Abstract
A method using selective ultraviolet radiation fractionation followed by polymerase chain reaction (PCR) can analyze specific cell subsets present on a microscope section. Direct ultraviolet radiation of fixed and stained tissue sections prevents subsequent amplification by PCR. An "umbrella" or dot placed physically over small numbers of pure cell populations selected by microscopic examination protects these cells from the ultraviolet inactivation. The DNA in these protected cells can be specifically amplified while no signal is derived from the unprotected surrounding cells. Specific amplification was demonstrated by detecting human papillomavirus sequences only if infected cells were protected. Similarly, loss of heterozygosity at the p53 locus was documented by selective dotting of normal or tumor cells. The method allows the specific and sensitive molecular genetic analysis of small numbers of cells histologically identified and selected under the microscope.
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- Bianchi A. B., Navone N. M., Conti C. J. Detection of loss of heterozygosity in formalin-fixed paraffin-embedded tumor specimens by the polymerase chain reaction. Am J Pathol. 1991 Feb;138(2):279–284. [PMC free article] [PubMed] [Google Scholar]
- Cimino G. D., Metchette K., Isaacs S. T., Zhu Y. S. More false-positive problems. Nature. 1990 Jun 28;345(6278):773–774. doi: 10.1038/345773b0. [DOI] [PubMed] [Google Scholar]
- Haase A. T., Retzel E. F., Staskus K. A. Amplification and detection of lentiviral DNA inside cells. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4971–4975. doi: 10.1073/pnas.87.13.4971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hadano S., Watanabe M., Yokoi H., Kogi M., Kondo I., Tsuchiya H., Kanazawa I., Wakasa K., Ikeda J. E. Laser microdissection and single unique primer PCR allow generation of regional chromosome DNA clones from a single human chromosome. Genomics. 1991 Oct;11(2):364–373. doi: 10.1016/0888-7543(91)90144-4. [DOI] [PubMed] [Google Scholar]
- Li H. H., Gyllensten U. B., Cui X. F., Saiki R. K., Erlich H. A., Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature. 1988 Sep 29;335(6189):414–417. doi: 10.1038/335414a0. [DOI] [PubMed] [Google Scholar]
- Nuovo G. J., MacConnell P., Forde A., Delvenne P. Detection of human papillomavirus DNA in formalin-fixed tissues by in situ hybridization after amplification by polymerase chain reaction. Am J Pathol. 1991 Oct;139(4):847–854. [PMC free article] [PubMed] [Google Scholar]
- Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
- Sarkar G., Sommer S. S. Shedding light on PCR contamination. Nature. 1990 Jan 4;343(6253):27–27. doi: 10.1038/343027a0. [DOI] [PubMed] [Google Scholar]
- Sarkar G., Sommer S. More light on PCR contamination. Nature. 1990 Sep 27;347(6291):340–341. doi: 10.1038/347340b0. [DOI] [PubMed] [Google Scholar]
- Shibata D., Brynes R. K., Nathwani B., Kwok S., Sninsky J., Arnheim N. Human immunodeficiency viral DNA is readily found in lymph node biopsies from seropositive individuals. Analysis of fixed tissue using the polymerase chain reaction. Am J Pathol. 1989 Oct;135(4):697–702. [PMC free article] [PubMed] [Google Scholar]
- Tsai Y. C., Nichols P. W., Hiti A. L., Williams Z., Skinner D. G., Jones P. A. Allelic losses of chromosomes 9, 11, and 17 in human bladder cancer. Cancer Res. 1990 Jan 1;50(1):44–47. [PubMed] [Google Scholar]
- Vogelstein B., Fearon E. R., Hamilton S. R., Kern S. E., Preisinger A. C., Leppert M., Nakamura Y., White R., Smits A. M., Bos J. L. Genetic alterations during colorectal-tumor development. N Engl J Med. 1988 Sep 1;319(9):525–532. doi: 10.1056/NEJM198809013190901. [DOI] [PubMed] [Google Scholar]