Detection of restriction enzyme-digested target DNA by PCR amplification using a stem-loop primer: application to the detection of hypomethylated fetal DNA in maternal plasma.
BACKGROUND
The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis and monitoring. Among the fetal markers that have been described, methylation markers are sex and polymorphism independent. Methylation-sensitive restriction endonucleases are commonly used to digest hypomethylated DNA molecules, and the hypermethylated molecules remain intact for detection. The positive detection of the cleaved hypomethylated molecules would be useful for certain targets but has not been reported.


METHODS
The use of a stem-loop primer in microRNA detection has previously been described. In this study, DNA assays were designed and performed on maternal plasma, which contained the hypomethylated placental serpin peptidase inhibitor, clade B (ovalbumin), member 5 (SERPINB5; maspin) gene in an excess background of hypermethylated maternal SERPINB5. Detection of the enzyme-digested placenta-derived hypomethylated SERPINB5 molecules was achieved by performing stem-loop extension followed by real-time PCR on maternal plasma. The placental origin of the stem-loop-extended SERPINB5 molecules was confirmed by genotyping.


RESULTS
From the real-time PCR results on maternal plasma, stem-loop-extended SERPINB5 promoter sequences were detectable in all 11 enzyme-digested predelivery maternal plasma samples. Postpartum clearance was demonstrated. In 9 cases in which the fetal and maternal SERPINB5 genotypes were distinguishable, the placental-specific genotypes were detected in all predelivery maternal plasma samples.


CONCLUSIONS
Detection of restriction enzyme-digested hypomethylated placental DNA molecules in maternal plasma by the use of a stem-loop primer represents a novel approach in fetal epigenetic marker detection. The analytical approach may also be generally applicable to the detection of restriction enzyme-digested nucleic acid fragments.
Tong
Yu K
YK
Li Ka Shing Institute of Health Sciences, Department of Chemical Pathology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR.
Chiu
Rossa W K
RW
Leung
Tak Y
TY
Ding
Chunming
C
Lau
Tze K
TK
Leung
Tse N
TN
Lo
Y M Dennis
YM
D004247:DNA / Q000097:blood*; D019175:DNA Methylation; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D005260:Female; D005333:Fetus; D005838:Genotype; D006801:Humans; D010920:Placenta / Q000378:metabolism; D010949:Plasma; D016133:Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods; D011401:Promoter Regions, Genetic; D015843:Serpins / Q000235:genetics*
D017931:DNA Primers; C085344:SERPIN-B5; D015843:Serpins; D004247:DNA; D004262:DNA Restriction Enzymes