Chromosome microdissection method

Details Chromosome microdissection method Chromosome microdissection method

1999-03-08

2001-05-08

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200

Reexamination Certificate

active

06228587

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to the microscopic dissection of chromosomes into specific regions that are then subjected to study. More specifically, it relates to the amplification of such microdissected chromosome regions so that such study may be more readily and intensely conducted.
BACKGROUND OF THE INVENTION
Chromosome microdissection is a recently developed molecular cytogenetic technique which has become increasingly important as a bridge connecting cytogenetics to molecular genetics. After a decade of effort, this approach has been developed into a useful and reproducible approach for several purposes, including: 1) the isolation of DNA from any cytogenetically recognizable region which can be used to generate DNA microclone libraries for molecular analysis and positional cloning; (see Guan, X -Y, Meltzer P. S., Cao, J. and Trent, J. M.: Rapid generation of region-specific genomic clones by chromosome microdissection: Isolation of DNA from a region frequently deleted in malignant melanoma.
Genomics
14: 680-684, 1992; and Leach, F. S., Nicolaides, N. C., Papadopoulos, N., Liu, B., Jen, J. Parsons, R., Peltomaki, P., Sistonen, P. Aaltonen, L. A., Nystrom-Lahti, M., Guan, X -Y, Zhang, J., Meltzer, P. S., Yu, J -W, Kao, F -T, Chen, D. J., Cerosaletti, K. M., Fournier, R. E. K., Todd, S., Lewis, T., Leach, R. J., Naylor, S. L., Weissenbach, J., Mecklin, J -P, Jarvinen, H., Petersen, G. M., Hamilton, S. R., Green, J., Jass, J., Watson, P., Lynch, H. T., Trent, J. M., de la Chapell, A., Kinzler, K. W., and Vogelstein, B.: Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer.
Cell
75: 1215-1225, 1993); 2) the generation of fluorescent in situ hybridization (FISH) probes for whole chromosome painting probes (see Guan, X -Y, Meltzer, P. S. and Trent, J. M.: Rapid construction of whole chromosome painting probes by chromosome microdissection.
Genomics
and 22: 101-107, 1994) and chromosome arm painting probes (Guan, X -Y, Zhang, H. E., Bitter, M., Jiang, Y., Meltzer, P. ., and Trent, J. .: Chromosome arm painting probes. Nature
Genet
12: 10-1996) for cytogenetic study; 3) combined with fluorescent in situ hybridization, microdissection has been applied to virtually detect virtually any kind of visible chromosome rearrangements (Guan, X -Y, Meltzer, P. S., Bittner, M., Trent, J. M.: Identification of cryptic sites of DNA sequence amplification in human breast cancer by chromosome microdissection.
Nature Genet
8: 155-161, 1994; and Guan, X -Y, Zhang, H. E., Horsman, D., Meltzer, P. S. and Trent, J. M.: Mapping a recurrent breakpoint on chromosome 6q11 in human follicular lymphoma by chromosome microdissection.
Blood
88: 1418-1422, 1996). More recently, 4) microdissection combined with hybrid selection has been applied to identify genes associated with homogeneously staining regions (HSR's) in human cancers (Su, Y. A., Meltzer, P. S., Guan, X -Y, and Trent, J. M.: Direct isolation of expressed sequences encoded within a homogeneous staining region by chromosome microdissection.
Proc Nat Aca Sci USA
91: 9121-9125, 1994; and Guan, X -Y, Xu, J., Anzick, S. L., Zhang, H. E., Trent, J. M., and Meltzer, P. S.: Direct selection of transcribed sequences from microdissected DNA: Isolation of genes within a commonly amplified region at 20q1-q13.2 in breast cancer.
Cancer Res
56: 3446-3450, 1996.)
The process of chromosome microdissection technique includes two parts, microdissection of a target chromosomal region under a microscope using a finely drawn glass needle and subsequent amplification of the dissected DNA fragments with a degenerate oligonucleotide primer by polymerase chain reaction (PCR). Briefly, 5-10 copies of target chromosomeal region are microdissected with glass needle and transferred to a PCR tube containing collection solution. Microdissected DNA fragments are then amplified by PCR. FISH with labeled microdissected PCR products is then routinely used in this protocol to evaluate the experimental result.
While the basic steps of microdissection, amplification and labeling the amplified DNA fragment is known to the art, it has also been recognized that certain microdissection techniques require large amounts of time to accomplish and are highly labor intensive. Thus, dissecting 20 or 30 DNA from a target region to obtain a sufficient template for PCR amplification is an onerous task, and repeated use of a microdissection needle increases the possibility of DNA contamination. As a consequence, it has been a desire of those in this art to provide a method of generating whereby amplification of microdissected chromosome fragments can be accomplished with greater ease and effectiveness, so that the number of microdissected fragments subjected to amplification is substantially decreased, thereby simultaneously decreasing the time spent in accomplishing the dissection as well as the possibility of contamination.
Indeed, the problem is addressed in U.S. Pat. No. 5,545,524 to Trent et al., which discloses a procedure in which a dissected DNA fragment is treated with topoisomerase I prior to amplification of the fragment. Although these patentees state that it is difficult to model the precise effect of Topo I on dissected DNA fragments, it is theorized highly coiled DNA impairs access of primer and DNA polymerase to the template, and that the use of Topo I promotes relaxation of the template DNA, thus permitting access to be more readily effected. Where access is more easily effected, amplification is more readily and easily accomplished.
SUMMARY OF THE INVENTION
The present invention is directed to greatly improving amplification of microdissected DNA fragments by treating the fragments with pepsin prior to amplification. In this manner the efficacy of the amplification is greatly improved, thereby reducing the number of DNA fragments required to later generate probes. In this manner the risks of contamination and time for microdissection to be effected are substantially reduced.


REFERENCES:
patent: 5545524 (1996-08-01), Trent et al.
Alberts et al., The Molecular Biology of the The Cell. 2nd ed. Garland Publishing, Inc. New York, 1989.*
Nenno et al., Genome, vol. 37, pp 1018-121, 1989.

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