In-Cell Polymerase Chain Reaction - Clinical Applications of PCR - page 55

Fig. 4. Gel electrophoresis of the third linked and nested in-cell polymerase chain
reaction product from an experiment involving the dilution of fixed and permeabilized
CD71 + cells derived from 0.5 mL of cord blood from a male pregnancy diluted in cells
derived from 0.5 mL of cord blood from a female pregnancy in volume ratios of 1:10
(lane 2), 1:20 (lane 3), 1:50 (lane 4), and 1:250 (lane 5). Lane 1 is a DNA marker (100-
bp ladder).
56 ( Fig. 3C ). The 1:1 mixture of male and female cells clearly shows the HLA -
DPB1 genotype of the male sample ( Fig. 3D ). This was also the case for other
HLA-DPB1 polymorphisms. Therefore, DNA sequencing of the PCR products
clearly demonstrates that no cross-association has occurred between male and
female PCR product sequences ( see Note 4 ). So in mixtures of male and female
cells, only the male HLA-DPB1 polymorphic DNA sequence is detected by
sequencing. In pure female samples, no linked PCR signal is obtained.
3.7. Studies of the Sensitivity of the In-Cell Linker PCR Method
To obtain data on the sensitivity of the in-cell linker PCR method fixed and
permeabilized CD71 + cells derived from 0.5 mL of cord blood from a male
pregnancy can be diluted in the same type of cells derived from 0.5 mL of cord
blood from a female pregnancy in volume ratios of 1:10, 1:20, 1:50, and 1:250.
Afterward, the three PCR steps described previously are performed with the
exception that the cells are lysed after the first PCR. Final PCR products are
visualized on agarose gels. A positive in-cell linker PCR signal can be seen
down to a ratio of 1:250 ( see Fig. 4 ). Because 6-7000 CD71 + cells were obtained
from 0.5 mL of cord blood, a positive PCR signal may be obtained from at
least approx 25 male cells in a male-female mixture of cells ( see Note 5 ).