Biomedical Engineering Reference
In-Depth Information
Fig. 2. Schematic drawing of the basic principle of the in-cell linker polymerase
chain reaction.
first PCR mixture and washing of the fixed cells will remove any female-de-
rived HLA-DPB1 PCR products diffused out of fixed female cells that would
be able to interfere with the following two nested PCR steps and the final DNA
sequencing, by linkage to TSPY PCR products diffused out of male cells. Dur-
ing the second nested PCR, the linked TSPY-DPB1 PCR product will be ampli-
fied further inside the fixed male cells (and outside cells in the supernatant).
After the second PCR, the PCR mixture is removed (and also used as a tem-
plate for a third PCR). Fixed cells are then resuspended and lysed in 1X PCR
buffer; a portion of this “cell lysate” is then used as template for the third
nested PCR using the primers nesTSPY5 and nesDPB1-3. This final PCR prod-
uct can be visualized on a gel and directly DNA sequenced.
3.2. Initial Control Experiments With Extracted Genomic DNA
Before conducting the actual in-cell PCR linker PCR experiments, the per-
formance of the co-amplification of the two PCR products and linkage has to
be evaluated using extracted genomic DNA from males and females in a tradi-
tional PCR setting (primer sequences in Fig. 1 ). The method is based on the
principle described by Diviacco et al. for constructing internal standards used
in quantitative PCR techniques ( 15 ) .
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