7) with 2 cosmids, U125A1 (PLP1) and U144A10 (control, hybridizes distal to the PLP1 locus) (7).
Each PLP1 exon was individually amplified by PCR in a 50-[micro]L reaction that contained 20-100 ng of DNA, 25 [micro]L of 2 x Taq PCR master mixture (Qiagen), 10 [micro]L of 5 x Q solution (Qiagen), and 0.
The 32 MLPA probes in the PLP1 reagent set are summarized in Fig.
MLPA analysis using DNA from a non-PMD male, a non-PMD female, a male with a PLP1 duplication (Coriell NA11005), a female carrier of a PLP1 duplication (CH21; Fig.
The CVs for MLPA compared with 2 different quantitative PCR assays in males with 1 or 2 PLP1 gene copies and females carrying 2 or 3 PLP1 gene copies, after calibration to female [MLPA and quantitative PCR 1 (21)] or male DNA [quantitative PCR 2 (20)] are shown in Table 1.
For 50 samples previously analyzed by quantitative real-time PCR for PLP1 gene duplications/ deletions (21), we performed MLPA to validate this assay.
DETECTION OF PLP1 GENE DELETIONS BY MLPA AND PCR Two samples from Fig.
MLPA ANALYSIS OF SAMPLES WITH PLP1 POINT VARIATIONS
Relative PLP1 copy numbers for each of the 7 PLP1 exons in DNA from 5 male samples with known PLP1 missense variations are shown in Fig.
Our results suggest that MLPA has important advantages over quantitative PLP1 PCR assays that determine gene copy number from analysis of 1 or 2 PLP1 exons (6, 7,10, 20, 21).
Interphase FISH can produce false-positive results if regions adjacent to, but not including, the PLP1 gene are duplicated and the FISH probe includes sequences outside the PLP1 gene.
In an unusual case of PMD, mosaicism in the lymphoblastoid cell lines of a PLP1 duplication carrier mother was demonstrated, but quantitative fluorescent multiplex PCR did not detect the duplication (7,29).