Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-05
2001-11-13
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S173300, C435S320100, C536S023100
Reexamination Certificate
active
06316193
ABSTRACT:
BACKGROUND
As efforts in high-throughput gene sequencing and discovery intensify, novel genes and gene transcripts are being identified at accelerated rates. There therefore exists a need for rapid and reliable methods for screening and isolating full-length nucleic acid sequences coding for such genes.
DESCRIPTION OF THE INVENTION
The present invention relates to nucleic acid molecules distributed into a plurality of containers. In one aspect of the invention, a population of nucleic acid molecules are distributed into multi-well plate containing a plurality of receptacles, containers, or depressions (“wells”), such a 24-well, a 96-well, or 384-well plate, etc. In another aspect of the invention, at least two different nucleic acid populations, obtainable from different sources, are distributed on a single multi-well plate. The multi-well plate can be used for a variety of purposes, including for detecting and obtaining full-length coding sequences, or, for screening for the presence or absence of one or more predetermined genes or nucleotide sequences. Detection, screening, etc. can be accomplished in any desired manner, including by polymerase chain reaction (PCR), differential display (e.g., see, Liang et al.,
Nucl. Acid Res.,
21:3269-3275, 1993; U.S. Pat. No. 5,599,672; or WO97/18454), mismatch repair (e.g., U.S. Pat. No. 5,656,430; U.S. Pat. No. 5,683,877; Wu et al., Proc. Natl. Acad. Sci., 89:8779-8783, 1992), hybridization with oligonucleotide probes, etc.
An object of the invention is an array of a cDNA population from a desired mRNA source, comprising: a multi-well plate containing a plurality of individual wells, each well comprising about 1000-10,000 cDNA clones in aqueous suspension, wherein said cDNA population comprises cDNA of a predetermined size; at least two wells in said plate comprise a different content of cDNAs; and said array of said cDNA population in all the wells of said plate is representative of substantially all mRNA of said predetermined size of said source. Optionally, wherein each well in said plate comprises a different content of cDNA; wherein said cDNA is inserted into a vector and said cDNA is operably linked to an expression control sequence; wherein said vector is a plasmid and said cDNA is operably linked to an expression control sequence; wherein each well comprises about 5000 cDNA clones.
An object of the invention is a method of identifying a desired cDNA having a nucleotide sequence, comprising: detecting said nucleotide sequence in at least one well of a plate as described above having a first array of a cDNA population. Optionally, whereby said detecting said nucleotide sequence is performing a polymerase chain reaction on said cDNA using specific oligonucleotide primers to said nucleotide sequence and observing a product of said reaction for each well; wherein said polymerase chain reaction is performed on an aliquot obtained from each well of said plate; identifying a first target well of said plate, which well contains a cDNA having a desired reaction product, wherein said desired product comprises said nucleotide sequence; detecting said nucleotide sequence in a second array of a cDNA population, comprising: a second multi-well plate comprising a plurality of wells, each well comprising about 10-100 cDNA clones, wherein said second array is an array of said cDNA in said first target well; whereby said detecting said nucleotide sequence is performing a polymerase chain reaction on said cDNA using said specific oligonucleotide primers to said nucleotide sequence and observing a product of said reaction for each well; wherein said polymerase chain reaction is performed on an aliquot obtained from each well of said second plate; further comprising: identifying a second target well of said second plate, which well contains a cDNA having a desired reaction product, wherein said desired product comprises said nucleotide sequence; further comprising detecting said cDNA having said nucleotide sequence in said second well by colony screening using a polymerase chain reaction or oligonucleotide hybridization.
An object of the invention is an array of a cDNA population, comprising: a multi-well plate comprising a plurality of wells, each well comprising about 10-100 cDNA clones in aqueous suspension, and said cDNA population is an array of a single well as described above. Optionally, wherein each well comprises about 50 cDNA clones.
An object of the present invention is an array of a cDNA population, comprising a plurality of plates, each plate comprising a plurality of wells, each well comprising about 10-100 cDNA clones in aqueous suspension, wherein said cDNA population comprises cDNA of a predetermined size and each well contains a different content cDNAs; and said plurality of plates is representative of substantially all mRNA of a predetermined size of said source. Optionally, wherein each well comprises about 50 cDNA clones.
An object of the invention is an array of at least two different cDNA populations in a single multi-well plate, each population prepared from a different source of mRNA, comprising: a multi-well plate containing a plurality of individual wells, each well comprising about 30,000-100,000 cDNA clones in aqueous suspension, wherein each different cDNA population comprises mRNA of a predetermined size; and at least two wells in said plate comprise a different content of cDNAs. Optionally, wherein each individual well comprises a content of cDNA from only one cDNA population; wherein said different source of mRNA is a different tissue type or a different cell type.
An object of the invention is an array of an aqueous suspension of at least two different cDNA populations in a single multi-well plate, each population obtainable from a different source of mRNA, comprising: a multi-well plate comprising a plurality of individual wells, wherein a subset of individual wells comprises a cDNA population in an aqueous suspension which is representative of substantially all mRNA of a predetermined size of a desired mRNA source, and the cDNA content of each individual well is different; and said plate contains at least two different said subsets of individual wells, each subset comprising a different cDNA population and each cDNA population is representative of substantially all mRNA of a predetermined size of a desired and different mRNA source. Optionally, wherein each individual well comprises about 1,000-120,000 cDNAs; wherein each individual well comprises about 30,000-100,000 cDNAs.
An object of the invention is an array of an aqueous suspension of a cDNA population obtainable from a desired mRNA source, comprising: a multi-well plate containing a plurality of individual wells, each individual well containing an aqueous suspension of a different content of said cDNA population, wherein said cDNA population comprises cDNA of a predetermined size and said cDNA population in all the wells of said plate is representative of substantially all mRNA of said predetermined size of said source. Optionally, wherein each individual well contains about 2,000-10,000 cDNAs; wherein each individual well contains about 5,000 cDNAs.
A In a preferred embodiment of the invention, a sample of RNA is extracted from one or more desired sources. The selection of the source will depend upon the objective, i.e., a source is preferably utilized which expresses the gene of interest, e.g., novel transiently expressed genes during development, cancer, or pathological conditions; genes involved in signal transduction; genes involved in immunological conditions; genes involved in the cell cycle; etc. Useful sources include, cell (primary, immortalized, transformed, cancerous, etc.) lines, tissues (e.g., normal, cancerous, biopsied, etc.), organs, whole organisms, etc.
The extracted RNA can be processed in any suitable way to achieve a desired objective. Polyadenylated mRNA can be separated from other RNAs by fractionation on oligo-dT substrates. Alternatively, RNA can be enriched/or fractioned by other methods. For instance, subtraction hybridization
He Wei-Wu
Jay Gilbert
Brusca John S.
Millen White Zelano & Branigan P.C.
Origene Technologies, Inc.
Siu Stephen
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