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D B Demers

Publications and source records attributed to D B Demers.

7 recordsLinked to original sources
PubMed1997

Prenatal determination of genotypes Kell and Cellano in at-risk pregnancies.

OBJECTIVE: To evaluate the accuracy of a DNA-based testing methodology in determining the KEL1 and KEL2 (Kell and Cellano) genotype of fetuses at risk for Kell or Cellano hemolytic disease. STUDY DESIGN: DNA was extracted from chorionic villus samples (CVS) or amniotic fluid (AF) cells, a portion of the Kell gene was amplified, the amplified product was cut with a restriction enzyme that recognizes the KEL1 nucleotide substitution, and the digested product was run on a polyacrylamide gel to separate the fragments. This analysis was routinely run on uncultured cells to provide rapid results. Testing of parental DNA was performed in conjunction with fetal analysis to ensure that their alleles were detectable with this DNA test. RESULTS: We determined the fetal KEL1 and KEL2 genotype in 1 CVS and 65 AF specimens. Forty-eight of them were determined to be KEL2, 17 were KEL1/2, and 1 was KEL1. Among the fetuses born to date, follow-up information was available on 14 of them, 11 KEL2 and 3 KEL1/2. In all 14 there was complete correlation between the DNA analysis and the serotype or clinical course. CONCLUSION: Determination of the fetal KEL1 and KEL2 genotype using this DNA-based method provides accurate and timely information that can aid the prenatal care of women sensitized to these Kell antigens.

Amniotic Fluid↗
PubMed1995

Enhanced PCR amplification of VNTR locus D1S80 using peptide nucleic acid (PNA).

Use of the polymerase chain reaction (PCR) to amplify variable numbers of tandem repeat (VNTR) loci has become widely used in genetic typing. Unfortunately, preferential amplification of small allelic products relative to large allelic products may result in incorrect or ambiguous typing in a heterozygous sample. The mechanism for preferential amplification has not been elucidated. Recently, PNA oligomers (peptide nucleic acids) have been used to detect single base mutations through PCR clamping. PNA is a DNA mimic that exhibits several unique hybridization characteristics. In this report we present a new application of PNA which exploits its unique properties to provide enhanced amplification. Rather than clamping the PCR, PNA is used to block the template making it unavailable for interstrand and intrastrand interactions while allowing polymerase to displace the PNA molecules and extend the primer to completion. Preferential amplification is reduced and overall efficiency is enhanced.

Base Sequence↗
PubMed1995

Molecular analysis of the RhD genotype in fetuses at risk for RhD hemolytic disease.

The objective of this study was to evaluate the accuracy of a DNA-based testing methodology in determining the RhD genotypes of fetuses at risk for RhD hemolytic disease. We designed a multiplex polymerase chain reaction-based test based on recent RhD and RhCE sequence information. To improve the accuracy of the results, two different portions of the RhD gene were examined. Deoxyribonucleic acid was extracted from fetal specimens, portions of the RhD gene were amplified by the polymerase chain reaction, and the amplified product was run on a polyacrylamide gel to look for the presence or absence of the RhD gene. We tested 67 amniotic fluid and two chorionic villus specimens to determine the fetal RhD genotype in pregnancies at risk for RhD hemolytic disease. Forty-seven of the 69 specimens were determined to be Rh-positive, and 22 were Rh-negative. Fifty of the 69 fetal specimens--31 Rh-positive and 19 Rh-negative--were serotyped at birth. In all 50, there was complete correlation between the DNA analysis and the serotyping results. RhD gene analysis is a rapid and reliable method that provides an accurate fetal genotype to aid in the prenatal care of RhD-alloimmunized women.

Erythroblastosis, Fetal↗
PubMed1995

DNA-based prenatal determination of the RhEe genotype.

BACKGROUND: Maternal antibodies to RhE may cause severe hemolytic disease. Based on recent RhD and RhCE sequence information, we have developed a DNA-based testing methodology to determine the RhEe genotype of fetuses at risk for RhE hemolytic disease from amniotic fluid (AF) or chorionic villus samples. CASE: RhEe testing was undertaken in a fetus at risk for RhE hemolytic disease. Maternal serum anti-E titers had risen between 12-15 weeks' gestation. Optical density (OD450) AF readings also rose slightly between 22-24 weeks' gestation. Both maternal serum titers and AF bilirubin measurements provided early indications that the fetus might have the RhE antigen. Using amniotic cells obtained at the first amniocentesis, DNA was extracted and analyzed for the RhE gene sequence. The use of two primer pairs from distinct sites in the RhCE gene, plus analysis of parental DNA, greatly minimized the possibility of false results. The fetus was determined to be Rhe/Rhe by molecular analysis. The DNA result was confirmed by serologic typing at birth. CONCLUSION: DNA-based RhEe genotyping of at-risk fetuses provides accurate and timely information that is useful in the management of RhE-sensitized pregnancies.

Adult↗
PubMed1988

Establishing paternity using minisatellite DNA probes when the putative father is unavailable for testing.

A paternity case involving a putative father who had died a few years earlier in an automobile accident was referred to the laboratory for testing. The child and his mother, the deceased's parents, and nine of the deceased's siblings were available for analysis. As previously reported, paternity testing using red blood cell groups, human leukocyte antigens (HLA), red blood cell enzymes, serum proteins, and immunoglobulin allotypes gave a cumulative paternity index of 43,300 and a combined probability of paternity equal to 99.998%. RFLP analysis using Hinf I and Sau 3A single digests and the minisatellite deoxyribonucleic acid (DNA) probes 15.1.11.4 and 6.3 showed no exclusion of paternity and gave nearly conclusive evidence that the putative father was the biological father of the child.

DNA Probes↗