Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-09
2003-11-04
Whisenant, Ethan (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091400, C435S091520, C435S252300, C435S325000, C536S023100
Reexamination Certificate
active
06641998
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to techniques for manipulating nucleic acids linked to a vector. The methods described can be used, for example, in screening nucleic acid libraries. The methods can also be adapted to provide kits for screening nucleic acid libraries.
2. Introduction
The invention comprises methods and corresponding kits for enriching the presence of one or more desired nucleic acids from a collection of many nucleic acids. By employing the appropriate vectors and primers, the desired nucleic acids are produced in a replication-competent form that can be introduced into a host cell or organism. Thus, for example, the methods can be used with an appropriate bacteriophage library in order to directly produce, from the DNA of the library, replication-competent plasmids containing a desired nucleic acid. The resulting plasmids can immediately be transformed into appropriate hosts. The methods advance the ability of one skilled in the art to rapidly identify and isolate a desired nucleic acid in a vector. By directly employing the nucleic acids of a library rather than the hosts bearing those nucleic acids, the methods circumvent the steps of plating, growing, and transferring millions of clones in order to screen for a desired nucleic acid sequence. Unlike other methods directly employing the nucleic acids of a library, no physical separation or binding procedures are required. Thus, practice of this invention simplifies the screening of genomic, cDNA, or other nucleic acid libraries compared to currently used methods.
3. Description of Related Art
Nucleic acid libraries consist of a collection of different nucleic acids from a particular source, which possess differing nucleic acid sequences. Each of the nucleic acids, called “inserts,” are operably linked to a vector with a particular nucleic acid sequence. The vector allows, inter alia, the nucleic acid inserts to be replicated in an appropriate host.
The nucleic acid molecules that make up a library are typically in circular or linear form. Plasmids are circular nucleic acid molecules that replicate in host organisms using an origin of replication and usually possess a gene that gives the host cell a selective advantage over other cells. Linear vectors, such as the bacteriophage lambda-derived vectors, may contain corresponding elements for replication and selection as well as elements encoding bacteriophage proteins necessary for propagation in bacteria.
Libraries of plasmid vectors typically contain the nucleic acid inserts at a defined location in the plasmid (“cloning site” or “multiple cloning site”). Linear vectors, such as lambda bacteriophage, will also typically contain the inserts at some fixed cloning site or multiple cloning site regions in the vector. The inserts are flanked by vector sequences of a particular design. For example, those skilled in the art have designed flanking regions to make “inserting” nucleic acids more convenient or easier. A number of vector designs have been developed and are well known in the art.
The libraries are generally constructed in order to facilitate the identification and isolation (cloning) of particular nucleic acid sequences, such as novel genes. Thus, it is often desirable to isolate one or more particular nucleic acids from a library for further study or use.
There are several ways to isolate a desired nucleic acid sequence from a library. Originally, in situ filter hybridization methods were used for this identification. (See, e.g., Sambrook, J., et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, N.Y. (1989)) However, filter hybridization methods require intensive labor and a significant amount of materials. For example, the libraries are typically grown or plated on an appropriate surface as individual clones and then transferred to a filter membrane. Identification and separation of a desired clone, containing the desired nucleic acid sequence, requires physically locating a positively hybridizing bacterial colony or phage-producing plaque. Each library needs a certain minimum surface area so that an individual colony or plaque can be differentiated from others. As larger surface areas are needed, the number of filters for hybridization also increase. A hypothetical library of 1 million clones typically requires numerous 100 mm diameter filter membranes in order to screen one copy of the entire library. In addition, the filter hybridization screening methods require multiple rounds of colony plating or phage infection, filter preparation, and hybridization steps. Generally, one skilled in the art can screen up to one million clones effectively, but it may take weeks or months to yield the desired clone.
Other procedures have eliminated the time consuming aspects of filter hybridization. Instead, these procedures combine conventional hybridization with chromatographic or magnetic physical separation techniques. There are numerous examples. One method for isolating a particular plasmid from a mixture of plasmids relies upon hybridization of circular double-stranded plasmid DNA to a RecA protein-coated biotinylated probe. A resulting triple-stranded complex may then bind to an agarose-streptavidin column and be physically separated from other plasmids present. (Rigas, B. et al., P.N.A.S. 83: 9591 (1986)) Another modification employs biotinylated homopyrimidine oligonucleotide probes to form complexes that bind to streptavidin-coated magnetic beads. (Ito, T. et al., Nucleic Acids Res. 20: 3524 (1992); Ito, T. et al., P.N.A.S. 89: 495 (1992)) Takabatake et al. describe a variation of this technique that employs a biotinylated purine-rich oligonucleotide probe to bind the desired nucleic acid molecule. (Takabatake, T. et al., Nucleic Acid Res. 20: 5853-5854 (1992)) One drawback with using only homopyrimidine and purine-rich probes is the limitation in the possible nucleic acid sequences for which screening can be done.
Many other methods also employ a physical binding and separation step. Methods for screening libraries using biotinylated probes and magnetic beads are discussed in U.S. Pat. No. 5,500,356. Another method of screening for nucleic acid sequences is described by Kwok, P. Y. et al. This method, which employs PCR-based screening procedures, uses an ELISA-based oligonucleotide-ligation assay (OLA) to detect the PCR products containing the desired sequence. (Kwok, P. Y., et al., Genomics 13: 935-941 (1992)) OLA employs a “reporter probe” and a phosphorylated/biotinylated “anchor” probe. Streptavidin binding to the biotinylated probe can then separate the desired nucleic acids. (Landegren, U., et al., Science 241:1077-1080 (1988)) Biotin-streptavidin systems also rely on physical binding efficiency and may have the added problems of incomplete biotinylation of the probes used, which results in non-biotinylated probes hybridizing and failing to be separated by the physical technique used, as well as limited accessibility of the biotin on the probe.
In library screening methods, the ability to increase the abundance of a particular nucleic acid relative to all other nucleic acids present in a library is limited by the effectiveness of the physical separation technique used. A general drawback to all of these techniques is their reliance on physical binding and separation steps, which are inefficient and complicated.
A method that employs PCR amplification from cDNA libraries for obtaining additional nucleotide sequence of a desired gene when only a partial sequence is known is discussed in PCT 213 publication WO 96/38591. In that method, a PCR reaction extends primers directed to the cDNA insert sequences on circular plasmids. The extended, double-stranded DNAs from the PCR can be purified and re-ligated to generate the same plasmid with the same insert. Thus, plasmids containing target inserts may be amplified from a cDNA library. However, this method requires circular plasmids. Furthermore, the method also involves time-consuming steps after the PCR amplification. The
Finnegan Henderson Farabow Garrett & Dunner LLP
Stratagene
Tung Joyce
Whisenant Ethan
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