Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-03-18
2002-12-24
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091200, C435S091510
Reexamination Certificate
active
06498024
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for selectively amplifying target RNA relative to non-target RNA in a sample of tester RNA.
BACKGROUND OF THE INVENTION
Disease is a deviation from the normal functioning of the body's organs or systems. This deviation can arise in a number of ways: by either an abnormal gene being switched on or by a normal gene being switched off; by chromosomal mutations or rearrangements which frequently result in abnormal or missing gene products in congenital conditions; or by the presence of an infectious agent in genetically normal individuals. In some cases, the total complement of mRNA, the products of gene expression, in an abnormal cell Will be different from that in a normal cell. In other cases, there may be no apparent difference in the level of gene expression, but the genetic lesion may be a subtle point mutation giving rise to a defective gene product.
Identification of differences in genetic expression or sequence between normal and abnormal cells is a powerful diagnostic and/or prognostic tool. It can also be the first step in understanding a disease by revealing its underlying mechanism. Thus, identification of genetic differences between normal and abnormal cells can provide a clear path to the design of new diagnostic tests, new drugs or gene therapy.
Methods for identifying and isolating sequences present or actively expressed in one cell but diminished or absent in another cell, referred to as “differential screening” and “difference cloning”, have been applied to both genomic DNA and mRNA. Difference cloning is based upon subtractive hybridization, a method for isolating “target” sequences from one DNA population, referred to in this application as “tester”, by using an excess of sequences from another DNA population, referred to in this application as “driver”. One method of subtractive hybridization mixes a restriction endonuclease-digested tester DNA with an excess of randomly sheared driver DNA Lamar, et al., “Y-Encoded, Species-Specific DNA in Mice: Evidence That the Y Chromosome Exists in Two Polymorphic Forms in Inbred Strains”, Cell, Vol. 37, pp. 171-177, (1984). The DNA mixture is denatured, hybridized and ligated into a compatible restriction site in a cloning vector. Only a tester DNA fragment reannealled to its complement would have both of the correct ends required for cloning. Conversely, any tester DNA fragments that anneal to complementary driver DNA fragments would not have both of the required ends. The low yield of cloned target sequences in this method is due primarily to the slow reannealling of dilute tester sequences to their complements. In addition, the enrichment of unique target sequences from a background of sequences common to the driver is limited by the initial excess of driver to tester.
Other methods of subtractive hybridization are directed toward the preparation of subtracted probes for differential screening of cDNA libraries by in situ colony blot hybridization. In differential screening, differentially expressed nucleic acids are not cloned, but are used as hybridization probes to identify and characterize unenriched cDNA clones. One method described by Kuze, et al., “A new vector and RNase H method for the subtractive hybridization”, Nucleic Acids Research, Vol. 17, No. 2, pp. 807, (1989). prepares subtracted RNA probes using hybridization to DNA, followed by digestion with RNase H to separate non-hybridized RNA from the hybrid. After the remaining RNA is purified, the subtractive hybridization process is repeated. Hybridization of immobilized DNA using the purified subtracted RNA probe indicates that the subtracted probes can be enriched at least 100 fold. Kuze et al. does not describe a way in which the sequences of the subtracted RNA itself may be cloned or amplified, or suggest a use for the subtracted RNA other than as a hybridization probe.
Some improvements to the subtractive hybridization methods, as applied to difference cloning, involve the use of nucleic acid amplification processes selectively to increase the copy number of a DNA segment having the target sequence. An improvement to subtractive hybridization described by Wieland, et al., “A method for difference cloning: Gene amplification following subtractive hybridization”, Proc. Natl., Acad. Sci. USA, Vol. 87, pp. 2720-2724, (1990). Uses the “polymerase chain reaction” (PCR) to increase the concentration of target sequences, and multiple steps of annealing tester DNA to excess driver DNA to further enrich the unique target sequences from a background of sequences common to the driver. in this procedure tester DNA fragments are first prepared for amplification by ligating to a “template” oligonucleotide. A mixture of prepared tester DNA and a 200-fold excess of randomly sheared driver DNA is denatured and reannealled to 90% completion, after which the remaining single-stranded DNA containing target sequences is purified from the double-stranded DNA containing the driver. After three rounds of denaturation, annealing and purification, the remaining tester DNA is then amplified in PCR using primers that anneal to the template sequences. The double-stranded PCR products are then cloned and sequenced. The method gave a 100- to 700-fold enrichment of target sequences.
Another improvement to subtractive hybridization described by Lisitsyn, et al., “Cloning the Differences Between Two Complex Gnomes”, Science, pp. 946-951, (1993). is a technique called “representational difference analysis” (RDA). RDA lowers the complexity of both tester and driver genomic DNA by using various restriction endonucleases, eg. BamHI, Bg/II and HindIII, to generate fragments of a particular length that can be efficiently amplified in PCR as “representations” of the genome. The tester and driver fragments are ligated to dephosphorylated oligonucleotide adaptors such that an adaptor sequence is ligated to the 5′-end of each strand. The adapted fragments are then amplified in separate PCR reactions using the adaptor as a primer to achieve kinetic enrichment of a population of “amplicons” that are below 1 kb in size. Finally, the tester and driver amplicons is digested with the same restriction endonuclease to remove the original adaptors.
The “difference analysis” step of RDA is based upon the kinetic and subtractive enrichment of the tester amplicons in a second PCR. It begins with ligating different dephosphorylated oligonucleotide adaptors, this time only to the tester amplicons. An excess of driver amplicons are then mixed with the adapted tester fragments, denatured, and allowed to anneal. A portion of the annealed fragments is then treated with a DNA polymerase to allow extension of driver and adapted tester strands using the complementary driver or adapted tester strands as template. The annealed and extended amplicons are then amplified in PCR using the adaptor as a primer. Driver strands annealed to complementary driver strands will not be extended or amplified. Driver strands annealed to complementary adapted tester strands will be extended, but will lack a 5′-terminal adaptor sequence that is necessary to form a template for exponential amplification in PCR. Only the adapted tester strands that anneal to their complementary adapted tester strands and are extended prior to PCR will contain sequences on both 5′ and 3′ ends to enable exponential amplification.
Following 10 cycles of PCR, a portion of the amplified products is treated with a nuclease to specifically degrade single-stranded nucleic acids. After inactivating the nuclease, a portion of the remaining nucleic acids is further amplified in PCR using the same primer. After 15 to 20 more cycles of PCR, the double-stranded DNA products are digested with the restriction endonuclease, and the process of difference analysis is repeated. After sufficient rounds (typically 3 to 4, for example) of RDA are performed, the double-stranded DNA products are finally cloned and analyzed.
Although RDA was originally developed for genomic DNA, a variation of
Malek Lawrence T.
Sooknanan Roy R.
Fulbright & Jaworski
Horlick Kenneth R.
SignalGene Inc.
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