of DNA produced in FL-PCR amplification is proportional to the quantity of the initial target sequence when strict experimental conditions are adhered to, and thus allelic ratios for any particular locus can be calculated from the final fluorescent yield (Ferre, 1992Go; Wells and Sherlock, 1998Go). Accordingly, disomy can be defined by an allelic ratio of 1:1, whereas a trisomy can either be defined as a tri-allelic pattern with an allelic ratio of 1:1:1 or a double dosage di-allelic pattern with an allelic ratio of 2:1. Previously published DNA fingerprinting systems developed for single cells, where the target DNA is in the order of 6 pg, have been plagued with several problems, including high rates of either preferential allelic amplification (PA) or allelic drop-out (ADO) (Sherlock et al., 1998Go; Findlay et al., 1998Go, 1999Go). ADO is defined as the total amplification failure of one allele at a heterozygous locus so that only one allele is detectable after the analysis of the PCR product, whereas PA is the under-representation of one of the two heterozygous alleles resulting in a distortion from the expected 1:1 allelic ratio. The effect of ADO, PA or both reduces the degree of reliability for quantitation of FL-PCR products at the single cell level.

A reliable single cell PCR DNA fingerprinting system would be a very powerful tool in PGD for unique identification of a DNA sample and a means to simultaneously detect specific gene defects and chromosomal aneuploidy. No other current technique has the potential for such a multitude of diagnoses. To this end, we developed a new single cell PCR DNA fingerprinting system based on multiplex FL-PCR amplification of five highly polymorphic microsatellite markers located on four different chromosomes and evaluated its performance on both buccal cells and blastomeres from cleavage stage IVF embryos.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Isolation of single human buccal cells
Buccal cell samples were collected by twirling a cytology brush (EndoScanPlus; Medico, USA) on the inner cheek for 30 s. Cells were collected into an 1.5 ml Eppendorf tube containing 750 µl of phosphate buffer solution (PBS) (Gibco Life Technologies, Australia), washed twice with PBS, and resuspended in 500 µl of PBS. A 10 µl aliquot was examined for single nucleated cells under an inverted microscope (Leica MS5) and several intact single cells aspirated into finely pulled 22.9 cm long glass Pasteur pipettes (Becton Dickinson, USA). Single cells were successively washed through three further 5 µl drops of PBS buffer and transferred together with 1–2 µl of PBS buffer into a sterile 0.2 ml PCR tube on ice. Using the same pipette, 1–2 µl of PBS buffer from the last wash droplet was transferred into a second sterile 0.2 ml PCR tube on ice to serve as a negative PCR control. Tubes were immediately frozen at –80°C prior to PCR analysis.

Embryo biopsy and FISH
Couples on the PGD programme at Monash IVF underwent standard IVF treatment including ovulation induction, surgical aspiration of the oocytes and sperm collection. Oocytes were fertilized by ICSI. On day 3 of embryonic development, cleavage stage embryos with 5–8 cells were considered suitable for biopsy. Embryos were incubated in Ca2+/Mg2+ free medium prior to zona drilling using acid Tyrode's solution and one or two cells were biopsied.

Isolation of human blastomeres from aneuploid embryos
Aneuploid embryos diagnosed by FISH at day 3 are regarded as genetically abnormal. Nine aneuploid embryos were obtained from patients with either advanced maternal age (>36 years) or repeated IVF failure (>3 cycles) who had a history of infertility. Under guidelines established by the Infertility Treatment Authority in Victoria, aneuploid embryos deemed to be unsuitable for transfer must be left to `succumb' on the bench for 24 h before being available for research. Succumbed embryos were treated with pronase (2 mg/ml in HEPES buffered human tubal fluid culture medium) for 1 min to dissolve the zona pellucida and transferred into Ca2+/Mg2+-free medium to dissociate the blastomeres. Single blastomeres were carefully washed through three 5 µl drops of PBS buffer and transferred together with 1–2 µl of PBS buffer into a sterile 0.2 ml PCR tube on ice.

Microsatellite markers
Five microsatellite markers were used in the pentaplex single cell DNA fingerprinting: D21S1413 (Findlay et al., 1998Go), D18S51 (Straub et al., 1993Go), D13S258 (Toth et al., 1998Go), D13S631 (Sherlock et al., 1998Go) and DXS8377 (Hu et al., 1996Go). Each microsatellite marker was selected for high heterozygosity (average 0.91). Based on available published allelic size ranges, appropriate fluorochrome tags (6-FAM, HEX and TET) were selected, where possible, to avoid overlapping profiles. Primers were synthesized and fluorescently labelled by Applied Biosystems, Australia. All primer pairs were diluted in molecular biology grade H2O (Sigma, Melbourne, Australia) to 200 pmol/µl stock solutions under sterile conditions and stored in aliquots of 100 pmol/µl at –20°C until use. For development of an octaplex single cell DNA fingerprinting system, primers for an additional three microsatellite markers were added to the pentaplex system.

Single cell multiplex FL-PCR
The optimized single cell multiplex FL-PCR developed for the five microsatellite markers consisted of the following: 2.5 µl of 10xTaq PCR Buffer (500 mmol/l KCl, 100 mmol/l Tris–HCl, pH 9.0 and 15 mmol/l MgCl2), 0.5 µl of 10 mmol/l dNTP (200 µmol/l), 0.3 µl of Taq polymerase (5 U/µl) (Amersham Pharmacia Biotech, Sydney, Australia), 11.20 µl molecular biology grade H2O and 10.5 µl of primer mix making a final volume of 25 µl. Multiplex FL-PCR was performed using a Hotstart on the 9700 Thermocycler PCR machine (Applied Biosystems). Reactions were subjected to 35 thermal cycles consisting of denaturation for 45 s at 94°C, annealing for 45 s at 60°C, and extension for 1 min at 72°C. With each single cell multiplex FL-PCR, positive and negative controls were always included to ensure that the PCR reaction mix was functional and none of the reagents were contaminated. Positive control tubes contained 10–20 cells in 1–2 µl of PBS buffer, whereas negative control tubes contained either 1–2 µl of PBS buffer from the last wash droplet and no cell.

Genescan analysis of DNA fingerprints
All PCR products were analysed using the ABI Prism 377 DNA Sequencer and associated Genescan 672 software (Applied Biosystems). PCR product (0.5–1.0 µl) was mixed with 1.54 µl of formamide, 0.15 µl loading buffer and 0.31 µl of Genescan TAMRA internal standard (Applied Biosystems). Samples were denatured at 95°C for 3 min, placed on ice and 2.5 µl loaded into the pre-formed wells of a 6% denaturing polyacrylamide gel. Samples were electrophoresed in 1xTris/borate/EDTA (TBE) buffer for 3.5 h at 3000 V and fragments automatically sized by Genescan software using the internal standard and a local Southern sizing alogrithm. Fluorescent product yield was calculated from integration of the peak area. Genescan profiles were generated showing the PCR products as coloured peaks dependent on the fluorescent dye used: TET (green), HEX (black) and 6-FAM (blue).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Development of a single cell pentaplex DNA fingerprinting system
A single cell DNA fingerprinting system based on the amplification of two chromosome 13 markers (D13S631 and D13S258), one chromosome 21 marker (D21S1413), one chromosome 18 marker (D18S51) and one X chromosome marker (DXS8377) was developed and optimized on buccal cells from a female subject. This subject was homozygous for D13S631 and D21S1413, and heterozygous for D13S258, DXS8377 and D18S51 (Figure 1AGo). The reliability and accuracy of this fingerprinting system was assessed on 50 single buccal cells. Since homozygous loci display a single peak following PCR, the two possible alleles are indistinguishable. Consequently, using these five microsatellite markers on 50 single buccal cells, only 400 of the theoretically possible 500 peaks could be analysed. Of these, 362 specific amplifications were observed establishing a 91% reliability rate (defined as the proportion of successful allelic amplifications). ADO and PA were assessed on the three heterozygous loci (300 alleles). There were 12 incidences of ADO (4%) and 18 incidences of PA (6%) observed (calculated as the area under the smaller peak divided by the area under the larger peak, <0.5). Based on the percentage of correct allelic amplifications (taking into account the incidence of ADO), an overall accuracy rate for this pentaplex DNA fingerprinting system was calculated at 95%. There was no significant difference (P > 0.05) observed between the individual loci for either the proportion of successful amplifications, total amplification failure, accuracy, ADO or PA (Figure 2Go). The vast majority of the DNA fingerprints (94%) were informative (amplification of at least three microsatellite markers, capable of identifying an individual), with 78% fully comprehensive (amplification of all five microsatellite markers).



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Figure 1. Single cell DNA fingerprinting. (A) DNA fingerprint of a single human female buccal cell. Homozygous loci (D21S1413 and D13S631) and heterozygous loci (DXS8377, D13S258 and D18S51). (B) DNA fingerprint of a single human buccal cell from a male subject with Down's syndrome. Homozygous loci (DXS8377 and D18S51), heterozygous loci (D13S258 and D13S631) and tri-allelic locus (D21S1413). Fluorochromes are as follows: green (TET), blue (6-FAM) and black (HEX). Red (TAMRA) peaks indicate internal mol. wt markers.

 


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Figure 2. Evaluation of the single cell pentaplex DNA fingerprinting system. The proportion of successful amplifications, amplification failure, accuracy, allele drop-out (ADO) and preferential amplification (PA) are shown for each individual tri- and tetranucleotide microsatellite marker. No significant difference was observed between the individual loci for any of the above parameters, P > 0.05.

 
The accuracy of this single cell DNA fingerprinting system for aneuploidy detection was also blindly assessed on single buccal cells collected from a male subject with Down's syndrome and a healthy female subject. Normal buccal cells (diploid) (n = 15) and trisomy 21 buccal cells (n = 25) were coded by an independent person, DNA fingerprinted, analysed and decoded. In 22 of the known Down's cells, a tri-allelic pattern for the chromosome 21 marker D21S1413 was observed. In the remaining three cells, two demonstrated ADO and one exhibited total amplification failure of the D21S1413 locus. The fingerprint from the subject with Down's syndrome showed one allele each for DXS8377 (male) and D18S51 (homozygous), two alleles each for D13S258 and D13S631 (heterozygous), and three alleles for D21S1413 diagnostic of trisomy 21 (Figure 1BGo). Based on the percentage of correct tri-allelic amplifications, taking into account the incidence of ADO, the overall accuracy for detection of the known trisomy by this pentaplex DNA fingerprinting system with only one chromosome 21 microsatellite marker was 92%.

DNA fingerprinting of blastomeres from aneuploid embryos
To determine if this system could also reliably and accurately DNA fingerprint embryonic cells, blastomeres from aneuploid embryos identified by FISH were subjected to PCR DNA fingerprinting. Nine slow developing aneuploid embryos were obtained, and following dissociation using pronase, a total of 21 blastomeres were isolated intact and transferred into PCR tubes (some additional blastomeres lysed or fragmented during dissociation). From all embryos, at least two sister blastomeres were available for PCR DNA fingerprinting. The aneuploid status of these embryos determined by independent FISH analysis (Table IGo) was unknown prior to fingerprinting analysis. Sixteen of the 21 blastomeres produced informative DNA fingerprints (amplification of at least three microsatelite markers) with 13 of the 16 informative DNA fingerprints being fully comprehensive (amplification of all five microsatellite markers). The remaining five blastomeres produced unacceptable DNA fingerprints (amplification of two or less microsatellite markers). Due to the aneuploid status of these embryos, calculating the number of possible allelic amplifications would be misleading. Consequently, the total number of microsatellite marker amplifications was employed to establish the rate of reliability. Using these five microsatellite markers on 21 blastomeres, a total of 81 microsatellite marker amplifications were observed out of a possible 105 (Table IGo), establishing a 77% reliability rate. There were five incidences of ADO (6.5%) and five incidences of PA (6.5%). Based on the percentage of correct microsatellite marker amplifications (taking into account the incidence of ADO), an overall accuracy rate for this pentaplex DNA fingerprinting system on blastomeres was calculated at 94%.


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Table I. Analysis of sister blastomeres from aneuploid embryos by fluorescent (FL)-PCR
 
This pentaplex DNA fingerprinting system has a high power of discrimination, making it possible to distinguish between different embryo cohorts. The allelic contributions of each couple were recognizable even in the absence of parental DNA fingerprints and confirmed that no DNA contamination had occurred during the dissociation and isolation of these cells. In addition, each individual sibling embryo in a couple's cohort was uniquely identifiable by its own individual DNA fingerprint derived from the contribution of one paternal and one maternal allele at each microsatellite locus, even with the infrequent occurrence of amplification failure and ADO (Figure 3Go).



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Figure 3. PCR DNA fingerprinting of sibling embryos from cohort 1 embryos (A–E). ADO was observed at locus D18S51 for embryo E with the presence of only one parental allele, while preferential amplification was observed at locus D18S51 for embryo B with a calculated allelic ratio of 0.32. Fluorochromes are as follows: green (TET), blue (6-FAM) and black (HEX). Red (TAMRA) peaks indicate internal mol. wt markers.

 
From the analysis of chromosomal numeracy, no tri-allelic patterns were observed in any of the blastomere DNA fingerprints. However, several double dosage di-allelic patterns (with a ratio calculated as the amount of fluorescent yield under the peak from the first allele correlated with the amount of fluorescent yield of the second allele, 1.7–2.3:1) were observed in some sister blastomeres, suggesting trisomy of the involved chromosomes (Table IGo). For example, in three blastomeres from E6 of cohort 3, all DNA fingerprints indicated a third copy of chromosome 13 which would be concordant with the original diagnosis of trisomy 13 by FISH. Single peaks or mono-allelic patterns were also observed in some sister blastomeres, suggesting monosomy of the chromosomes involved (Table IGo). For example, in two blastomeres from E8 of cohort 1, both DNA fingerprints indicated the presence of only one copy of chromosome 21 and was concordant with monosomy 21 diagnosed by FISH in two other sister blastomeres. However, in most cases, it was not possible with the analysis of only one or two microsatellite markers per chromosome to differentiate between a double dosage di-allelic pattern and the occurrence of PA or between a mono-allelic pattern and the occurrence of ADO/parental homozygosity.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A reliable and accurate single cell PCR DNA fingerprinting system based on the fluorescent amplification of five highly polymorphic microsatellite markers was successfully developed and used to DNA fingerprint IVF embryos. DNA fingerprints obtained from single buccal cells and blastomeres showed strong and accurate allelic amplification with virtually no non-specific background interference to confound interpretation. However, the reliability of DNA fingerprints obtained from blastomeres (as assessed by the proportion of successful microsatellite marker amplifications) was lower than that observed from buccal cells (as assessed by the proportion of successful allelic amplifications). The smaller number of blastomeres and/or differences in cell quality may account for the higher proportion of reliable and comprehensive DNA fingerprints derived from buccal cells compared to blastomeres. Buccal cell samples were taken directly from the cheek lining and single cells frozen at –80°C within an hour of isolation. In contrast, blastomeres were dissociated from morphologically low grade aneuploid embryos that were left to succumb on the bench for up to 24 h. It has been reported that the reliability of PCR amplification decreases as the embryo quality decreases (Findlay et al., 1999Go). In addition, blastomeres from arrested or fragmented embryos have also been shown to yield much lower amplification efficiencies (Ray et al., 1998Go) and this is presumably due to partial or total nuclear DNA degeneration in some cells (Cui and Matthews, 1996Go). On this basis, we would expect the pentaplex DNA fingerprinting system to exhibit higher amplification efficiency on fresh biopsied blastomeres from PGD cases. Nevertheless, this pentaplex single cell PCR DNA fingerprinting system performed significantly better than other previously published systems (Sherlock et al., 1998Go; Findlay et al., 1998Go, 1999Go) that have reported ADO rates as high as 56%, PA rates as high as 25%, and reliability rates (total number of successful amplifications) in the order of only 75% on several cell types.

A diploidy status at any particular locus was determined by the presence of two alleles with an expected allelic ratio of 1:1, a monosomy by the presence of only one allele and a trisomy by the presence of three alleles with the expected ratio of 1:1:1 (tri-allelic) or two alleles with an expected allelic ratio of 2:1 (double dosage di-allelic). There were five incidences of double dosage di-allelic patterns observed in the DNA fingerprints of blastomeres in this study. Even though it is not possible to differentiate between double dosage di-allelic and PA, this single cell DNA fingerprinting system displayed a PA rate of only 6.5% and therefore it was more than likely that a third copy of the chromosome was present. There were also five incidences of discordance between sister blastomeres (Table IGo). This could either be explained by embryonic mosaicism resulting from mitotic cell division errors, or by the occurrence of PA at some of these loci, distorting the allelic ratios. Overall, due to the possibility of parental homozygosity, ADO and PA, it is essential that three or more microsatellite markers per chromosome under analysis are included in a single cell DNA fingerprinting system for confident aneuploidy detection.

PCR DNA fingerprinting has many unique advantages including confirmation of parental allelic contribution to the embryo, the identification of extraneous DNA contamination that could cause a misdiagnosis and detection of uniparental disomy in an embryo if parental DNA fingerprints were available. The newly developed pentaplex DNA fingerprinting system has high discriminating power for identification with a 1 in 630 probability that any two siblings will share an identical DNA fingerprint. Using this pentaplex DNA fingerprinting system it was possible to separate each sibling embryo in the cohorts by their unique allelic fingerprints. Thus, this system could potentially be used to track IVF embryos from the time of transfer to term and identify the viable embryo that produced the pregnancy in a multiple transfer. It is also possible to combine mutation detection for PGD for single gene disorders with the amplification of microsatellite markers for aneuploidy detection (Blake et al., 1999Go). Many couples presenting for PGD for single gene disorders have either experienced the birth of an affected child or undergone prenatal testing resulting in termination of pregnancy or several pregnancies. Hence, these women are usually of advanced maternal age and have a greater chance of producing embryos with chromosomal aneuploidies. It would be most unfortunate if a single gene disorder-free pregnancy resulted in a chromosomally aneuploid fetus, for example Down's syndrome. Consequently, it would be possible to combine a pentaplex chromosome 21 specific DNA fingerprinting system with mutation detection and offer simultaneous mutation and chromosome 21 screening for couples of advanced maternal age presenting primarily for PGD of a single gene disorder.

The incorporation of additional microsatellite markers on clinically relevant chromosomes to this pentaplex DNA fingerprinting system is a feasible prospect and is currently under development. To date, we have successfully added a further three microsatellite marker primers to produce an octaplex DNA fingerprinting system (Figure 4Go). Initial studies on 15 single buccal cells showed high reliability and accuracy (>90%) and low ADO and PA (<10%) and produced DNA fingerprints that were easily interpretable with virtually no background interference or non-specific amplification. This octaplex DNA fingerprinting system (two markers for chromosomes 13, 18, 21 and X) is therefore exceptionally robust. The physical limits of single cell DNA fingerprinting are unknown, although the further addition of four microsatellite markers could be possible in view of the successful development of this octaplex system. A more complex and comprehensive single cell PCR DNA fingerprinting system based on compatible microsatellite marker primers for clinically relevant chromosomes (three microsatellite markers per chromosome) could potentially offer PGD couples the possibility of a combined detection system for single gene disorders and chromosomal aneuploidy. The availability of more comprehensive fingerprinting systems would also allow a larger study to be undertaken to assess the origin, nature and incidence of chromosomal mosaicism in IVF embryos from different patient groups.



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Figure 4. Octaplex single cell DNA fingerprinting of a single human female buccal cell. Fluorochromes are as follows: green (TET), blue (6-FAM) and black (HEX). Red (TAMRA) peaks indicate internal mol. wt markers

 


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Lyn Gras and Jennifer Mansfield for performing embryo biopsy and FISH analysis. This study was supported by a research grant from Monash IVF Pty Ltd, Melbourne, Australia.


    Notes
 
3 To whom correspondence should be addressed at: Centre for Early Human Development, Monash Institute of Reproduction and Development, Level 3, 27–31 Wright St, Clayton 3168, Victoria, Australia. E-mail: mandy.katz{at}med.monash.edu.au Back

Submitted on August 24, 2001


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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accepted on October 24, 2001.





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