Evaluation of seven PCR-based assays for the analysis of microchimerism.
OBJECTIVE
The presence of small numbers of cells of donor origin in the circulation of recipients of organ transplants (microchimerism) may correlate with immunologic tolerance. As part of our ongoing studies on microchimerism, we evaluated the utility of seven PCR-based assays for the detection of the less abundant DNA in paired mixtures (100 ng total DNA).


METHODS
DNA samples were screened to identify pairs informative for one or more PCR assays. DNA mixtures from the informative pairs were then analyzed using at least one assay. The assays were based on the X-Y homologous region; a Y chromosome microsatellite locus; three autosomal microsatellite loci; the D1S80 minisatellite locus; and sequence specific oligonucleotide probe (SSOP) analysis of the HLA DRB1 locus.


RESULTS
About 0.1% of male DNA against a background of female DNA was detectable using primers for the X-Y homologous region, but the sensitivity was increased to 0.0001% using nested primers for the Y chromosome microsatellite marker. Analysis of the minor DNA component was difficult with the three autosomal microsatellite assays because of the presence of shadow bands. Similar problems with the D1S80 assay were resolved using more stringent PCR conditions, and the sensitivity was 0.1%. Using the DRB1 locus, we were able to detect 1% DNA in the mixed samples.


CONCLUSIONS
These studies show that: (a) nested PCR for the Y chromosome is the most sensitive assay for the detection of microchimerism; (b) D1S80 is a useful marker for microchimerism; (c) additional optimization of analytical conditions is required if autosomal microsatellite markers and the SSOP assay are to be used for microchimerism analysis.
Sahota
A
A
Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis 46202-5251, USA. asahota@iupui.edu
Yang
M
M
McDaniel
H B
HB
Sidner
R A
RA
Book
B
B
Barr
R
R
Brahmi
Z
Z
Jindal
R M
RM
D005260:Female; D006684:HLA-DR Antigens / Q000235:genetics; D059811:HLA-DRB1 Chains; D006801:Humans; D016030:Kidney Transplantation / Q000276:immunology; D016031:Liver Transplantation / Q000276:immunology; D008297:Male; D018895:Microsatellite Repeats / Q000235:genetics; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D018183:Transplantation Chimera / Q000235:genetics*; D014998:Y Chromosome / Q000235:genetics
D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D015345:Oligonucleotide Probes