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Clinical Chemistry 48: 2115-2123, 2002;
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(Clinical Chemistry. 2002;48:2115-2123.)
© 2002 American Association for Clinical Chemistry, Inc.

In-Cell PCR Method for Specific Genotyping of Genomic DNA from One Individual in a Mixture of Cells from Two Individuals: A Model Study with Specific Relevance to Prenatal Diagnosis Based on Fetal Cells in Maternal Blood

T. Vauvert Hviid1

1 Department of Clinical Biochemistry 339, H:S Hvidovre Hospital, Copenhagen University Hospital, 30 Kettegaard Allé, DK-2650 Hvidovre, and Department of Clinical Biochemistry, H:S Rigshospitalet, Copenhagen University Hospital, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark.

Address for correspondence: Department of Clinical Biochemistry, KB 4111, H:S Rigshospitalet, Copenhagen University Hospital, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. Fax 45-3545-4160; e-mail thomas.hviid{at}rh.dk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: During recent years, much attention has been paid to the possibility of using fetal cells circulating in the pregnant woman’s blood for prenatal diagnosis of genetic or chromosomal abnormalities. Although successes have been achieved in enrichment procedures for fetal cells from maternal blood samples, the use of such an approach for genotyping by molecular biology techniques in a more routine setting has been hampered by the large contamination of maternal nucleated blood cells in the cell isolates. Therefore, a new method based on in-cell PCR is described, which may overcome this problem.

Methods and Results: Mixtures of cells from two different individuals were fixed and permeabilized in suspension. After coamplification of a DNA sequence specific for one of the individuals and the DNA sequence to be genotyped, the two PCR products were linked together in the fixed cells positive for both DNA sequences by complementary primer tails and further amplification steps. In a model system of mixtures of male and female CD71-positive cells from umbilical cord blood attached to immunomagnetic particles, a Y-chromosome-specific sequence (TSPY) was linked to a polymorphic HLA-DPB1 sequence only in the male cells, leading to the correct HLA-DPB1 genotyping of the male by DNA sequencing of a nested, linked TSPY-HLA-DPB1 PCR product.

Conclusion: This approach might be usable on mixed cell populations of fetal and maternal cells obtained after conventional cell-sorting techniques on maternal peripheral vein blood.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It has been known for many years that fetal cells circulate in the maternal blood, but during the last decade, a growing effort has been made to isolate these few fetal cells and, by the use of new DNA amplification techniques and in situ hybridization techniques, perform prenatal genetic diagnoses. Some success has been achieved but only in smaller research studies. For example, noninvasive prenatal diagnoses of trisomy 21 by fluorescence in situ hybridization (1) and of thalassemia by PCR (2) have been made on fetal erythroblasts isolated from 10–20 mL of maternal blood. In these pilot studies, advanced equipment and time-consuming procedures such as fluorescence-activated cell-sorting analysis and micromanipulation have been used, and it does not seem straightforward to transfer these approaches to routine analyses.

The main problem is a high degree of maternal blood cell contamination after the initial cell isolation procedures. Therefore, the final identification or collecting of single fetal cells is very demanding and time-consuming. Furthermore, a truly specific fetal antigen marker seems not to have been identified yet (3)(4), which might lead to more simple and specific isolation and identification procedures of the few fetal cells in a maternal blood sample.

This study explores a different approach based on variations of the PCR technique to achieve fetal-specific genotyping that might work under the current status of only a low percentage of fetal cells or even a few fetal cells in a background of thousands of maternal blood cells after initial cell isolation steps. Although recent studies indicate that after some cell isolation procedures the amount of fetal erythroblasts might be as high as 10–50% of the enriched cell population, individual micromanipulation is needed for further analyses (5)(6)(7)(8).

The method presented here is based on in-cell PCR, an approach that has been used in certain immunologic studies of amplifying and linking rearranged immunoglobulin heavy and light chain V genes within single cells (9)(10) and intracellular detection of HIV mRNA and DNA (11). Here, studies of the feasibility of such an approach in a model system of CD71+ cells from umbilical cord blood samples are reported. Blood cells from female and male pregnancies were mixed. After capture of CD71+ fetal erythroblasts attached to magnetic beads coated with anti-CD71 monoclonal antibodies, the cells underwent steps of fixation and permeabilization. Thereafter, with the fixed cells in suspension, a Y-chromosome-specific PCR, which functions as a fetal-specific marker DNA sequence, and a PCR of the sequence of interest (including polymorphisms; in the model system used here, the HLA class II gene DPB1) were performed. On the basis of complementary primer tails, a linked PCR product was amplified in male cells. After nested PCR steps, a nested and linked PCR product of expected fragment length could be visualized after gel electrophoresis. Direct DNA sequencing of the PCR product revealed the HLA-DPB1 genotype of the male cells.

Further studies will clarify whether this new approach of fetal in-cell-specific PCR is sensitive enough to be used on cell isolates from 10–20 mL of maternal blood to determine the fetal genotype. In cases of female fetuses, any fetal-specific polymorphism may be used for designing primers for a fetal-specific, PCR-driven marker sequence. Information about fetal-specific polymorphisms may easily be obtained from genomic DNA in a small maternal serum or plasma sample (12).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
samples
Genomic DNA was extracted from whole peripheral blood according to established methods (proteinase K digestion and salting-out) (13). The study was approved by the local Research and Ethics Committee. Umbilical cord blood samples were collected in tubes containing citrate immediately after delivery and processed within 12 h. Adult peripheral blood samples were collected in tubes containing citrate and processed immediately.

overview of the general principle
The main steps in the procedure are outlined briefly here. A detailed description follows in the sections below. Figs. 1 and 2 illustrate the general principle of the in-cell linker PCR method used. In the model system, CD71+ cells are isolated from mixtures of male and female umbilical cord blood with use of anti-CD71 monoclonal antibody-coated immunomagnetic particles (Dynabeads; Dynal A/S). The different steps of the procedure, which include several changes of the buffer the cells are suspended in, could then be performed easily by immobilization of the beads binding the cells by use of a magnet. This has been described previously by Chapal et al. (10) in another in-cell PCR procedure. After fixation and permeabilization, the cells are resuspended in PCR buffer with the primers BIO5TSPY, A3TSPYHL, NY3DPB1, and A5DPB1HL. During the first PCR, two PCR products will be amplified in male cells: a sequence of the gene TSPY and a sequence of exon 2 of HLA-DPB1, as shown in Fig. 1 . TSPY (CYS14) is a Y-chromosome-specific, single-copy gene encoding a testis-specific protein (14) and was chosen as the male ("fetal")-specific marker sequence. The HLA-DPB1 sequence was chosen as an example of a polymorphic gene of interest because it is a well-characterized gene and ~80 alleles have been reported to date (e.g., HLA Informatics Group; http://www.anthonynolan.com/HIG) (15); therefore, the possibility that any two samples have some polymorphic differences in this sequence is large. Thus, HLA-DPB1 sequences from either the male or female cells can easily be defined in most cases. At the end of the first PCR, the TSPY and the HLA-DPB1 PCR products will begin to form a linked PCR product because of complementary primer tail sequences as shown in Figs. 1 and 2 . These tail sequences have no marked homology to any known human genome sequences. Differences in the primer concentrations added will partly force this to happen. This first PCR and linkage of PCR products are supposed to occur to a great extent inside the fixed male cells. In the fixed female cells, only the HLA-DPB1 sequence will be amplified from the genomic DNA. After the first PCR, the PCR mixture is removed from the cells, and the cells are washed once in 1x PCR buffer and resuspended in the second PCR mixture containing the nested primers NTPY5 and N2DPB1 (shown in Fig. 1 ). Removal of the first PCR mixture and washing of the fixed cells will remove any female-derived HLA-DPB1 PCR products diffused out of fixed female cells that would be able to interfere with the following two nested PCR steps and the final DNA sequencing, by linkage to TSPY PCR products diffused out of male cells. It can be argued that female HLA-DPB1 PCR products, which may diffuse out of female cells, may enter male cells, and vice versa with TSPY PCR products. This is addressed in the Discussion.



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  		Figure 1. Primer sequences and generation of a linked PCR product.

All primer sequences of the model system of the in-cell linker PCR principle are shown as parts of the two PCR products of, respectively, HLA-DPB1 and TSPY amplified in the first PCR step. Below is the formation of the linked PCR product illustrated (n = number of nucleotides). An extra adenosine is included between the A5DPB1HL (or A3TSPYHL) primer sequence and the tail-linker sequence. This is because the Taq polymerase incorporates an adenosine on the 3' end of the new DNA strand.



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  		Figure 2. Schematic drawing of the basic principle of the in-cell linker PCR procedure (see Materials and Methods for a detailed description).

During the second nested PCR, the linked TSPY-DPB1 PCR product will be amplified further inside the fixed male cells (and outside cells in the supernatant). After the second PCR, the PCR mixture is removed (and used as a template for a third PCR). Fixed cells are then resuspended and lysed in 1x PCR buffer; a portion of this "cell lysate" is then used as template for the third nested PCR using the primers nesTSPY5 and nesDPB1-3. This final PCR product can be visualized on a gel and directly DNA sequenced (Fig. 3 ).



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  		Figure 3. Cell lysates or supernatants were used as templates for the third nested PCR, with the primers nesTSPY5 and nesDPB1-3, to produce the final products visualized on the gel.

(A), gel electrophoresis of the third linked and nested in-cell PCR product from an experiment using a 1:1 mixture of cells from umbilical cord blood from a male and a female pregnancy. Lanes: 1, 100-bp ladder marker; 2, male genomic DNA from the second PCR; 3, female HLA-DPB1 class II PCR product; 4, male HLA-DPB1 class II PCR product; 5, supernatant from male cells; 6, supernatant from a 1:1 mixture of male and female cells; 7, supernatant from female cells; 8, supernatant from male cells; 9, male genomic DNA; 10, 2 µL of male cell lysate; 11, 2 µL of cell lysate from a 1:1 mixture of male and female cells; 12, 2 µL of female cell lysate; 13, 2 µL of male cell lysate; 14, 10 µL of male cell lysate; 15, 10 µL of cell lysate from a 1:1 mixture of male and female cells; 16, 10 µL of female cell lysate; 17, 10 µL of male cell lysate; 18, negative PCR control (10 µL of water). (B–D), DNA sequencing results (electropherograms) of the PCR products shown in lanes 3, 4, and 11 in panel A, respectively. The HLA-DPB1 polymorphisms at codons 55 and 56 obtained from the lysate of the 1:1 mixture of male and female cord blood cells (D) are the ones of the male (C).

initial control experiments with extracted genomic dna from males and females as the template
Before conducting the actual in-cell PCR linker PCR experiments, the performance of the coamplification of the two PCR products and linkage was evaluated using extracted genomic DNA from males and females in a traditional PCR setting. The method is based on the principle described by Diviacco et al. (16) for constructing internal standards used in quantitative PCR techniques. Primer sequences are listed in Fig. 1Up . Briefly, 200 ng of male or female genomic DNA was made up to a final volume of 50 µL containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 200 µM deoxynucleotide triphosphates (dNTPs), 50 pmol each of primers BIO5TSPY and NY3DPB1, 20 pmol each of primers A5DPB1HL and A3TSPYHL, and 2.5 U of Taq polymerase (Life Technologies). PCR conditions were denaturation for 5 min at 94 °C and 35 cycles of annealing for 90 s at 55 °C, extension for 90 s at 72 °C, and denaturation for 45 s at 94 °C. One µL of this first PCR product was used as template in a second PCR: final volume of 50 µL with the same buffer conditions as the first PCR, except for 1.5 mM MgCl2 and 25 pmol each of primer NTPY5 and primer N2DPB1. PCR conditions were 94 °C for 2 min; 30 cycles of 94 °C for 45 s, 55 °C for 1 min, 72 °C for 3 min; and a final extension of 72 °C for 5 min.

In a separate line of experiments to determine a lower sensitivity cutoff for the coamplification linker PCR procedure using male genomic DNA, a third PCR was conducted. Serial dilutions of male genomic DNA from 0.01 ng (corresponding to ~1 cell) and to 5 ng (~500 cells) were used as templates. Furthermore, the second PCR was modified in such a way that to the 50-µL volume of the first PCR, 50 µL of the second PCR mixture was added with a final amount of each of the primers NTPY5 and N2DPB1 of 100 pmol and 5 U of Taq polymerase. Five µL of this second PCR product was used as template in the third PCR: final volume of 50 µL containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 µM dNTP, 40 pmol each of primers nesTSPY5 and nesDPB1-3, and 2.5 U of Taq polymerase (Life Technologies). PCR conditions were denaturation for 2 min at 95 °C, followed by 40 cycles of denaturation for 45 s at 95 °C, annealing for 60 s at 55 °C, and extension for 2 min at 72 °C. Amplified DNA fragments were analyzed by electrophoresis on 2% agarose gels and stained with ethidium bromide.

isolation of cd71-positive umbilical cord blood cells
One or 0.5 mL of umbilical cord blood (or in a few experiments, adult peripheral vein blood) was processed separately. CD71+ cell isolation was performed from male or female samples alone or from mixtures (e.g., 1:1 or 1:9) of male and female samples always made-up to a volume of 1.0 or 5.0 mL. The isolation of CD71+ cells was performed as described by the manufacturer of the immunomagnetic beads. Briefly, 1.2 x 107 anti-CD71 Dynabeads (Dynal A/S) were added to 1 mL of cord blood and incubated at 4 °C with rotation for 20 min. Washing buffer [1x phosphate-buffered saline (PBS) containing 20 mL/L fetal calf serum] in a volume of 2–3 mL was added, and the tube was placed in a magnetic particle concentrator for 2–3 min. The supernatant was discarded, and the captured cells were washed three times in washing buffer and finally resuspended in 200 µL of 1x PBS.

fixation and permeabilization of cells in suspension
Initial sets of fixation experiments were performed with 1% paraformaldehyde (17)(18) and permeabilization with proteinase K (11), but no positive results were obtained with these methods. Therefore, an IntraStain reagent set was evaluated (Dako A/S). This procedure worked well and was performed as recommended by the manufacturer. Again briefly, CD71+ cells bound to Dynabeads in 200 µL of 1x PBS were placed in the magnet, and the supernatant was discarded. One hundred µL of IntraStain Reagent A for fixation was added, and the cells were resuspended. Incubation was for 15 min at room temperature. To the cell suspension, 1.4 mL of 1x PBS was added and mixed carefully. The supernatant was then discarded with use of the magnet, and 100 µL of IntraStain Reagent B for permeabilization was added. After 15 min of incubation at room temperature, 1.4 mL of 1x PBS was added, the tube was placed in the magnet, and the supernatant was removed. The fixed and permeabilized cells were washed three times in 100 µL of 1x PCR buffer.

in-cell linker pcr of y-chromosome-specific dna sequence and hla-dpb1 dna sequence
Fixed and permeabilized cells (12–15 000 cells from 1 mL of blood) derived from the CD71+ cell isolation procedure were resuspended in 50 µL of PCR mixture containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 200 µM dNTP, 100 pmol each of primers BIO5TSPY and NY3DPB1, 40 pmol each of primers A5DPB1HL and A3TSPYHL, and 5 U of Taq polymerase (Life Technologies). PCR conditions were denaturation for 5 min at 94 °C, followed by 35 cycles of annealing for 90 s at 55 °C, extension for 90 s at 72 °C, and denaturation for 45 s at 94 °C. After the first PCR, the tube was placed in a magnet (magnetic particle concentrator), and the supernatant was removed. One hundred µL of 1x PCR buffer was added for 1 min and removed, followed by the addition of 50 µL of the second PCR mixture (final volume of 50 µL with the same buffer conditions as the first PCR except for 2.5 mM MgCl2 and 100 pmol each of primers NTPY5 and N2DPB1). PCR conditions were 95 °C for 2 min, followed by 35 cycles of 94 °C for 45 s, 55 °C for 90 s, and 72 °C for 90 s. For some of the pure female samples, 2 µL of supernatant from the first PCR was used as template in a second PCR with 1.5 mM MgCl2, 20 pmol each of N2DPB1 and 5NDPB1 (Table 1 ), and 2.5 U of Taq polymerase (same PCR program). This was to obtain an HLA-DPB1 exon 2 sequence of the female samples for later DNA sequencing. After the second PCR, the supernatant was removed using the magnet and stored at -20 °C. The cells were resuspended in 50 µL of 1x PCR buffer and placed at 100 °C for 8 min. We used 2 or 10 µL of the cell lysates or supernatants as template for the third nested PCR [total of 50 µL of PCR mixture containing 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.8 µL/mL Nonidet P40, 200 µM dNTP, 20 pmol each of primers nesTSPY5 and nesDPB1-3, and 2.5 U of Taq polymerase (MBI Fermentas)]. PCR conditions were denaturation for 2 min at 95 °C, followed by 35 cycles of denaturation for 45 s at 95 °C, annealing for 60 s at 55 °C, and extension for 2 min at 72 °C. Amplified DNA fragments were analyzed by electrophoresis on 2% agarose gels and stained with ethidium bromide.


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  		Table 1. List of primers for PCR amplification.1

dna sequencing
Dye primer (Cy-5' labeling) technology was used. A Thermo Sequenase fluorescence-labeled primer cycle sequencing method with 7-deaza-dGTP (Amersham Pharmacia Biotech) was used and performed according to the manufacturer’s instructions. Two sequencing primers were used: DPB1SEK5, Cy-5'-GCG CCT CCG CTC ATG TCC GCC CCC T-3'; and TSPYSEK5, Cy-5'-GTA AGT AAC TGA TGG GCA GCT CGG CT-3'.

pilot studies of the sensitivity of the in-cell linker pcr method and studies of blood samples from pregnant women
To obtain data on the sensitivity of the in-cell linker PCR method, fixed and permeabilized CD71+ cells derived from 0.5 mL of cord blood from a male pregnancy was diluted in the same type of cells derived from 0.5 mL of cord blood from a female pregnancy in volume ratios of 1:10, 1:20, 1:50, and 1:250. Afterward, the three PCR steps described above were performed with the exception that the cells were lysed after the first PCR. Final PCR products were visualized on agarose gels.

In a first line of experiments to reveal whether the method used for enrichment of CD71+ cells described above would lead to detection of fetal-specific DNA sequences (a Y-chromosome-specific, single-copy gene, SRY, a testis-determining factor) (19) if the starting material was peripheral vein blood samples from pregnant women. Samples of 5 mL of blood from four pregnant women (12, 13, 24, and 28 weeks of gestation) were obtained in EDTA tubes. A male blood sample was included as a positive control. The samples were centrifuged for 10 min at 600g, and the plasma phase was removed. The cell pellet was resuspended in 10 mL of 1x PBS containing 20 mL/L fetal calf serum and divided into two aliquots of 5 mL each. To each 5-mL aliquot, 2 x 107 anti-CD71 Dynabeads were added, and the cell isolation procedure was performed as described above except that the incubation step with Dynabeads was performed overnight. Each aliquot, after the last washing step, was resuspended in 40 µL of 1x PCR buffer and divided into two samples of 20 µL (therefore, each 5-mL blood sample from one woman ended up in four PCR samples). Samples were placed at 100 °C for 8 min. To this, 30 µL of a first PCR mixture was added: 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 0.8 µL/mL Nonidet P40, 2.0 mM MgCl2, 400 µM dNTP, 50 pmol each of primers Y1NY5 and Y2NY3 (see Table 1Up ), and 2.5 U of Taq polymerase (MBI Fermentas). PCR conditions were denaturation for 2 min at 95 °C; 35 cycles of denaturation for 60 s at 95 °C, annealing for 60 s at 57 °C, extension for 2 min at 72 °C; and a final extension of 72 °C for 10 min. After this first PCR, 50 µL of a second PCR mixture containing nested primers was added to the first PCR mixture, giving a total of 100 µL: 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 0.8 µL/mL Nonidet P40, 1.5 mM MgCl2, 200 µM dNTP, 40 pmol each of nested primers BY5NES and Y3NESNY (see Table 1Up ), and 5 U of Taq polymerase (MBI Fermentas). The PCR program was as in the first PCR, except that 40 cycles of amplification were used. PCR products were visualized on agarose gels.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
control experiments with extracted genomic dna
The two-step PCR procedure with coamplification and linkage of the TSPY and HLA-DPB1 PCR products followed by nested PCR in a traditional PCR set-up produced clear bands after gel electrophoresis corresponding to the predicted length of the linked PCR product (531 bp) in only the male samples (triplicate experiments; data not shown). These results confirmed that the basic methodology worked. Dilutions of male genomic DNA showed that a concentration down to 0.05 ng, corresponding to genomic DNA from ~7 cells, produced a positive PCR signal.

optimization of the method for fixation and permeabilization of cd71+ cells in suspension
Microscopic examination of the enriched CD71+ cell population coupled to immunomagnetic particles (Dynabeads) after fixation and permeabilization using the IntraStain reagents showed intact cells in conjunction with the beads. Furthermore, as described below, positive PCR results were obtained with these procedures. From 1 mL of umbilical cord blood, 12–15 000 cells were obtained. Further characterization of the enriched cell population was not done because this had no consequences for the model test experiments undertaken in this study.

Initial experiments with 1% paraformaldehyde and proteinase K treatment did not produce any positive PCR results, and early experiments using centrifugation steps instead of Dynabeads for shifts of buffer or washing did not produce PCR signals; furthermore, there was a trend that most cells were lost during these centrifugation steps.

the in-cell linker pcr procedure
Results from an in-cell linker PCR experiment performed on CD71+ cells derived from, respectively, a male and a female pregnancy and a 1:1 mixture of cord blood are shown in Fig. 3AUp . Only pure male or mixed samples produced a linked and nested PCR product of the predicted size of 495 bp. Linked PCR products were present in the PCR supernatant after the third PCR step in samples containing fixed male cells (Fig. 3AUp , lanes 5 and 6). There were no clear differences in the results of the third PCR based on, respectively, supernatant or cell lysate from the second PCR. The linked and nested PCR products could not be visualized in ethidium bromide-stained gels after the second PCR. A third (and a second nested) PCR was necessary. This was also the case in other in-cell PCR studies (9)(10).

dna sequencing of in-cell linker pcr products from single and mixed suspensions of cells
Direct DNA sequencing results (electropherograms) of the PCR products described in the preceding section (and in Fig. 3AUp ) are shown in Fig. 3Up , B–D. Two HLA-DPB1 polymorphisms, which were found to distinguish the male and female samples, are shown as examples. The DPB1 exon 2 polymorphisms are well described (HLA Informatics Group; http://www.anthonynolan.com/HIG) and are located, respectively, in codons 55 and 56. The female sample has GAT at codon 55 and GAG at codon 56 (Fig. 3BUp ), whereas the male sample shows GCT at codon 55 and GCG at codon 56 (Fig. 3CUp ). The 1:1 mixture of male and female cells clearly shows the HLA-DPB1 genotype of the male sample (Fig. 3DUp ). This was also the case for other HLA-DPB1 polymorphisms. Therefore, DNA sequencing of the PCR products clearly demonstrates that no cross-association has occurred between male and female PCR product sequences.

studies of the sensitivity of the procedure and investigation of maternal blood samples
When a suspension of CD71+ cells coupled to Dynabeads derived from 0.5 mL of male cord blood was serial diluted into a constant volume of female cell suspensions derived from 0.5 mL of cord blood, a positive in-cell linker PCR signal could clearly be seen down to a ratio of 1:250 (Fig. 4A ). Because 6–7000 CD71+ cells were obtained from 0.5 mL of cord blood, a positive PCR signal may be obtained at least from ~25 male cells in a male-female mixture of cells.



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  		Figure 4. Serial dilution of a suspension of CD71+ cells followed by nested in-cell PCR (A), and detection of the Y-chromosome-specific DNA in CD71+ cells from maternal blood (B).

(A), gel electrophoresis of the third linked and nested in-cell PCR product from an experiment involving the dilution of fixed and permeabilized CD71+ cells derived from 0.5 mL of cord blood from a male pregnancy diluted in cells derived from 0.5 mL of cord blood from a female pregnancy in volume ratios of 1:10 (lane 2), 1:20 (lane 3), 1:50 (lane 4), and 1:250 (lane 5). Lane 1 is a DNA marker (100-bp ladder). (B), detection of a Y-chromosome-specific DNA sequence using nested PCR in one of four PCR mixtures (lane 15) from a CD71+-enriched cell fraction from 5 mL of maternal blood (24 weeks of gestation; sample D, lanes 14–17). The woman gave birth to a boy. Lane 1, DNA marker (100-bp ladder); lanes 2–5, maternal blood sample A (28 weeks of gestation, female pregnancy); lanes 6–9, maternal blood sample B (12 weeks of gestation, female pregnancy); lanes 10–13, maternal blood sample C (13 weeks of gestation, female pregnancy); lane 18, negative control (water).

From one of the maternal blood samples (24 weeks of gestation), it was possible to detect a Y-chromosome-specific DNA sequence using nested PCR on a CD71+-enriched cell fraction (a 254-bp nested PCR product; Fig. 4BUp ). The woman gave birth to a boy. No positive results were obtained from the other maternal blood samples, but this was in agreement with the fact that all three women gave birth to girls.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results from the present study point toward a new, alternative way to handle the relatively high background of maternal blood cells after fetal cell enrichment procedures for prenatal diagnosis of genetic diseases with maternal peripheral blood as starting material. The present study shows that it is possible to genotype a specific gene of interest derived from one individual in a mixture of cells from two individuals. This new method is based on in-cell PCR and is dependent on the presence of a well-characterized and specific DNA sequence or polymorphism, which has to be present only in the cells from the individual being genotyped for the gene sequence of interest. In the present model system, a Y-chromosome-specific DNA sequence (TSPY, encoding a testis-specific protein) was used as the specific marker sequence. Naturally, in one-half of all pregnancies, another fetal-specific marker DNA sequence must be used, such as the Rhesus D gene in cases of rhesus-negative mothers and rhesus-positive fetuses. Many paternal/fetal specific DNA polymorphisms will, however, be usable in an allele-specific PCR set-up. Furthermore, recent studies indicate the existence of more general specific fetal mRNA marker sequences that may be used in a RT-PCR-modified version of the in-cell PCR procedure presented here (20)(21). A fetal-specific marker DNA (or mRNA) sequence can be rapidly and easily detected in a small maternal serum or plasma sample (12)(22) by DNA chip technology. This could form the first step for a possible noninvasive prenatal diagnosis procedure based on the principle presented in the present study. The DNA gene sequence could be any sequence with a mutation leading to a serious inherited disease. Genetic diseases involving chromosomal abnormalities such as trisomy 21 or 18 might also be diagnosed by quantitative PCR techniques (23).

In the present study, a model system of PCR coamplification of a TSPY DNA sequence and a polymorphic HLA-DPB1 DNA sequence and linkage of these two PCR products, and further nested PCR amplification of this linked PCR product, inside fixed and permeabilized male CD71+ cells was examined. It was shown that in mixtures of male and female cells derived from umbilical cord blood, only the male HLA-DPB1 polymorphic DNA sequence was detected by sequencing. In pure female samples, no linked PCR signal was obtained. Furthermore, dilutions of male genomic DNA showed that a concentration down to 0.05 ng, corresponding approximately to genomic DNA from seven cells, produced a positive PCR signal. This indicates, together with another result from the present study, that a positive PCR signal may be obtained from ~25 male cells in a male-female mixture of cells and that a positive, in-cell PCR signal might be obtained from the few fetal cells in a background of maternal cells (isolated from 10–15 mL of peripheral vein blood from a pregnant woman). However, some further optimization may be necessary. It was also interesting that a fetal-specific DNA sequence could be detected with the use of the CD71+ cell isolation procedure on peripheral vein blood from a few pregnant women. Interestingly, in the in-cell PCR study by Chapal et al. (10), the best results of in-cell PCR amplification and linkage of the two PCR products were in the following order, according to the number of cells included: 500 > 5000 > 50 000. This could indicate that the in-cell PCR efficiency is dependent on the number of fixed cells included in the reaction and may decrease with increasing cell number. There might be an optimum between the numbers of PCR-specific male cells, PCR-unspecific female cells, and magnetic beads. Interestingly, in the dilution experiment a clearer band of linked PCR product was observed in the high dilution (1:250) of male cells in female cells compared with the low dilutions. The consequence might be that isolated cells from maternal blood samples have to be diluted to obtain an in-cell linker PCR signal. As the number of fetal cells in the background of maternal cells might be low in a low total number of cells, however, this dilution effect will only be in favor of positive results with the use of maternal blood samples as starting material.

In a possible routine implementation of nested PCRs, extreme care must be taken to avoid PCR contamination. Each PCR step probably should be performed in different physical localities. However, in different kinds of routine gene diagnostic testing, nested PCR analyses are currently performed. The most pressing problem with the in situ or in-cell PCR technique, both in theory and apparently sometimes in practice, is false-positive results because of diffusion of PCR products out of and/or into the fixed and permeabilized cells (17). This would lead to a linkage of male (or fetal) marker sequences (in the model system the TSPY PCR product) and female (or maternal) PCR products of the gene sequence of interest (in the model system, HLA-DPB1 PCR products) in the present procedure. However, this seems not to be a problem in the presented model system; only male HLA-DPB1 polymorphisms were detected in the mixture of male and female cells. In the literature, diffusion problems seem mainly to occur during in situ PCR in tissue sections, whereas the problem seems minimized with fixed cells in suspension (17)(24). One way proposed in the literature to reduce possible problems with diffusion is to use, e.g., biotin coupled to the primers or to include biotin- or digoxigenin-substituted nucleotides. The idea is that a more "bulky" PCR product accumulated inside the fixed cells will not diffuse out of the cells to the same extent as nonlabeled PCR products (17). In the present study, one of the TSPY primers was labeled with biotin. Actually, a third (but second nested) PCR with supernatant from the second PCR with male or mixed cells as template produced a linked PCR product of expected size (see Fig. 3AUp ). Therefore, PCR products diffuse out of fixed cells in the model system presented here, but DNA sequencing of the PCR product showed the male DPB1 polymorphisms and, thus, no signs of cross-association between male- and female-derived PCR products.

Another modification of the procedure could be to shorten the primer tail sequences in the first PCR and increase the length of the primers in the two following nested PCR steps. A high annealing temperature could thus be used in the second and third PCR, whereby no "false" de novo formation of linked PCR products would occur.

More studies, especially of attempts to apply the presented approach of in-cell linker PCR on maternal blood samples, are needed to clarify whether it has any future in a clinical setting. However, the basic concept described here may stimulate such studies. Interestingly, recent studies indicate improvements in the final fetal/maternal cell ratios after further developments of previous cell enrichment procedures (7)(8). Additional studies are needed to connect the in-cell linker PCR procedure described here with such procedures of fetal cell enrichment from maternal peripheral vein blood. The advantage of such a new strategy is the omission of the time-consuming steps of microscopy of many preparations and/or micromanipulation techniques and their replacement with an easier, automated molecular biology-based procedure.


   Acknowledgments
 
We thank Lone G. Nielsen for careful technical assistance on certain parts of the study.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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