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.
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.


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.


CONCLUSIONS
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.
Hviid
T Vauvert
TV
Department of Clinical Biochemistry 339, H:S Hvidovre Hospital, Copenhagen University Hospital, 30 Kettegaard Allé, DK-2650 Hvidovre, Denmark. thomas.hviid@rh.dk
D015703:Antigens, CD / Q000378:metabolism; D000944:Antigens, Differentiation, B-Lymphocyte / Q000378:metabolism; D041322:Chromosomes, Human, Y / Q000235:genetics; D004247:DNA / Q000235:genetics*; D005260:Female; D005312:Fetal Blood / Q000166:cytology; D015894:Genome, Human; D005838:Genotype; D006682:HLA-DP Antigens / Q000235:genetics; D059868:HLA-DP beta-Chains; D006801:Humans; D008297:Male; D009035:Mothers; D016133:Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D011990:Receptors, Transferrin; D012680:Sensitivity and Specificity
D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; C068126:CD71 antigen; D006682:HLA-DP Antigens; D059868:HLA-DP beta-Chains; C071170:HLA-DPB1 antigen; D011990:Receptors, Transferrin; D004247:DNA