Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-29
2002-10-15
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S024300, C536S024330, C536S026600, C536S023100
Reexamination Certificate
active
06465182
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to improved methods for detecting and mapping genetic abnormalities associated with various diseases. In particular, it relates to the use of nucleic acid hybridization methods for comparing copy numbers of particular nucleic acid sequences in a collection of sequences relative to the copy number of these sequences in other collections of sequences.
Many genomic and genetic studies are directed to the identification of differences in gene dosage or expression among cell populations for the study and detection of disease. For example, many malignancies involve the gain or loss of DNA sequences resulting in activation of oncogenes or inactivation of tumor suppressor genes. Identification of the genetic events leading to neoplastic transformation and subsequent progression can facilitate efforts to define the biological basis for disease, improve prognostication of therapeutic response, and permit earlier tumor detection. In addition, perinatal genetic problems frequently result from loss or gain of chromosome segments such as trisomy 21 or the micro deletion syndromes.
Cytogenetics is the traditional method for detecting amplified or deleted chromosomal regions. More recent methods permit assessing the amount of a given nucleic acid sequence in a sample using molecular techniques. These methods (e.g., Southern blotting) employ cloned DNA or RNA probes that are hybridized to isolated DNA. Southern blotting and related techniques are effective even if the genome is heavily rearranged so as to eliminate useful karyotype information. However, these methods require use of a probe specific for the sequence to be analyzed. Thus, it is necessary to employ very many individual probes, one at a time, to survey the entire genome of each specimen, if no prior information on particular suspect regions of the genome is available.
Comparative genomic hybridization (CGH) is a recent approach to detect the presence and identify the location of amplified or deleted sequences. See, Kallioniemi et al., Science 258: 818-821 (1992) and U.S. Pat. No. 5,665,549). CGH reveals increases and decreases irrespective of genome rearrangement. In one implementation of CGH, genomic DNA is isolated from normal reference cells, as well as from test cells (e.g., tumor cells). The two nucleic acid sequences are differentially labeled and then hybridized in situ to metaphase chromosomes of a reference cell. The repetitive sequences in both the reference and test DNAs are either removed or their hybridization capacity is reduced by some means. Chromosomal regions in the test cells which are at increased or decreased copy number can be quickly identified by detecting regions where the ratio of signal from the two DNAs is altered. For example, those regions that have been decreased in copy number in the test cells will show relatively lower signal from the test DNA than the reference compared to other regions of the genome. Regions that have been increased in copy number in the test cells will show relatively higher signal from the test DNA.
Improved CGH techniques have also been described. For instance, CGH applied to arrays allows for more precise localization of chromosome abnormalities than use of a metaphase spreads as the target (see U.S. Pat. No. 5,830,645).
Despite these improvements, there is a constant need for improved methods of genetic analysis that provide fast, reliable results. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods for quantitatively comparing the copy number of a nucleic acid sequence in a first collection of labeled nucleic acid molecules relative to the copy number of that same sequence in a second collection of labeled nucleic acid sequences. The method comprises labeling the nucleic acid molecules in the first collection and the nucleic acid molecules in the second collection with first and second labels, respectively. The first and second labels should be distinguishable from each other. The collections are contacted to a plurality of target oligonucleotides (a microarray) under conditions such that nucleic acid hybridization to the target elements can occur. The two collections can be contacted to the target elements either simultaneously or serially.
The two collections of labeled nucleic acid sequences are prepared by specifically amplifying sequences that hybridize specifically to the target oligonucleotides from source. This amplification produces a representative collection of nucleic acid sequences, meaning that the amplification is both quantitative and results in a collection of reduced complexity. As explained below, a representative collection of nucleic acid sequences is one in which the relative abundance of particular sequences in the source nucleic acids is maintained in the labeled nucleic acids used in the assays of the invention (i.e. is quantitative). In addition, the collection of labeled nucleic acid sequences has much lower complexity as compared to the source nucleic acid molecules. The reduced complexity is advantageous because the rate of hybridization is enhanced, as compared to hybridization using highly complex collections of labeled nucleic acid sequences.
The target oligonucleotides and the labeled nucleic acid sequences may be, for example, RNA, DNA, or cDNA. The nucleic acid sequences may be derived from any organism. Usually the nucleic acid in the target elements and the labeled nucleic acid sequences are from the same species.
The target elements are typically arranged in separate discrete locations on a solid surface. The target oligonucleotides in a target element are those for which comparative copy number information is desired. For example, the oligonucleotides may originate from a chromosomal location known to be associated with disease, may be selected to be representative of a chromosomal region whose association with disease is to be tested, or may correspond to genes whose transcription is to be assayed.
After contacting the labeled nucleic acid sequences to the target elements the amount of binding of each, and the binding ratio is determined for each target element. Typically the greater the ratio of the binding to a target element the greater the copy number ratio of sequences in the two labeled nucleic acid sequences that bind to that element. Thus comparison of the ratios among target elements permits comparison of copy number ratios of different sequences in the labeled nucleic acid sequences.
The methods are typically carried out using techniques suitable for fluorescence in situ hybridization. Thus, the first and second labels are usually fluorescent labels.
In a typical embodiment, one collection of labeled nucleic acid sequences is prepared from a test cell, cell population, or tissue under study; and the second collection of labeled nucleic acid sequences is prepared from a reference cell, cell population, or tissue. Reference cells can be normal non-diseased cells, or they can be from a sample of diseased tissue that serves as a standard for other aspects of the disease. For example, if the reference nucleic acid is genomic DNA isolated from normal cells, then the copy number of each sequence in that collection relative to the others is known (e.g., two copies of each autosomal sequence, and one or two copies of each sex chromosomal sequence depending on gender). Comparison of this to DNA prepared from a test cell permits detection in variations from normal.
Alternatively the reference collection of labeled nucleic acid sequences may be prepared from genomic DNA from a primary tumor which may contain substantial variations in copy number among its different sequences, and the test may be prepared from genomic DNA of metastatic cells from that tumor, so that the comparison shows the differences between the primary tumor and its metastasis. Further, both collections may be prepared from normal cells. For example comparison of mRNA populations between normal cells of different tissues permits detection of d
Albertson Donna
Baldocchi Russell
Collins Colin
Gray Joe
Pinkel Dan
Horlick Kenneth R.
Spiegler Alexander H.
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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