Amplification by the polymerase chain reaction of hypervariable regions of the human genome for evaluation of chimerism after bone marrow transplantation

Abstract

We combined the polymerase chain reaction (PCR) with oligonucleotide hybridization as a novel and sensitive technique to evaluate posttransplant chimerism. Specific oligonucleotides for hybridization were synthesized homologous to tandemly repetitive core sequences of regions with a variable number of tandem repeats (VNTRs). Polymorphisms at such loci result from allelic differences in the number of repeats. Primers flanking the repeat region of each of the corresponding VNTRs were used for amplification. Recipient and donor pretransplant DNA and recipient posttransplant DNA were amplified. The resultant fragments were analyzed after gel electrophoresis either by hybridization in-gel or after Southern transfer. To confirm our findings, we also performed standard assays of restriction fragment length polymorphisms (RFLPs). Evaluation of 13 selected cases indicated mixed chimerism (4), complete chimerism (5), recurrence of leukemia (2), and endogenous repopulation of hematopoiesis (2) after marrow transplantation. Sensitivity of the method was determined by mixing various proportions of recipient and donor DNA; the limit of detection of the minor component in a mixture was 0.1%. PCR data correlated with RFLP data in all cases except two in which PCR proved more sensitive than RFLP. PCR amplification of VNTRs combined with oligonucleotide hybridization is a novel technique for documenting posttransplant chimerism and has advantages over RFLP analysis: high sensitivity, use of small amounts of DNA (250 ng), ease of preparation of DNA, elimination of need for restriction enzymes, and the ability to complete studies in 2 days.

A simple and effective approach for detecting maternal cell contamination in molecular prenatal diagnosis

“…Since the PCR technique has the potential to detect DNA at the level of a few cells, MCC may be a serious source for misdiagnosis. PCR allows detection of a minor cell population at levels of 0.1%, as demonstrated for evaluation of chimerism after bone marrow transplantation (Ugozzoli et al, 1991). It has been suggested that levels of contamination as little as 1 ml of maternal blood per ml of amniotic fluid could interfere with PCR results (Rebello et al, 1994).…”

“…Uncultured amniotic cells present a higher level of maternal contamination than cultured cells (Winsor et al, 1996), while cultured chorionic villi present the highest level of maternal contamination (Saura et al, 1994). However, CVS is still the first choice for prenatal diagnosis in pregnancies at high risk (van den Berg et al, 2000). Therefore, it is obligatory to reveal the potential MCC in fetal tissues.…”

Hemoglobin Loves Park [β68 (E12) Leu→Phe]: Report of five cases including one originating from a de novo mutation

“…Haplotypes were assigned after examining nine restriction endonuclease sites within the bglobin gene cluster [HincII (e), XmnI (5 0 to G g), HindIII (within G g and A g), HincII (within and 3 0 to b), AvaII (within b), and HinfI (3 0 to b)] [9]. The study of the segregation in the family of four VNTRs (APO B, DXS52, D1S111, and D1S80) was made using a PCR-based methodology [10,11]. False paternity was excluded by using the AmplFSTR 1 Profiler Plus TM (PCR Amplification Kit) according the manufacturer's instructions (ABI Prism 3100 DNA Genetic Analyzer; Applied BioSystems).…”

Quantitative analysis of chimerism using a short tandem repeat method on a fluorescent automated DNA sequencer

“…These STR loci were chosen on the basis of their high frequency in the population (Edwards et al, 1992). The sequences of primers used in the study have been published by other investigators (Ugozzoli et al, 1991;Anker, Steinbrueck & Donis-Keller, 1992;Kimpton, Walton & Gill, 1992;Sullivan et al, 1993;Edwards & Gibbs, 1994;Hammond et al, 1994;Hochmeister et al, 1994;Kimpton et al, 1994;Walsh et al, 1996;de Weger et al, 2000). Oligonucleotides for PCR amplification were ordered from BioSource INC (BioSource, Rockville, MD, USA).…”