Biomedical Engineering Reference
In-Depth Information
5.
Interestingly, in the in-cell PCR study by Chapal et al.
(
17
)
, the best results of
in-cell PCR amplification and linkage of the two PCR products were in the fol-
lowing order according to the number of cells included: 500 > 5000 > 50,000.
This could indicate that the in-cell PCR efficiency is dependent on the number of
fixed cells included in the reaction and may decrease with increasing cell num-
ber. There might be an optimum between the numbers of PCR-specific male cells,
PCR-unspecific female cells, and magnetic beads. Interestingly, in dilution
experiments a clearer band of linked PCR product was observed in the high dilu-
tion (1:250) of male cells in female cells compared with the low dilutions.
Acknowledgments
The author thanks Lone G. Nielsen for careful technical assistance on cer-
tain parts of this work.
References
1. Komminoth, P. and Long, A. A. (1993) In-situ polymerase chain reaction. An
overview of methods, applications and limitations of a new molecular technique.
Virchows Arch. B. Cell Pathol. Incl. Mol. Pathol.
64,
67-73.
2. Long, A. A., Komminoth, P., Lee, E., and Wolfe, H. J. (1993) Comparison of
indirect and direct in-situ polymerase chain reaction in cell preparations and tis-
sue sections. Detection of viral DNA, gene rearrangements and chromosomal
translocations.
Histochemistry
99,
151-162.
3. Patterson, B. K., Till, M., Otto, P., et al. (1993) Detection of HIV-1 DNA and
messenger RNA in individual cells by PCR- driven in situ hybridization and flow
cytometry.
Science
260,
976-979.
4. Timm, E. A. J., Podniesinski, E., Duckett, L., Cardott, J., and Stewart, C. C. (1995)
Amplification and detection of a Y-chromosome DNA sequence by fluorescence
in situ polymerase chain reaction and flow cytometry using cells in suspension.
Cytometry
22,
250-255.
5. Hviid, T. V. (2002) In-cell PCR method for specific genotyping of genomic DNA
from one individual in a mixture of cells from two individuals: a model study with
specific relevance to prenatal diagnosis based on fetal cells in maternal blood.
Clin. Chem.
48,
2115-2123.
6. Elias, S., Price, J., Dockter, M., et al. (1992) First trimester prenatal diagnosis of
trisomy 21 in fetal cells from maternal blood.
Lancet
340,
1033.
7. Cheung, M. C., Goldberg, J. D., and Kan, Y. W. (1996) Prenatal diagnosis of
sickle cell anaemia and thalassaemia by analysis of fetal cells in maternal blood.
Nat. Genet.
14,
264-268.
8. Bianchi, D. W., Simpson, J. L., Jackson, L. G., et al. (2002) Fetal gender and
aneuploidy detection using etal cells in maternal blood: analysis of NIFTY I data.
National Institute of Child Health and Development Fetal Cell Isolation Study.
Prenat. Diagn.
22,
609-615.
9. Lo, Y. M. D. (2005) Recent advances in fetal nucleic acids in maternal plasma.
J. Histochem. Cytochem.
53,
293-296.
