Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-02
2002-08-13
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024330
Reexamination Certificate
active
06432650
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the amplification of chromosomal DNA in situ. In particular, this invention relates to the amplification of chromosomal DNA in situ prior to microdissection.
BACKGROUND OF THE INVENTION
Chromosome microdissection is an extremely useful molecular cytogenetic tool for the characterization and analysis of chromosomes. For example, microdissection has become a very popular method for making both whole-chromosome and region-specific painting probes for use in Fluorescence in situ Hybridization (FISH). In addition, microdissection has been applied to the isolation and characterization of region specific cDNAs.
While useful, microdissection does possess serious drawbacks, however. For instance, it is generally necessary to dissect multiple copies of a target chromosome or chromosome region to produce a probe sufficiently complex for FISH. Although band-specific probes have been made from single chromosome fragments, painting regions larger than this requires as many as 50 chromosome copies to be dissected for sufficient probe coverage. The need to dissect more than one copy of a target chromosome complicates the process of microdissection as it can be difficult to precisely locate the same chromosomal region when making multiple scrapes of a single band. As a result, the painting probe covers a wider region than desired. When probes are made for adjacent bands this can result in overlapping signals, which complicates analysis. Thus, the ability to make chromosome paints from single scrapes of a band, arm, or chromosome is highly desirable.
There is thus a need to amplify chromosomal DNA in situ in order to increase the amount of DNA associated with a chromosome or chromosome region to facilitate chromosome analysis by microdissection.
SUMMARY OF THE INVENTION
In order to meet these needs, the present invention is directed to the amplification of chromosomal DNA in situ to increase the amount of DNA associated with a chromosome or chromosome region. The chromosomes may be isolated from any chromosome-containing organism including birds (avian), reptiles, amphibians, plants, and mammals such as humans, mice and rats, etc. The chromosomes include mini chromosomes.
The amplification of chromosomal DNA in situ allows Fluorescence in situ Hybridization (FISH) painting probes to be made from single dissected chromosome fragments. Furthermore, the amplification of chromosomal DNA in situ permits the synthesis of cDNA libraries from low copy mRNAs. The amplification of chromosomal DNA in situ facilitates comparative hybridization and microdissection of tumor sections. In addition, the hybridization of cDNA libraries to chromosomes normalizes the frequency of the constituent cDNA sequences, i.e. increases the ratio of the less prevalent to more prevalent expressed sequences.
The present invention is directed to a method of preparing chromosomes for microdissection, comprising the steps of: a) fixing cells on a surface wherein the cells comprise chromosomes and the chromosomes include chromosomal DNA and b) amplifying the chromosomal DNA on the surface. The chromosomal DNA may be amplified by PCR including DOP-PCR.
The present invention is further directed to a method of microdissecting chromosomes, comprising the steps of: a) fixing cells on a surface wherein the cells comprise chromosomes and the chromosomes include DNA; b) amplifying the DNA in situ, and c) microdissecting the chromosomes. The DNA may be amplified by PCR including DOP-PCR in step b).
The present invention is further directed to a method of amplifying chromosomal DNA in situ, comprising the steps of: a) fixing cells on a surface wherein the cells comprise chromosomes and the chromosomes include DNA; b) preparing a PCR reaction buffer; c) adding the PCR reaction buffer to the fixed cells of step a); and d) amplifying the DNA by PCR in situ. The PCR amplification step d) may be DOP-PCR. The PCR reaction buffer will include DNA polymerase, eg. thermostable DNA polymerase.
The present invention is further directed to a method of amplifying chromosomal DNA in situ, comprising: a) providing cells embedded in paraffin wherein the cells comprise chromosomes and the chromosomes include DNA; b) preparing a PCR reaction buffer; c) combining the PCR reaction buffer with the paraffin embedded cells of step a) and d) amplifying the DNA by PCR in situ. The DNA in step d) may be amplified by DOP-PCR. This method may be used in comparative genomic hybridization (CGH) to compare the DNA content of cells, e.g. cancer cells vs. normal cells. For example, after DNA amplification in situ, CGH may be used on DNA isolated from tumor cells labeled with a fluorochrome of one color (e.g., green), and DNA from normal cells, usually but not necessarily obtained from a non-neoplastic region adjacent to the tumor, labeled with a different color (e.g., red). After hybridizing and washing steps, the chromosomes are analyzed for differential hybridization to compare and contrast cancerous and non-cancerous cells.
The present invention is further directed to a method of generating chromosome region-specific nucleic acids for a chromosomal region of interest comprising the steps of: a) obtaining chromosomes containing chromosomal DNA; b) amplifying the chromosomal DNA in situ, and c) microdissecting the chromosomal region of interest to provide a microdissected chromosome fragment containing microdissected chromosomal DNA. The amplification step b) may further comprise a polymerase chain reaction. The method may further comprise step e) labeling the amplified microdissected chromosomal DNA. The invention is further directed to labeled chromosomes and chromosome probes, and labeled chromosome region-specific nucleic acids for a chromosome region-specific nucleic acid produced by the method of the invention.
The DNA may be labeled using any conventional techniques, which permit detection. Examples of suitable labels include biotin-avidin immunofluorescent, digoxigenin, chromogenic, and radioisotopic labels and direct chemical labeling with fluorochromes.
The present invention is further directed to a method of localizing a chromosomal region of interest in a chromosome sample having nucleic acid sequences, comprising the steps of: a) providing a chromosome region-specific probe generated by: (i) amplifying the chromosomal DNA in situ, (ii) microdissecting the chromosomal region of interest to provide a microdissected chromosome fragment; (iii) amplifying the microdissected chromosome fragment; and (iv) labeling the amplified fragment to provide the probe; and b) contacting the chromosome sample with the probe under conditions favorable for hybridization between the probe and complementary nucleic acid sequences in the sample and c) determining the existence and location of hybridization in the chromosome sample.
The present invention is further directed to a method of screening a library of nucleic acid clones for a clone of a chromosomal region of interest comprising the steps of: a) providing a chromosome region-specific probe generated by: (i) amplifying the chromosomal DNA in situ, (ii) microdissecting the chromosomal region of interest to provide a microdissected chromosome fragment; (iii) amplifying the microdissected chromosome fragment; and (iv) labeling the amplified fragment to provide the probe; b) providing the library of clones to be screened; c) contacting each clone with the probe under conditions favorable for nucleic acid hybridization; and d) determining whether and in which clone hybridization has occurred.
The present invention is further directed to a method of amplifying a cDNA library, comprising: a) fixing cells on a surface wherein the cells comprise chromosomes and the chromosomes include genomic DNA; b) amplifying the genomic DNA on the surface to form amplified genomic DNA; c) hybridizing the cDNA library to the genomic DNA to form DNA hybrids; and d) amplifying the DNA hybrids on the surface to form an amplified cDNA library. In the method, the DNA in steps b) and d) may be amplified b
Christian Allen T.
Coleman Matthew A.
Tucker James D.
Campbell Eggerton A.
Chunduru Suryaprabha
Lee Ann M.
The Regents of the University of California
Thompson Alan H.
LandOfFree
Amplification of chromosomal DNA in situ does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Amplification of chromosomal DNA in situ, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Amplification of chromosomal DNA in situ will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2880974