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The applicability of different PCR‐based methods for fetal RHD and K1 genotyping: a prospective study
Author(s) -
Faas B. H. W.,
Maaskantvan Wijk P. A.,
von dem Borne A. E. G. Kr.,
van der Schoot C. E.,
Christiaens G. C. M. L.
Publication year - 2000
Publication title -
prenatal diagnosis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.956
H-Index - 97
eISSN - 1097-0223
pISSN - 0197-3851
DOI - 10.1002/1097-0223(200006)20:6<453::aid-pd858>3.0.co;2-0
Subject(s) - genotyping , biology , multiplex polymerase chain reaction , microbiology and biotechnology , polymerase chain reaction , exon , multiplex , amniotic fluid , variants of pcr , genotype , genetics , gene , fetus , pregnancy
The applicability of different PCR‐based assays for fetal RHD and K1 genotyping using DNA isolated from uncultured amniotic fluid cells has been tested prospectively: cord blood serotyping served as a control. For RHD genotyping, DNA was amplified with PCRs specific for RHD exon 7, the 3′‐non‐coding region and intron 4, using standard conditions. The results of these three separate assays were compared to those of a newly‐developed multiplex PCR, simultaneously amplifying six regions of RHD. The PCRs analysing the 3′‐non‐coding region or intron 4 often yielded false‐negative results or no results at all. Results of the exon 7 PCR and of the multiplex PCR always corresponded with postnatal serotyping, the multiplex PCR having the advantage of analysing six RHD ‐specific exons simultaneously. For K1 genotyping, two different PCR‐based assays, both analysing the presence of T578C in the KEL gene, were applied. With the first method, a consensus 740‐bp product of the KEL gene was amplified and subsequently specifically digested. As we were not able to obtain any PCR product from amniotic fluid DNA, we developed a new K1 ‐specific PCR, amplifying a fragment of 91 bp only in cases of K1 ‐positivity. With this PCR, all K1 genotyping results ( n =30) correctly predicted the phenotypes. We conclude that fetal RHD and K1 genotyping can be performed reliably with DNA from uncultured amniotic fluid cells. Copyright © 2000 John Wiley & Sons, Ltd.