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(12) Patent Application Publication (10) Pub. No.: US 2004/0009477 A1 Fernandez Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2004/0009477 A1 Fernandez Et Al US 20040009477A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0009477 A1 Fernandez et al. (43) Pub. Date: Jan. 15, 2004 (54) METHODS FOR PRODUCING LIBRARIES ation of application No. 09/647,651, now abandoned, OF EXPRESSIBLE GENE SEQUENCES filed as 371 of international application No. PCT/ US99/07270, filed on Apr. 2, 1999, which is a con (75) Inventors: Joseph M. Fernandez, Carlsbad, CA tinuation-in-part of application No. 09/054,936, filed (US); John A. Heyman, Rixensart on Apr. 3, 1998, now abandoned. (BE); James P. Hoeffler, Anchorage, AK (US); Heather L. Marks-Hull, (30) Foreign Application Priority Data Oceanside, CA (US); Michelle L. Sindici, San Diego, CA (US) Apr. 2, 1999 (US)....................................... US99/07270 Correspondence Address: Publication Classification LISA A. HAILE, Ph.D. GRAY CARY WARE & FREDENRICH LLP 51)1) Int. Cl.Cl." .............................. C12O 1/68 ; C12P 19/34 Suite 1100 (52) U.S. Cl. ............................................... 435/6; 435/91.2 43.65 Executive Drive (57) ABSTRACT San Diego, CA 92.121-2133 (US) The present invention comprises a method for producing (73) Assignee: INVITROGEN CORPORATION libraries of expressible gene Sequences. The method of the invention allows for the Simultaneous manipulation of mul (21) Appl. No.: 09/990,091 tiple gene Sequences and thus allows libraries to be created in an efficient and high throughput manner. The expression (22) Filed: Nov. 21, 2001 vectors containing verified gene Sequences can be used to Related U.S. Application Data transfect cells for the production of recombinant proteins. The invention further comprises libraries of expressible gene (63) Continuation of application No. 09/843,281, filed on Sequences produced using the method of the invention and Apr. 25, 2001, now abandoned, which is a continu expression vectors used in the construction of Said libraries. Patent Application Publication Jan. 15, 2004 US 2004/0009477 A1 Phase I Mici lov-Tac-1 plugs, acid to TOPO-cioning . PCR Amplify each ORF --> -o-y indi cinnpelent cells. to remove its stipp codon heat shock acid growth incoun. is batcard plate 6-- Analyze on low melt gel Pick 8 colonies from each transformation Biorobot 9600 Plasmid Prep Isolation Phase V. Expression QC b SE as s Expression Figure US 2004/0009477 A1 Jan. 15, 2004 METHODS FOR PRODUCING LIBRARIES OF invention method comprises the following Steps: amplifying EXPRESSIBLE GENE SEQUENCES a plurality of gene Sequences, purifying the amplified gene Sequences, inserting each of the purified gene Sequences into FIELD OF THE INVENTION an expression vector, and Verifying the Size and orientation 0001. The invention disclosed herein relates to the fields of the inserted gene Sequence. of genomics and molecular biology. More Specifically the 0009. In the first step, the gene sequences that are to be invention relates to new high through-put methods of mak expressed are amplified. By “amplification’ it is meant that ing libraries of expressed gene Sequences and the libraries the copy number of the gene sequence(s) is increased. One made using Said methods. commonly used method of amplification is the polymerase chain reaction (PCR). In brief, starter DNA is heat-denatured BACKGROUND OF THE INVENTION into Single Strands. Two Synthetic oligonucleotides, one 0002 Recent breakthroughs in nucleic acid sequencing complementary to Sequence at the 3' end of the Sense Strand technology have made possible the Sequencing of entire of DNA segment of interest and the other complementary to genomes from a variety of organisms, including humans. the sequence at the 3' end of the anti-sense strand of a DNA The potential benefits of a complete genome Sequence are Segment of interest, are added in great excess to the DNA many, ranging from applications in medicine to a greater Sequence to be amplified and the temperature is lowered to understanding of evolutionary processes. These benefits 50-60° C. The specific oligonucleotides hybridize with the cannot be fully realized, however, without an understanding complementary Sequences in the DNA and then Serve as of how and where these newly Sequenced genes function. primers of DNA chain synthesis, which requires the addition of a Supply of deoxynucleotides and a temperature-resistant 0.003 Traditionally, functional understanding started with DNA polymerase, Such as Taq polymerase, which can recognizing an activity, isolating a protein associated with extend the primers at temperatures up to 72 C. When that activity, then identifying and isolating the gene, or Synthesis is complete, the whole mixture is heated further genes, encoding that protein. Each gene of interest was (up to 95° C) to melt the newly formed DNA duplexes. identified, isolated and expressed separately, a relatively When the temperature is lowered again, another round of time consuming process. Synthesis takes place, Since an excess of primer is still 0004 Recently, breakthroughs in high through-put DNA present. Repeated cycles of Synthesis and melting quickly Sequencing technology have allowed massive amounts of amplify the Sequence of interest. A more detailed description gene Sequence information to become available to the pub of PCR can be found in Erlich, Ed, PCR Technology: lic. Yet methods of expressing these sequences to produce Principles and Applications for DNA Amplification, W. H. the proteins encoded by them for study have still required Freeman and Co., 1992 and Erlich, et al., Eds., Polymerase that each Sequence be manipulated one at a time. Accord Chain Reaction, Cold Spring Harbor Laboratory, 1989, both ingly, a need exists for the development of methods for the of which are incorporated by reference herein. rapid, Simultaneous expression of large numbers of gene 0010 Starter DNA can come from a variety of sources. It Sequences. The invention described herein addresses this can be total genomic DNA from an organism, for example, and related needs as will become apparent upon inspection or can be cDNA that has been synthesized from cellular of the Specification and the appended claims. mRNA using reverse transcriptase. Sources of suitable RNA BRIEF DESCRIPTION OF THE INVENTION include normal and diseased tissues, cellular extracts, and the like. 0005 The present invention comprises a method for producing libraries of expressible gene Sequences. The 0011. In practicing the method of the invention, the method of the invention allows for the simultaneous desired gene Sequences can come from any Source. The manipulation of multiple gene Sequences and thus allows examples presented below show the amplification of all libraries to be created in an efficient and high through-put open reading frames (ORFs) from a single organism, Sac manner. The expression vectors containing verified gene charomyces cerevisiae, for example. By “open reading Sequences can be used to transfect cells for the production of frame” it is meant a segment of DNA that exists between a recombinant proteins. The invention method utilizes known Start codon and a stop codon and is likely to represent a gene. techniques in Such a way as to create an efficient high The examples presented below further show the amplifica through-put means of producing libraries of expressible tion of a group of human genes thought to be important in gene Sequences. the development of cancer. 0006 The invention further comprises libraries of 0012 Public databases exist that contain the entire or expressible gene Sequences produced using the method of partial genome of a particular organism, for example yeast the invention and expression vectors used in the construction (Saccharomyces cerevisiae), prokaryotes (Bacillus Subtilis, of Such libraries. E. coli, Borrelia burgdorferi, Helicobacter pylori, Myco plasma genitalium, and the like), fish (Fugu rubripes), BRIEF DESCRIPTION OF THE FIGURE mammals (human, mouse), plants (rice, cotton) and the like. Well known databases include GenBank, Unigene, EMBL, 0007 FIG. 1 shows a schematic representation of the IMAGE and TIGR, for example. Public databases such as vaccinia topoisomerase type I cloning method used in the these can be used a Source of gene Sequences for use in the practice of the invention. method of the invention. BRIEF DESCRIPTION OF THE INVENTION 0013 The primers employed in the amplification step are 0008. The present invention comprises a method for Specific for each desired gene Sequence and include a variety producing libraries of expressible gene Sequences. The of unique features. For example, the 5"Sense” primer Starts US 2004/0009477 A1 Jan. 15, 2004 with the sequence 5'-CACCATG . (the start codon is 0017 Promoters may be, furthermore, either constitutive underlined). The CACC Sequence is added as a Kozak or, more preferably, regulatable (i.e., inducible orderepress consensus that aids in translational efficiency. When the ible). Inducible elements are DNA sequence elements which gene Sequence being amplified represents a full-length gene, act in conjunction with promoters and bind either repressors the 3"antisense” codon is preferably designed to make the (e.g. lacO/LAC Iq repressor System in E. coli) or inducers amplification product end at the 3rd position of the last (e.g. gal1/GAL4 inducer System in yeast). In either case, codon of the gene being amplified, plus a Single adenine transcription is virtually “shut off until the promoter is residue. This facilitates the fusion of the coding region derepressed or induced, at which point transcription is in-frame with a heterologous peptide Sequence Such as an “turned-on”. epitope tag, an affinity purification tag, and the like (see 0018. Examples of constitutive promoters include the int below). The gene Sequence need not encode a full-length promoter of bacteriophage 2, the bla promoter of the 3-lac Sequence, however, as the invention methods are equally tamase gene Sequence of pBR322, the CAT promoter of the Suitable for any gene Sequence, including Expressed chloramphenicol acetyl transferase gene Sequence of Sequence Tags (ESTS). The primers can be Synthesized and pPR325, and the like.
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