American Society of Hematology

Fig. 1.

$Fig. 1. Terminal restriction fragment length analysis on granulocyte DNA. / The donor's TRF minus the patient's TRF gives rise to the dTRF. TRF was calculated and compared between the duplicate samples. The average difference between each duplicate was calculated to be 104 base pairs (bp), and the SD was 88 bp. The lower limit of detection of the method was therefore selected as the average (104 bp) + 3 SD or 369 bp. Differences in TRF of fewer than 369 bp were considered not significant and ascribed to inherent experimental variability. One to 5 (median = 3) replicate experiments were performed with DNA from each of the 17 patient/donor pairs for a total of 50 TRF pairs (in 2 cases, only 1 experiment was done owing to limited DNA quantities). (A) Representative TRF analysis of patient/donor pairs 5, 10, and 14. High MW DNA was digested with HinfI and RsaI restriction enzymes and size-fractionated on a 0.8% agarose gel. Recipient and donor samples were loaded in duplicate lanes 1-4 (pair 5), 5-8 (pair 10), and 9-12 (pair 14), and a radiolabeled 1-kb DNA ladder MW marker was included (lanes M). In situ hybridization was performed with a P32 end-radiolabeled oligonucleotide probe comprising the telomere-specific sequence (TTAGGG)3. The signal was detected by scanning the gel by means of the Phosphorimager analysis system (Molecular Dynamics, Sunnyvale, CA). Mean TRF length was assigned to the MW corresponding to the distance of peak signal intensity from the origin of the electrophoresis. The MW was then calculated by means of the Imagequant (Molecular Dynamics) and Fragment (Molecular Dynamics) software. (B) dTRF data summary of each patient/donor pair. Each column represents the average dTRF of 1 to 5 (median 3) replicate experiments for each pair. The top and bottom ends of the vertical lines depict the maximum and minimum dTRF values obtained in each patient/donor pair. Single experiments were performed on pairs 6 and 8 because of limited amounts of DNA. The 2 thin dotted horizontal lines depict the experimental limit of detection, which was 369 bp as described in “Materials and methods.” When the average difference was computed across experiments for each individual patient, 15 of 17 recipients had dTRF more than 0.369 kb. Of the 15 recipients, 8 had dTRF of more than 1.0 kb and 7 had dTRF 0.3 to 1.0 kb. One of the remaining 2 recipients had dTRF of −0.1 kb, and the other, dTRF of 0.2 kb, both less than 0.369 kb.$

Terminal restriction fragment length analysis on granulocyte DNA.

The donor's TRF minus the patient's TRF gives rise to the dTRF. TRF was calculated and compared between the duplicate samples. The average difference between each duplicate was calculated to be 104 base pairs (bp), and the SD was 88 bp. The lower limit of detection of the method was therefore selected as the average (104 bp) + 3 SD or 369 bp. Differences in TRF of fewer than 369 bp were considered not significant and ascribed to inherent experimental variability. One to 5 (median = 3) replicate experiments were performed with DNA from each of the 17 patient/donor pairs for a total of 50 TRF pairs (in 2 cases, only 1 experiment was done owing to limited DNA quantities). (A) Representative TRF analysis of patient/donor pairs 5, 10, and 14. High MW DNA was digested with HinfI and RsaI restriction enzymes and size-fractionated on a 0.8% agarose gel. Recipient and donor samples were loaded in duplicate lanes 1-4 (pair 5), 5-8 (pair 10), and 9-12 (pair 14), and a radiolabeled 1-kb DNA ladder MW marker was included (lanes M). In situ hybridization was performed with a P³² end-radiolabeled oligonucleotide probe comprising the telomere-specific sequence (TTAGGG)₃. The signal was detected by scanning the gel by means of the Phosphorimager analysis system (Molecular Dynamics, Sunnyvale, CA). Mean TRF length was assigned to the MW corresponding to the distance of peak signal intensity from the origin of the electrophoresis. The MW was then calculated by means of the Imagequant (Molecular Dynamics) and Fragment (Molecular Dynamics) software. (B) dTRF data summary of each patient/donor pair. Each column represents the average dTRF of 1 to 5 (median 3) replicate experiments for each pair. The top and bottom ends of the vertical lines depict the maximum and minimum dTRF values obtained in each patient/donor pair. Single experiments were performed on pairs 6 and 8 because of limited amounts of DNA. The 2 thin dotted horizontal lines depict the experimental limit of detection, which was 369 bp as described in “Materials and methods.” When the average difference was computed across experiments for each individual patient, 15 of 17 recipients had dTRF more than 0.369 kb. Of the 15 recipients, 8 had dTRF of more than 1.0 kb and 7 had dTRF 0.3 to 1.0 kb. One of the remaining 2 recipients had dTRF of −0.1 kb, and the other, dTRF of 0.2 kb, both less than 0.369 kb.

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