The presence of human-specific DNA within the blood and organs of transplanted mice was confirmed by polymerase chain reaction (PCR) amplifying an 850-bp fragment of the α-satellite region of the human chromosome 17 using primers corresponding to the primer pair 17a1/17a2 as described by Warburton et al (1991) (link). The primers were elongated to 25 nucleotides each for use at higher annealing temperatures. The sequences are shown in Table 1 Oligonucleotides and probes used for PCR ![]()
M each of the respective nucleotides, 250 nM of each primer, 2 mM MgCl2, and 250 ng of genomic DNA template. Following an initial DNA denaturation and Taq activation at 94°C for 10 min, 35 1-min cycles of denaturation at 94°C and annealing/extension at 60°C were performed followed by a final elongation step at 72°C for 10 min. Amplified DNA fragments were electrophoresed through 1.75% agarose gels and subsequently visualised through ultraviolet light after staining with ethidium bromide. Genomic DNA samples from both a human breast carcinoma line (MaCa 3366) as a positive control and NOD/SCID mouse liver tissue as a negative control were processed in parallel. Routinely used PCR was evaluated by scoring band intensities expressed by one to three plus. One plus corresponded to very weak bands and three plus corresponded to very intense bands comparable to that of the positive control with 100% human cells.
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