Quantitative PCR method to enumerate DNA copy number

Details Quantitative PCR method to enumerate DNA copy number Quantitative PCR method to enumerate DNA copy number

1999-05-18

2001-01-30

Brusca, John S. (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S023100, C536S024300

Reexamination Certificate

active

06180349

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the field of nucleic acid chemistry and, more particularly, to methods of quantifying the copy number of a locus of interest. The present methods allow for rapid screening of samples to determine copy number, hence the methods can be used in various clinical applications including, establishing correlations between copy number and pathology, and rapidly screening patients to identify those having a disease or those which are susceptible to acquiring a disease.
BACKGROUND OF THE INVENTION
In a variety of different fields of biological research, methods for quantitating nucleic acid sequences has become an increasingly important tool. For example, measurement of gene expression has been used in several different applications to monitor biological responses to various stimuli. An important step in the molecular genetic analysis of human disease, especially cancer and tumors, is often the enumeration of DNA copy number in particular regions of the genome.
Several different approaches are currently available to make quantitative determinations of nucleic acids. Chromosome-based techniques, such as comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH) facilitate efforts to cytogenetically localize genomic regions that are altered in tumor cells. Regions of genomic alteration can be narrowed further using loss of heterozygosity analysis (LOH), in which tumor DNA is analyzed and compared with normal DNA for the loss of a heterozygous polymorphic marker. The first experiments used restriction fragment length polymorphisms (RFLPs) (1, 2), or hypervariable minisatellite DNA (3). In recent years LOH has been performed primarily using PCR amplification of microsatellite markers and electrophoresis of the radiolabeled (4) or fluorescently labeled PCR products (5, 6) and compared between paired normal and tumor DNAs.
These chromosomal methods, however, have several shortcomings. For example, LOH, requires heterozygosity at the markers being analyzed and it is not possible to differentiate between deletions and amplifications with the method. Both FISH and LOH are slow and labor intensive. CGH is an excellent tool for scanning the whole genome, but it is limited to 5 Mb resolution at best.
A number of other methods have also been developed to quantify nucleic acids (Southern, E. M., J. Mol. Biol., 98:503-517, 1975; Sharp, P. A., et al., Methods Enzymol. 65:750-768, 1980; Thomas, P. S., Proc. Nat. Acad. Sci., 77:5201-5205, 1980). More recently, PCR and RT-PCR methods have been developed which are capable of measuring the amount of a nucleic acid in a sample. One approach, for example, measures PCR product quantity in the log phase of the reaction before the formation of reaction products plateaus (Kellogg, D. E., et al., Anal. Biochem. 189:202-208 (1990); and Pang, S., et al., Nature 343:85-89 (1990)). A gene sequence contained in all samples at relatively constant quantity is typically utilized for sample amplification efficiency normalization. This approach, however, suffers from several drawbacks. The method requires that each sample have equal input amounts of the nucleic acid and that the amplification efficiency between samples be identical until the time of analysis. Furthermore, it is difficult using the conventional methods of PCR quantitation such as gel electrophoresis or plate capture hybridization to determine that all samples are in fact analyzed during the log phase of the reaction as required by the method.
Another method called quantitative competitive (QC)-PCR, as the name implies, relies on the inclusion of an internal control competitor in each reaction (Becker-Andre, M., Meth. Mol. Cell Biol. 2:189-201 (1991); Piatak, M. J., et al., BioTechniques 14:70-81 (1993); and Piatak, M. J., et al., Science 259:1749-1754 (1993)). The efficiency of each reaction is normalized to the internal competitor. A known amount of internal competitor is typically added to each sample. The unknown target PCR product is compared with the known competitor PCR product to obtain relative quantitation. A difficulty with this general approach lies in developing an internal control that amplifies with the same efficiency of the target molecule.
Another problem common to a variety of PCR quantitation methods is that probes must be tailored for each locus to be interrogated. Yet another shortcoming is that often a single locus or reference marker is used as a control. The risk inherent in methods relying on a single reference marker as a control is that the quantity of DNA may differ from the normal value, thus preventing a precise measurement of the locus being tested. This is of particular concern in the case of studies with tumors because genomic instability is a common feature of tumors. Current methods also lack the precision necessary to consistently distinguish between one and two copies of DNA.
SUMMARY OF THE INVENTION
The present invention provides rapid and inexpensive methods for determining the copy number of essentially any region of a genome, especially the genome of an eukaryote. The methods generally involve monitoring the formation of amplification product using real time amplification detection systems to quantify the amount of test locus and reference loci in a test subject and the amount of test locus and reference loci in at least one control subject. The methods can be used to interrogate the copy number of loci containing simple sequence repeats. Since such sequences are ubiquitous in eukaryotic genomes, the present methods have wide-ranging applicability. The methods of the present invention can be used as diagnostic and prognostic tools and in correlating abnormal copy number values for specific loci with disease and effectiveness of different treatment options.
In certain methods, the determination of copy number for a polynucleotide locus in a sample includes four steps. First, a test polynucleotide locus in a nucleic acid sample from a test subject is amplified to determine a value for the quantity of the test locus in the sample from the test subject. Second, a plurality of reference polynucleotide loci in a second nucleic acid sample from the test subject are amplified to determine a value for the quantity of the reference loci in a second sample from the test subject. Third, the test polynucleotide locus from a control subject is amplified to determine a value for the quantity of the test locus in the sample from the control subject. Finally, a plurality of reference polynucleotide loci from a second nucleic acid sample from the control subject is amplified to determine a value for the quantity of the reference loci in the second sample from the control subject. The values determined in these four steps are then utilized to determine a measure of the copy number of the test locus in the sample from the test subject.
In some instances, the four values just described, i.e., the values for the test locus and reference loci in the test subject and control subject, are values for the extent of amplification necessary for amplification product formed from the test locus or reference loci to reach a threshold value. Thus, in such methods, the value for the test locus in the test subject is a value for the extent of amplification required for amplification product of the test locus to reach a threshold level; likewise, the value for the reference loci in the test subject is a value for the extent of amplification required for amplification product of the reference loci to reach the threshold level. Similarly, for the control subject, the value for the test locus in the control subject is a value for the extent of amplification required for amplification product of the test locus in the control subject to reach the threshold level, and the value for the reference loci in the control subject is a value for the extent of amplification required for amplification product of the reference loci in the control subject to reach a threshold level. In some methods, the values for the extent of amplificati

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