Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-06-21
2001-05-22
McKelvey, Terry (Department: 1635)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
Reexamination Certificate
active
06235503
ABSTRACT:
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
Not Applicable
TECHNICAL FIELD
The invention is in the field of genetic analysis. The invention relates to methods for isolation of polynucleotides comprising nucleic acid sequences which are differentially expressed, differentially present, or differentially arranged in two or more different cells, cell populations or cell types, utilizing techniques of subtractive hybridization and selective amplification.
BACKGROUND OF THE INVENTION
The ability to detect differences between two populations of nucleic acid sequences is important to characterizing the molecular basis of various pathological states, for example neoplasia, infectious and degenerative diseases, viral infections and hereditary predisposition to disease. Increasingly, the technique of subtractive hybridization is being used to identify polynucleotides comprising sequences that are present in a first population of nucleic acid sequences but absent, present in a different concentration, or arranged differently in a second population.
Sargent and David,
Science
222: 135-139 (1983) used subtractive hybridization to isolate cDNAs representing mRNA molecules preferentially expressed at the gastrula stage of development of the frog embryo. Gastrula cDNA was hybridized to RNA from unfertilized eggs and the cDNA that failed to hybridize was cloned. These cloned sequences represented mRNAs that were differentially expressed in the frog gastrula. Similarly, Hedrick et al.,
Nature
(London) 308: 149-153 (1984) cloned a T-cell receptor molecule by hybridizing cDNA from antigen-specific T-cells with RNA from B-cells and collecting the non-hybridized cDNA. Despite these early successes, it soon became evident that this method is limited in practice to detection of differentially-expressed mRNA representing 0.01% or more of the total mRNA population. Furthermore, in cases where the method is practical, selection of the differentially expressed cDNA (as single-stranded material) is achieved by hydroxyapatite chromatography, which is cumbersome and results in losses of valuable material. Finally, this technique did not provide a method to detect differences in genome organization, such as deletion, gene amplification, or rearrangement.
Adaptations of the subtractive hybridization technique have been developed which allow the identification and isolation of polynucleotides representing sequence differences between different genomes. Lamar and Palmer,
Cell
37: 171-177 (1984) used a selective cloning approach to isolate Y chromosome-specific sequences in the mouse. Hybridizations were conducted using restriction enzyme-digested male DNA as tracer and sonicated female DNA as driver. Of the duplexes obtained after annealing, only those with both strands derived from male DNA contain sequences unique to the Y chromosome and possess a restriction enzyme recognition site at each end. Such duplexes were cloned preferentially into a vector containing compatible restriction enzyme-generated ends.
Kunkel et al.,
Proc. Natl. Acad. Sci. USA
82: 4778-4782 (1985) and Nussbaum et al.,
Proc. Natl. Acad. Sci. USA
84: 6521-6525 (1987) described the isolation of fragments containing sequences deleted from the human X chromosome by hybridization of restriction enzyme-digested DNA from cells that were polysomic for the X chromosome with an excess of sheared DNA from cells harboring one or more X chromosome deletions, using conditions in which the rate of reassociation was enhanced. Selective cloning using a vector with compatible restriction enzyme-generated ends was used for the isolation of sequences absent in the X chromosome deletions.
Strauss and Ausubel,
Proc. Natl. Acad. Sci. USA
87: 1889-1893 (1990) described a technique for isolating a polynucleotide comprising DNA that is absent in a yeast deletion mutant. In this method, denatured wild-type DNA is allowed to anneal with biotin-labeled DNA from the deletion mutant, and biotin-containing duplexes (which contain sequences common to the mutant and wild-type) are removed from solution by binding to avidin-coated beads. The process is repeated for several cycles, with addition of fresh biotinylated wild-type DNA to the mutant DNA remaining unbound at the end of each cycle. Finally, single-stranded material is amplified by a polymerase chain reaction to generate a probe enriched in sequences missing in the deletion mutant. Of course, this method can only be used to isolate a genomic region that is defined by a deletion mutant, and its applicability to genomes more complicated than that of yeast has not been tested. A similar procedure using biotin-based separation for isolation of differentially expressed cDNAs was described by Lebeau et al.,
Nucleic Acids Research
19: 4778 (1991).
Wieland et al.,
Proc. Natl. Acad. Sci. USA
87: 2720-2724 (1990) described a method for isolating polynucleotides comprising sequences present in a “tester” DNA population that are absent in a “driver” population. In this method, the tester DNA is labeled with biotin, then subjected to several rounds of hybridization with excess driver DNA. After each round, single-stranded DNA is collected by hydroxyapatite chromatography. After the final round, the small amount of nonhybridized biotinylated DNA (unique to the tester population) is purified by avidin affinity chromatography, amplified by a polymerase chain reaction and cloned to generate a probe for sequences unique to the tester population.
Recently, a technique known as Representational Difference Analysis (RDA) has been developed, which allows the isolation of DNA fragments that are present in one population of DNA sequences but absent in another population of DNA sequences. Lisitsyn et al.,
Science
259: 946-951 (1993); Lisitsyn et al.,
Meth. Enzymology
254: 291-304 (1995); U.S. Pat. No. 5,436,142; U.S. Pat. No. 5,501,964; Lisitsyn et al.,
Nature Genetics
6: 57-63 (1994). This method allows one to search for fragments present in a “tester” population of DNA sequences that are not present in a related “driver” population. Such unique fragments are denoted “target” sequences. In the first step of RDA, “representations” of both populations are obtained. These representations consist of lower-complexity subsets of the original sequence populations. In the most widely-practiced embodiment of the technique, a representation is obtained by separately subjecting both populations to digestion with a restriction endonuclease, ligating a first set of adapters to the ends of the fragments so generated, and amplifying by a polymerase chain reaction (PCR) using primers complementary to the first set of adapters, under conditions in which only relatively short fragments (less than 2 kilobase pairs) are amplified. The first adapters are then removed from the amplified fragments of both populations by restriction enzyme digestion and a second set of adapters (having a different sequence than the first set) is attached, by ligation, to amplified fragments from the tester DNA population only.
The adapter-containing amplified fragments from the tester population are then combined with an excess of amplified fragments from the driver population, (which lack adapters) and the mixture is incubated under denaturing and annealing conditions, followed by another round of PCR amplification using primers complementary to the second set of adapters. During the annealing step, several types of duplex will be formed. Because driver fragments are present in excess, the vast majority of fragments containing sequences common to both tester and driver populations will form either driver-driver duplexes (containing no adapter) or tester-driver duplexes (containing a single adapter on the strand derived from the tester fragment). Fragments containing sequences that are unique to the tester population are capable of self-annealing to generate duplexes possessing an adapter at each end. Consequently, during the PCR step subsequent to annealing, tester-tester duplexes will be amplified exponentially. On the other h
Lindemann Garrett W.
Schueler Paula A.
McKelvey Terry
Roche Diagnostics Corporation
Roche Diagnostics Corporation
Waite Kenneth J.
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