CA2215870A1 - Method for the identification of essential genes and therapeutic targets - Google Patents
Method for the identification of essential genes and therapeutic targets Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1082—Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Abstract
The present invention relates to a method of identifying essential genes in a genome, based on an insertional mutagenesis of a population of cells or of DNA molecules and subjecting this population of cells or DNA molecules to an amplification process, whereby this total population of cells or DNA molecules which statistically represents at least one full insertionally mutated genome is amplified with at least two primer pairs and the extension products analysed, in order to distinguish essential genes from dispensable genes. The present invention is especially suited to the functional analysis of microbial genomes, and especially to haploid genomes.
Description
CA 0221~870 1997-09-19 TITLE OF THE INVENTION
METHOD FOR THE IDENTIFICATION OF ESSENTIAL
GENES AND THERAPEUTIC TARGETS
The present invention relates to the identification of essential genes in a given genome. More specifically, the invention relates to the identification of essential genes in a diploid organism in which homozygocity conversion is erricie nl or in a haploid organism. The 10 present invention also relates to the identification of therapeutic targets and more specifically to therapeutic targets in bacteria.
BACKGROUND OF THE INVENTION
The human genome project as well as genome projects 15 of model organisms have opened the area of genomics. Although thousands of genetic sequences are available in data bases, only a small minority thereof have a recognized function. It has become apparent that biological functions cannot be solely deduced by computer approaches and that even in integrated format, databases present significant 20 limitations.
Large amounts of data, from the partial or complete DNA
sequences of microbial genomes are also rapidly accumulating in databases. There is heightened expectations that the increasingly powerful computer analyses will be able to yield biological function from 25 these DNA sequence. However, it is becoming clear that even for microbial genomes, the sole information in databases will not be sufficient 11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 to deduce the biological function. Thus, it becomes apparent that whole genome or genome-based analysis of biological function could provide significant results. Indeed, such analysis could be the next phase in microbial genomics, particularly as it pertains to finding novel therapeutic 5 targets in bacteria.
It has become apparent that expression of a subset of genes is essential for survival of the eukaryotic and prokaryotic cells;
mutations in these genes give rise to a lethal phenotype. Recently, the number of lethal loci has been estimated in a number of life forms 10 serving as model organisms for genome projects: Drosophila (3,600 essential genes), Caenorhabditis (3,000), Arabidopsis (500), Saccharomyces (900). Bacterial genomes comprise gene numbers which vary from approximately 500 to more than 8000. The number of essential genes in such genomes is unknown but can be esli",aled as being between 100 to 150 in smaller genomes, such as that of Haemophilus influenzae (1.83 Mb), to more than 500 in larger bacterial genomes, such as that of Pseudomonas aeruginosa (5.9 Mb). The potential and ramifications of using these essential genes and their products as novel therapeutic targets is enormous for the pharmaceutical industry and could 20 open a new era in antimicrobial research. In addition, the identification of essential genes in higher life forms could provide important fundamental and practical information relating to cellular homeostasis, cancer and the like.
Powerful genetic techniques such as allelic replacement 25 and gene knockouts have been developed. These technologies are effective but can only be applied to selected and candidate genes of 11 229-79.DRF 1 9 Sep. 1 997 - 1 6h 1 2 CA 0221~870 1997-09-19 interest. Applying these genetic techniques to whole genomes, even in the context of bacterial genomics, represents a highly inefficient and costly task and novel whole-genome based techniques and gene-screening assays must therefore be developed.
Comprehensive, rapid and, simple screening of bacterial genomes for essential genes has not been possible because of the inability to identify mutants having an attenuated or no significant growth within pools of mutagenized bacteria. It is also impractical to separately assess the significance of essential versus non-essential genes from each of the several thousand mutants necessary to screen a bacterial genome. Although genome-wide functional analysis appears to offer the best approach for the identification of dispensable versus essential genes, no simple, rapid and effficient identification method therefor has been forthcoming. Genome-based analyses provide primarily a functional classification rather than a detailed understanding of each gene. This is a critical aspect in microbial genomics in which one can identify therapeutic targets by identifying essential genes.
USP 5,612,180 teaches a genetic footprinting method which, in essence, is a functional screen of genes under different selective conditions. A PCR-based method which identifies genes essential for survival of a cell, under the selective growth conditions used is taught. Briefly, insertional mutagenesis is carried out on the genome to be tested. The method is then based on the use of one set of primers for the PCR-based genetic footprinting: one primer binding to the insertional mutagen, the other being chosen arbitrarily as a unique sequence in the targeted region. This genetic footprinting method is unfortunately 11229-79.DRF 1 9 Sep. 1 997 -16h12 CA 0221~870 1997-09-19 restricted to the identification of essential genes under a specific selection scheme. Furthermore, it lacks in providing a positive control of amplification originating solely from the targeted region (not from the insertional mutagen). Moreover, it is dependant on the discrimination of small differences in the extension products. Finally, it is based on the comparison of amplification products originating from two different sub-populations (selected vs non-selected).
There therefore remains a need to provide a simple and effficient method of identifying essential genes in a genome under non-selective conditions. There also remains a need to provide a simple and efficient method of identifying genes which are essential under specific conditions, the method providing an amplified signal originating solely from the non-mutagenised targeted region and in which amplification products from a single sub-population of cells are analysed. The present invention seeks to meet these and other needs.
The description refers to a number of documents, the content of which is herein incorporated by reference.
SUMMARY OF THE INVENTION
Accordingly, the present invention seeks to provide an essential gene test (EGT), an effficient and economical approach to define the function of thousands of sequences containing a complete open reading frame (ORF) or parts thereof, or known and/or unknown genes encoding hypothetical proteins or products. The EGT test is particularly effective at defining which sequences in databases contain an essential or a non-essential (dispensable) gene. In one embodiment the EGT
1 1 229-79. DRF 1 9 Sep. 1 997 -1 6h 1 2 CA 0221~870 1997-09-19 assay is based on the premise that a mutation inactivating an essential gene should give rise in vivo, to a lethal phenotype irrespective of the growth conditions.
The present invention also seeks to provide an EGT test 5 which enables the categorization of gene sequences as encoding essential and dispensable genes under selective conditions, the categorization being based on the analysis of a single sub-population of cells ("one tube population").
Furthermore, the present invention seeks to provide an 10 EGT test based on the detection of two basic types of extension products originating from two primer pairs.
By enabling an idei,liricalion of essential genes in organism, the EGT assays permits the identification of therapeutic targets in this organism. The present invention more preferably seeks to provide 15 therapeutic targets in haploid organisms, particularly bacteria.
Nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature 20 Commission.
The present description refers to a number of routinely used recomb,nant DNA (rDNA) technology terms. Nevertheless, definitions of selected examples of such rDNA terms are provided for clarity and consistency.
11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 As used herein, "isolated nucleic acid molecule", refers to a polymer of nucleotides. Non-limiting examples thereof include DNA
and RNA molecules purified from their natural environment.
The term "recombinant DNA" as known in the art refers 5 to a DNA molecule resulting from the joining of DNA segments. This is often referred to as genetic engineering.
The term "DNA segment", is used herein, to refer to a DNA molecule comprising a linear stretch or sequence of nucleotides.
This sequence when read in accordance with the genetic code, can 10 encode a linear stretch or sequence of amino acids which can be referred to as a polypeptide, protein, protein fragment and the like.
The terminology "amplification pair" or "primer pair"
refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic 15 acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction. Other types of amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below. As commonly known in the art, the oligos are 20 designed to bind to a complementary sequence under selected conditions.
The nucleic acid (i.e. DNA or RNA) for practicing the present invention may be obtained according to well known methods.
Oligonucleotide probes or primers of the present 25 invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed.
1 1229-79.DRF 1 9 Sep. 1 997 -1 6h1 2 CA 0221~870 1997-09-19 In general, the oligonucleotide probes or primers are at least 12 nucleotides in length, preferably between 15 and 24 nucleotides, and they may be adapted to be especially suited to a chosen nucleic acid amplification system. As commonly known in the art, the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hydrizidation thereof with its targeted sequence (see below, and in Sambrook et al., 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).
"Nucleic acid hybridization" refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al., 1989, supra and Ausubel et al., 1989 supra) and are commonly known in the art. In the case of a hybridization to a nitrocellulose filter, as for example in the well known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at 65~C with a labeled probe in a solution containing 50% formamide, high salt ( 5 x SSC or 5 x SSPE), 5 x Denhardt's solution, 1% SDS, and 100 ,ug/ml denatured carried DNA ( i.e. salmon sperm DNA). The non-specifically binding probe can then be washed off the filter by several washes in 0.2 x SSC/0.1% SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42~C
(moderate stringency) or 65~C (high stringency). The selected temperature is based on the melting temperature (Tm) of the DNA hybrid.
11229-79.DRF 19 Sep. 1997 - 16h12 CA 0221~870 1997-09-19 Of course, RNA-DNA hybrids can also be formed and detected. In such cases, the conditions of hybridization and washing can be adapted according to well known methods by the person of ordinary skill. High stringency conditions will be preferably used (Sambrook et al.,1989, 5 supra).
Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the like. Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann. Reports Med. Chem. 23:295 and Moran et al., 1987, Nucleic acid molecule. Acids Res., 14:5019. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
The types of detection methods in which probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Although less prepared, labelled proteins could also be used to detect a particular nucleic acid sequence to which it binds. Other detection methods include kits containing probes on a dipstick setup and the like.
Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labelled according to numerous well known methods (Sambrook et al.,1989, supra). Non-limiting examples of labels include 3H,14C,32p, and 35S. Non-limiting examples of detectable markers include ligands, 11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides. It will become evident to the person of ordinary skill that 5 the choice of a particular label dictates the manner in which it is bound to the probe.
As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples thereof include kinasing the 5' ends of the probes using gamma 10 32p ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E. coli in the presence of radioactive dNTP (i.e. uniformly labelled DNA
probe using random oligonucleotide primers in low-melt gels), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like.
As used herein, "oligonucleotides" or "oligos" define a molecule having two or more nucleotides (ribo or deoxyribonucleotides).
The size of the oligo will be dictated by the particular situation and ultimately by the particular use thereof, and adapted accordingly by the person of ordinary skill. An oligonucleotide can be synthetised chemically or derived by cloning according to well known methods.
As used herein, a "primer" defines an oligonucleotide which is capable of annealing to a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA
synthesis under suitable conditions.
Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See 11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 generally Kwoh et al., 1990, (Am. Biotechnol. Lab. 8:14-25). Numerous amplification techniques have been described and can be readily adapted to suit the particular needs of a person of ordinary skill. Non-limiting examples of amplification techniques include polymerase chain reaction 5 (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Q~ replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177;
Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., 1989, supra).
10 Preferably, amplification will be carried out using PCR.
Polymerase chain reaction (PCR) is carried out in accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195;
4,683,202; 4,800,159; and 4,965,188 (the disclosures of all three U.S.
Patent are incorporated herein by reference). In general, PCR involves, 15 a treatment of a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected. An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers 20 sufficiently complementary to each strand of the specific sequence to hybridize therewith. The extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers. Following a sufficient number of rounds of synthesis of extension products, the sample is analysed to assess 25 whether the sequence or sequences to be detected are present.
Detection of the amplified sequence may be carried out by visualization 1 1229-79.DRF 1 9 Sep. 1 997 -1 6h1 2 CA 0221~870 1997-09-19 following EtBr staining of the DNA ~ol'ow;"g gel electrophoresis, or using a detectable label in accordance with known techniques, and the like. For a review on PCR techniques (see PCR Protocols, A Guide to Methods and Amplifications, Michael et al., Eds, Acad. Press, 1990).
Ligase chain reaction (LCR) is carried out in accordance with known techniques (Weiss,1991, Science 254:1292). Adaptation of the protocol to meet the desired needs can be carried out by a person of ordinary skill. Strand displacement amplification (SDA) is also carried out in accordance with known techniques or adaptations thereof to meet the particular needs (Walker et al., 1992, Proc. Natl. Acad. Sci. USA
89:392-396; and ibid., 1992, NucleicAcids Res. 20:1691-1696.
As used herein, the term "gene" is well known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide. A "structural gene" defines a DNA sequence which is transcribed into RNA and translated into a protein having a specific amino acid sequence thereby giving rise the a specific polypeptide or protein. It will be readily recognized by the person of ordinary skill, that the nucleic acid sequences of the present invention can be incorporated into anyone of numerous established kit formats which are well known in the art.
The term "vector" is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned. Numerous types of vectors exist and are well known in the art.
The term "expression" defines the process by which a structural gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more.
11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 The terminology "expression vector" defines a vector or vehicle, as described above, but designed to enable the expression of an inserted sequence following transformation into a host. The cloned gene (inserted sequence) is usually placed under the control of control element 5 sequences such as promoter sequences. The placing of a cloned gene under such control sequences is often referred to as being "operably linked" to control elements or sequences.
Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a 10 prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
As used herein, the designation "functional derivative"
15 denotes, in the context of a functional derivative of a sequence, whether nucleic acid or amino acid sequence, a molecule that retains a biological activity (either functional or structural) that is substantially similar to thatof the original sequence. This functional derivative or equivalent may be a natural derivative or may be prepared synthetically. Such derivatives 20 include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The same applies to derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the 25 sequence is generally maintained. When relating to a protein sequence, the substituting amino acid has chemico-physical properties which are 11229-79.DRF 19 Sep. 1997 - 16h12 CA 0221~870 1997-09-19 similar to that of the substituted amino acid. The similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylicity and the like. The term "functional derivatives" is intended to include "fragments", "segments", "variants", "analogs" or"chemical 5 derivatives" of the subject matter of the present invention.
Thus, the term "variant" refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention.
The functional derivatives of the present invention can 10 be synthesized chemically or produced through recombinant DNA
technology. All these methods are well known in the art.
As used herein, "chemical derivatives" is meant to cover additional chemical moieties not normally part of the subject matter of the invention. Such moieties could affect the physico-chemical characteristic 15 of the derivative (i.e. solubility, absorption, half life and the like, decrease of toxicity). Such moieties are exemplified in Remington's Pharmaceutical Sciences (1980). Methods of coupling these chemical-physical moieties to a polypeptide are well known in the art.
The term "allele" defines an alternative form of a gene 20 which occupies a given locus on a chromosome.
As commonly known, a "mutation" is a detectable change in the genetic material which can be transmitted to a daughter cell. As well known, a mutation can be, for example, a detectable change in one or more deoxyribonucleotide. For example, nucleotides can be 25 added, deleted, substituted for, inverted, or transposed to a new position.
Spontaneous mutations and experimentally induced mutations exist. The 11229-79.DRF 19 Sep. 1997 - 16h12 CA 0221~870 1997-09-19 result of a mutations of nucleic acid molecule is a mutant nucleic acid molecule. A mutant polypeptide can be encoded from this mutant nucleic acid molecule.
As used herein, the term "purified" refers to a molecule 5 having been separated from a cellular component. Thus, for example, a "purified protein" has been purified to a level not found in nature. A
"substantially pure" molecule is a molecule that is lacking in all other cellular components.
The mutagenesis of the DNA or of the cells is carried out 10 in accordance with well-known methods (Sambrook et al., 1989, supra), such that the total DNA population or cell population has statistically at least an insertion mutation in each and every gene of the genome.
Essentially, the one tube collection of mutants obtained by mutagenesis covers the complete genome. A typical mutagenesis experiment can yield mutants at frequencies varying from 10,000 clones to more than 1,000,000 clones. Such mutants can be recovered in a single tube.This mutagenesis scheme is based on the premise that the genome size is known, that mutagenesis is a random event and that a typical gene has an average size of 1 kilobase. For example and on a statistical basis, the 20 5.9 Mb Pseudomonas aeruginosa genome would require a minimum of 5,900 mutants to cover the genome at least once. This is herein defined as a 1 X genome coverage. Thus, a collection of 17,500 mutants (3 X), 29,500 mutants (5 X) or 59,000 mutants (10X) could be utilized for screening in a typical EGT assay for this particular microorganism. Of 25 course, the person of ordinary skill could also screen more than 10X.
11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 As used herein, the designation "therapeutic target"
refers to any gene or product thereof that when blocked by known or novel molecules will affect the growth of the organism coding for the target.
As used herein, the designation "Non-selective conditions" refers to in vitro and/or in vivo growth conditions wherein all the paramaters and factors which are required for optimal growth are present. Non-limiting examples of such parameters/factors include growth media nutrients, temperature, pH, cell line, and the like. Under such conditions, one would expect the organism to be maintained prior to the mutagenesis step.
As used herein, the designation "Selective conditions"
refers to conditions which are defined by the nature of the experiment done in vitro and/or in vivo and in which one specific parameter or factor or set of conditions are modified (in comparison to non-selective conditions) to determine if essentials genes or gene products can be identified in that particular condition. A non-limiting example of a selective condition includes growth at a restrictive temperature.
It will be clear to the person of ordinary skill, that insertional mutagenesis of an essential gene, within the context of a cell, will result in the death of that cell. Consequently, the genome of this particular cell will not be available as a substrate for the amplification process in accordance with the EGT method of the present invention.
The DNA molecule analysed may be a gene, a fragment thereof cloned into a vector or preferably a genome.
11229-79.DRF 19 Sep. 1997 - 1 6h12 CA 0221~870 1997-09-19 As used herein, the terminology "target region" defines a DNA region for which preliminary sequence data is sufficiently available to enable the design of a first primer pair which will, under appropriate conditions, give rise to a recognizable extension product. The target region is determined and defined by the available sequence data available for the particular genome analysed, and by the limits in the amplification method used. For PCR, for example, the conditions permit extension products to reach about 2000 nucleotides. The target region should thus be between about 50 to about 2000 nucleotides. Preferably between about 200 and about 1000. Since sequence information can be clustered, some genes might have several target regions. In any event, the mutagenesis conditions should be adapted so as to enable an insertional mutagenesis of all targeted regions. In essence, a person of ordinary skill will adapt the mutagenesis scheme so as to permit saturation mutagenesis of the DNA to be analysed.
Although in a preferred embodiment, the present invention is adapted for use with a whole genome, a DNA molecule inserted into a vector can also be used in accordance with the present invention. In such an embodiment, the vector should permit an expression of the DNA molecule in order to permit an ~ssessment of the essentiality of the gene product. In such a scheme, it will be understood that only dominant insertional mutation can provoke the lethality since, presumably, a copy of a wild type or homologous copy of the gene which is present on the vector, is present in the host cell. Consequently, it will be clear to the person of ordinary skill that although the present invention is not limited to haploid genomes, the method of the present invention is 11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 favorably used in a context of a haploid organism, and more preferably a haploid microorganism. Organisms in which conversion to homozygocity is efficient and/or complete are also covered by the scope of the present invention. In a preferred embodiment therefore, prokaryotic genomes and lower eukaryotic genomes such as the haploid genomes of parasites and protista are used. Non-limiting examples of such lower embryotic genomes include that of tachyzoite form of Toxoplasma gondii, of Plasmodia, Schistosoma and Leishmania species, as well as those of fungi such as that of Candida, Aspergillus, Neospora and other disease causing (in plants, in animals and in humans) relevant fungi are especially preferred genomes. In addition, all disease causing agents such as Influenzae, Hl\/, Herpes and other viruses may also be used in the context of the present invention.
It shall be understood that although the saturation insertional mutagenesis of the present invention is carried out by a shotgun approach (without specifically directing the insertion to specific sequences), a rational design of insertion mutation could also be carried out, especially with DNA molecules inserted into vectors.
Since the design of the first pair of primers depends on known sequence data from the genome to be analysed, it follows that minimum stretches of sequence data must be available in order to enable the EGT method of the present invention. Preferably, contiguous nucleic acid sequence data of approximately twelve nucleotides, to approximately twenty-four nucleotides in the targeted region must be available.
Although in a preferred embodiment, the method of the present invention relates particularly to genomes of organisms which do 11 229-79.DRF 19 Sep. 1997 - 1 6h12 CA 0221~870 1997-09-19 not contain or contain few introns, the present invention could be adapted by a person of ordinary skill for intron-containing genomes. Briefly, the level of mutagenesis would have to be increased in order to enable saturation to occur. Saccharomyces cerevisiae is one non-limiting 5 example of an organism which contains introns.
Numerous insertional mutagenesis method are known in the art. It will be clear to the person of ordinary skill that the method should be adapted to enable the insertion of the sequence which is complementary to that of a primer binding thereto (generally described 10 herein as primer 3).
The term "saturation mutagenesis" as used herein with reference to a genome, refers to an insertion mutagenesis in substantially every gene thereof and/or every target region thereof. Based upon statistical analysis and well known methods, at least 90%, preferably, 95% and more preferably 100% of the genes and/or target regions will have been mutagenised. Briefly, to esli,nate the conditions to permit the aiming of a complete population of mutagenised genes, the statistical analysis utilised is based on a number of criterions: 1) a completely random insertion of the insertion element (i.e. a mobile element); 2) an average size of 1 Kb for a typical gene in a prokaryote genome; 3) knowledge a priori of the genome size (Megabases). For example, a complete 1 X coverage of the P. aeruginosa 5.9 Mb genome would require a minimum of 6000 clones after the mutagenesis experiment.
Preferably, a minimum of 5 X coverage of the genome should be used by using 60,000 clones. When relating to DNA molecules present on a 11229-79.DRF 19 Sep. 1997 - 16h12 CA 0221~870 1997-09-19 vector, saturation mutagenesis refers preferably to the insertion element being present at every nucleotide position thereof.
Mutational methods include, without being limited thereto, insertional mutations in which a DNA molecule is inserted without 5 loss of native sequences, or substitutional mutations in which the DNA
molecule inserted replaces native DNA molecule of the targeted region.
It shall be understood that the choice of a particular insertional element can be adapted to particular needs, provided that it is absent from the genome which is to be analysed, that it is sufficiently long 10 to permit the generation of a primer which binds thereto (hence the need for known sequence data of about 12 contiguous nucleotides for the primer target on the genome, and disrupts the gene or target region it is inserted into. In a preferred embodiment, the insertional mutagenesis is provided by a insertional element such as transposons (i.e. Tn5, Tn10, 15 Tn916, Ty). In such cases, the insertional mutagenesis will be carried out with the insertional elements in accordance with known methods.
Insertional mutagenesis of DNA can also be carried out by using the integrases protein of retroviruses to mediate the insertion of a selected primer into a target region. Following amplification, the 20 amplified product or extension product can be detected. In a preferred embodiment they can be sized-fractionated by gel electrophoresis as well known in the art. In another embodiment the extension products can be detected after separation on columns and the like. Hybridization capture and the triplex DNA technology are non-limiting examples of technologies 25 which could be used to detect the amplified products (Lanbiewicz et al., 11229-79.DRF 19 Sep. 1997 - 16h12 CA 0221~870 1997-09-19 1997, Nucl. Acids Res.25; 2037-38; and Ito et al.,1992, Proc. Natl, Acad.
Sci 89: 495-8).
A kit for identifying essential genes in a genome contains at least three oligonucleotide primers, constituting at least two 5 primer pairs, a mutated genome, and solutions for enabling hybridization between the mutated genome sequences and the oligonucleotide primers and for enabling amplification of the extension product. Oligonucleotide primers can be suspended in solution or provided separately in Iyophilized form. The components of the kit can be packaged together in 10 a common container, the kit typically including an instruction sheet for carrying out a specific embodiment of the method of the present invention. Additional optional components of the kit include detection probes, and means for carrying out a detection step (for example, a probe or primer is labelled with a detectable marker).
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawings:
Figure 1 shows a sumll,a, i~ed schematic representation of the essential gene test (EGT) according to the present invention; and Figure 2 shows a more detailed view of the EGT shown 25 in Figure 1.
11229-79.DRF 19 Sep. 1997 - 16h12 CA 0221~870 1997-09-19 Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments with reference to the accompanying drawing which is exemplary and should not be interpreted 5 as limiting the scope of the present invention.
DETAILED DESCRIPTION
Insertional Mutagenesis of the Targeted Genome First, insertional mutagenesis must be performed so as 10 to cover most if not all genes of a particular genome in a population of cells. Under these conditions, one would expect the one tube mutagenized population to cover the spectrum of each and every gene coded by a particular organism.
Insertional Mutagen In one embodiment in which a bacterial genome is targeted, a bacterial population is mutagenized using for example a mobile element having a high frequency of transposition (Tn5, Tn10, Tn916, IS elements or any other known mobile genetic element) creating insertional mutations at diverse sites. Depending on the conditions and 20 mobile element utilized, one may produce a single tube population containing cells having an insertion in essentially all the genes. Any particular type of mutagenesis scheme including insertion elements, PCR
mutagenesis, random insertion of DNA by synthetic or biological methods would be amenable to genetic analysis by the EGT test or assay.
1 1229-79.DRF 19 Sep. 1 997 -16h12 CA 0221~870 1997-09-19 The assay can also be applied to any simple organisms such as viruses. The EGT has excellent potential in disease causing viruses from plants, from animals and from humans. Non-limiting examples include the potato blight virus in plants, the equine encephalitis 5 virus in animals and the cytomegalovirus in humans. Additional examples include single eukaryotic cells of fungi and of yeasts causing diseases such as mycoses and include Candida, Cryptococcus, Histoplasma, Blastomyces, Coccioides, Aspergillus, Fusarium, and Trychophyton, and the like. Thus, the EGT assay could be applied to all disease causing 10 organisms (See the listing of the Manual of Clinical Microbiology, 1995, ASM Press). The person of ordinary skill will adapt the EGT accordingly.
For the targeting of the yeast genome the insertional element Ty is a representative example of an insertional mutagen which can be used in accordance with the present invention. In addition, the EGT assay can be 15 utilized to dissect metabolic and genetic pathways by assessing mutagenized populations in different in vitro and in vivo conditions.
Amplification A sample of the mutagenized population is then submitted to nucleic acid amplification. In a preferred embodiment, the 20 amplification is carried out by PCR using either cells directly or by preparing an aliquot of DNA. A collection of two primers specific to the sequence under investigation (from a genomic database and assumed to encode an essential or dispensable gene where only part of the ORF is known) and defining a first primer pair, gives rise to an amplification 25 product of a defined size. A third primer specific to the insertional mutagen is also used. This three primer assay will give specific 11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 amplification products defining a sequence as essential or dispensable.
The EGT assay is performed as summarized in Figure 1 using a wild-type and a mutagenized population. The role of a particular sequence as essential or dispensable is visu~ ed as the presence (non-essential) or 5 depletion of defined satellite amplification products (essential) (Fig. 1).
A more detailed representation is shown in Fig. 2.
Intcr~,ret~lion of the results of EGT assay The primer pairs selected from the sequence of interest defines an amplification product that will be present both in essential 10 genes and in dispensable genes irrespective of the growth conditions since in the context of a population of cells, individual cells having no insertions in the targeted sequence of interest will always be present.
Thus, the first primer pair serves as an internal control for the assay conditions. If the insertion occurs in a dispensable gene, the second 15 primer pair, constituted by a primer specific to the targeted sequence and one specific to the insertional mutagen, gives rise to a specific extension product and a series of additional band products. Thus, in addition to the expected product originating from the first primer pair, additional amplification products will be visible. The difference in the size of the 20 additional product will reflect the distance between the target region of thethird primer (the insertion "point") and that of the first primer (or second primer). In contrast, insertion of an element in an essential gene will not yield an amplification product (lethal phenotype) and the only visualized amplification product will be generated by the amplification of 25 mutagenized cells containing no insertions in the essential sequence of interest (originating from the first primer pair).
11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 As alluded to above, the EGT assay enables automation. For example, by using fluorescent primers (labelled with distinct fluorochromes) the EGT assay could be used in conjunction with the ABI GENESCAN.
The following examples are offered by way of illustration and not by way of limitation.
E~CAMPLE 1 EGT assay on two Pseudomonas aeruginosa genes The EGT assay was applied to the Pseudomonas aeruginosa strain PAO1 5.9 Mb genome in the following way. First, a library of insertion mutants was constructed with the miniTn5 Km insertion element using standard methods. A collection of 60,000 clones (10 X
genome coverage) obtained were pooled into a single tube.
A first primer pair of 21-mers specific and internal to the ftsZ gene sequence (ftsZ1 :5'-ATC ACC ATC CCG MC GAG MG-3') and (ftsZ2:5'-TAT CCA GGT MT CCA GGT CAT-3') give a 669 bps amplified PCR product.The PCR conditions for DNA amplification were carried out in accordance with the manufacturer's recommendations (Perkin Elmer Cetus and Applied Biosystems). In a typical EGT assay, one would expect the 669 bps to be present irrespective of the mutagenesis or growth conditions.
The EGT assay was performed for ffsZ by using the following primers :(KanaputR1: 5'-GCG GCC TCG AGC MG ACG
TTT-3') and (KanaputF4: 5'-TTG GTT GTA ACA CTG GCA GAG-3') in combination with one and\or the two above-mentioned primers (ftsZ1 and 1 1229-79.DRF 1 9 Sep. 1 997 -1 6h12 CA 0221~870 1997-09-19 ftsZ2). The result of the EGT assay showed a product of 669 bps and no satellite bands, irrespective of the mutagenesis scheme. Thus, only the first primer pair gave rise to an extension product. Thus, ftsZ is therefore defined as an essential gene by the EGT method.
The EGT assay was tested with the ampC gene using primers (ampcF1: 5'- CAT CGC TTC CAC ACT GCT-3') and (ampcR1:
5'-TGC CGG GM CAC TTG CTG CTC-3') constituting a first primer pair giving rise to a PCR product of 592 bps irrespective of the mutagenesis.
When used in conjunction with the KanaputR1 and KanaputF1 primers, a PCR product of 592 bps (positive control) and additional DNA bands (due to insertions in the ampC gene) could be visualized in the agarose ethidium bromide stained gel. Thus, the EGT assay would define the ampC gene as non-essential.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
11229-79.DRF 19Sep. 1997-16h12
METHOD FOR THE IDENTIFICATION OF ESSENTIAL
GENES AND THERAPEUTIC TARGETS
The present invention relates to the identification of essential genes in a given genome. More specifically, the invention relates to the identification of essential genes in a diploid organism in which homozygocity conversion is erricie nl or in a haploid organism. The 10 present invention also relates to the identification of therapeutic targets and more specifically to therapeutic targets in bacteria.
BACKGROUND OF THE INVENTION
The human genome project as well as genome projects 15 of model organisms have opened the area of genomics. Although thousands of genetic sequences are available in data bases, only a small minority thereof have a recognized function. It has become apparent that biological functions cannot be solely deduced by computer approaches and that even in integrated format, databases present significant 20 limitations.
Large amounts of data, from the partial or complete DNA
sequences of microbial genomes are also rapidly accumulating in databases. There is heightened expectations that the increasingly powerful computer analyses will be able to yield biological function from 25 these DNA sequence. However, it is becoming clear that even for microbial genomes, the sole information in databases will not be sufficient 11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 to deduce the biological function. Thus, it becomes apparent that whole genome or genome-based analysis of biological function could provide significant results. Indeed, such analysis could be the next phase in microbial genomics, particularly as it pertains to finding novel therapeutic 5 targets in bacteria.
It has become apparent that expression of a subset of genes is essential for survival of the eukaryotic and prokaryotic cells;
mutations in these genes give rise to a lethal phenotype. Recently, the number of lethal loci has been estimated in a number of life forms 10 serving as model organisms for genome projects: Drosophila (3,600 essential genes), Caenorhabditis (3,000), Arabidopsis (500), Saccharomyces (900). Bacterial genomes comprise gene numbers which vary from approximately 500 to more than 8000. The number of essential genes in such genomes is unknown but can be esli",aled as being between 100 to 150 in smaller genomes, such as that of Haemophilus influenzae (1.83 Mb), to more than 500 in larger bacterial genomes, such as that of Pseudomonas aeruginosa (5.9 Mb). The potential and ramifications of using these essential genes and their products as novel therapeutic targets is enormous for the pharmaceutical industry and could 20 open a new era in antimicrobial research. In addition, the identification of essential genes in higher life forms could provide important fundamental and practical information relating to cellular homeostasis, cancer and the like.
Powerful genetic techniques such as allelic replacement 25 and gene knockouts have been developed. These technologies are effective but can only be applied to selected and candidate genes of 11 229-79.DRF 1 9 Sep. 1 997 - 1 6h 1 2 CA 0221~870 1997-09-19 interest. Applying these genetic techniques to whole genomes, even in the context of bacterial genomics, represents a highly inefficient and costly task and novel whole-genome based techniques and gene-screening assays must therefore be developed.
Comprehensive, rapid and, simple screening of bacterial genomes for essential genes has not been possible because of the inability to identify mutants having an attenuated or no significant growth within pools of mutagenized bacteria. It is also impractical to separately assess the significance of essential versus non-essential genes from each of the several thousand mutants necessary to screen a bacterial genome. Although genome-wide functional analysis appears to offer the best approach for the identification of dispensable versus essential genes, no simple, rapid and effficient identification method therefor has been forthcoming. Genome-based analyses provide primarily a functional classification rather than a detailed understanding of each gene. This is a critical aspect in microbial genomics in which one can identify therapeutic targets by identifying essential genes.
USP 5,612,180 teaches a genetic footprinting method which, in essence, is a functional screen of genes under different selective conditions. A PCR-based method which identifies genes essential for survival of a cell, under the selective growth conditions used is taught. Briefly, insertional mutagenesis is carried out on the genome to be tested. The method is then based on the use of one set of primers for the PCR-based genetic footprinting: one primer binding to the insertional mutagen, the other being chosen arbitrarily as a unique sequence in the targeted region. This genetic footprinting method is unfortunately 11229-79.DRF 1 9 Sep. 1 997 -16h12 CA 0221~870 1997-09-19 restricted to the identification of essential genes under a specific selection scheme. Furthermore, it lacks in providing a positive control of amplification originating solely from the targeted region (not from the insertional mutagen). Moreover, it is dependant on the discrimination of small differences in the extension products. Finally, it is based on the comparison of amplification products originating from two different sub-populations (selected vs non-selected).
There therefore remains a need to provide a simple and effficient method of identifying essential genes in a genome under non-selective conditions. There also remains a need to provide a simple and efficient method of identifying genes which are essential under specific conditions, the method providing an amplified signal originating solely from the non-mutagenised targeted region and in which amplification products from a single sub-population of cells are analysed. The present invention seeks to meet these and other needs.
The description refers to a number of documents, the content of which is herein incorporated by reference.
SUMMARY OF THE INVENTION
Accordingly, the present invention seeks to provide an essential gene test (EGT), an effficient and economical approach to define the function of thousands of sequences containing a complete open reading frame (ORF) or parts thereof, or known and/or unknown genes encoding hypothetical proteins or products. The EGT test is particularly effective at defining which sequences in databases contain an essential or a non-essential (dispensable) gene. In one embodiment the EGT
1 1 229-79. DRF 1 9 Sep. 1 997 -1 6h 1 2 CA 0221~870 1997-09-19 assay is based on the premise that a mutation inactivating an essential gene should give rise in vivo, to a lethal phenotype irrespective of the growth conditions.
The present invention also seeks to provide an EGT test 5 which enables the categorization of gene sequences as encoding essential and dispensable genes under selective conditions, the categorization being based on the analysis of a single sub-population of cells ("one tube population").
Furthermore, the present invention seeks to provide an 10 EGT test based on the detection of two basic types of extension products originating from two primer pairs.
By enabling an idei,liricalion of essential genes in organism, the EGT assays permits the identification of therapeutic targets in this organism. The present invention more preferably seeks to provide 15 therapeutic targets in haploid organisms, particularly bacteria.
Nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature 20 Commission.
The present description refers to a number of routinely used recomb,nant DNA (rDNA) technology terms. Nevertheless, definitions of selected examples of such rDNA terms are provided for clarity and consistency.
11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 As used herein, "isolated nucleic acid molecule", refers to a polymer of nucleotides. Non-limiting examples thereof include DNA
and RNA molecules purified from their natural environment.
The term "recombinant DNA" as known in the art refers 5 to a DNA molecule resulting from the joining of DNA segments. This is often referred to as genetic engineering.
The term "DNA segment", is used herein, to refer to a DNA molecule comprising a linear stretch or sequence of nucleotides.
This sequence when read in accordance with the genetic code, can 10 encode a linear stretch or sequence of amino acids which can be referred to as a polypeptide, protein, protein fragment and the like.
The terminology "amplification pair" or "primer pair"
refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic 15 acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction. Other types of amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below. As commonly known in the art, the oligos are 20 designed to bind to a complementary sequence under selected conditions.
The nucleic acid (i.e. DNA or RNA) for practicing the present invention may be obtained according to well known methods.
Oligonucleotide probes or primers of the present 25 invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed.
1 1229-79.DRF 1 9 Sep. 1 997 -1 6h1 2 CA 0221~870 1997-09-19 In general, the oligonucleotide probes or primers are at least 12 nucleotides in length, preferably between 15 and 24 nucleotides, and they may be adapted to be especially suited to a chosen nucleic acid amplification system. As commonly known in the art, the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hydrizidation thereof with its targeted sequence (see below, and in Sambrook et al., 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).
"Nucleic acid hybridization" refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al., 1989, supra and Ausubel et al., 1989 supra) and are commonly known in the art. In the case of a hybridization to a nitrocellulose filter, as for example in the well known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at 65~C with a labeled probe in a solution containing 50% formamide, high salt ( 5 x SSC or 5 x SSPE), 5 x Denhardt's solution, 1% SDS, and 100 ,ug/ml denatured carried DNA ( i.e. salmon sperm DNA). The non-specifically binding probe can then be washed off the filter by several washes in 0.2 x SSC/0.1% SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42~C
(moderate stringency) or 65~C (high stringency). The selected temperature is based on the melting temperature (Tm) of the DNA hybrid.
11229-79.DRF 19 Sep. 1997 - 16h12 CA 0221~870 1997-09-19 Of course, RNA-DNA hybrids can also be formed and detected. In such cases, the conditions of hybridization and washing can be adapted according to well known methods by the person of ordinary skill. High stringency conditions will be preferably used (Sambrook et al.,1989, 5 supra).
Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the like. Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann. Reports Med. Chem. 23:295 and Moran et al., 1987, Nucleic acid molecule. Acids Res., 14:5019. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
The types of detection methods in which probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Although less prepared, labelled proteins could also be used to detect a particular nucleic acid sequence to which it binds. Other detection methods include kits containing probes on a dipstick setup and the like.
Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labelled according to numerous well known methods (Sambrook et al.,1989, supra). Non-limiting examples of labels include 3H,14C,32p, and 35S. Non-limiting examples of detectable markers include ligands, 11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides. It will become evident to the person of ordinary skill that 5 the choice of a particular label dictates the manner in which it is bound to the probe.
As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples thereof include kinasing the 5' ends of the probes using gamma 10 32p ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E. coli in the presence of radioactive dNTP (i.e. uniformly labelled DNA
probe using random oligonucleotide primers in low-melt gels), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like.
As used herein, "oligonucleotides" or "oligos" define a molecule having two or more nucleotides (ribo or deoxyribonucleotides).
The size of the oligo will be dictated by the particular situation and ultimately by the particular use thereof, and adapted accordingly by the person of ordinary skill. An oligonucleotide can be synthetised chemically or derived by cloning according to well known methods.
As used herein, a "primer" defines an oligonucleotide which is capable of annealing to a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA
synthesis under suitable conditions.
Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See 11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 generally Kwoh et al., 1990, (Am. Biotechnol. Lab. 8:14-25). Numerous amplification techniques have been described and can be readily adapted to suit the particular needs of a person of ordinary skill. Non-limiting examples of amplification techniques include polymerase chain reaction 5 (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Q~ replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177;
Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., 1989, supra).
10 Preferably, amplification will be carried out using PCR.
Polymerase chain reaction (PCR) is carried out in accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195;
4,683,202; 4,800,159; and 4,965,188 (the disclosures of all three U.S.
Patent are incorporated herein by reference). In general, PCR involves, 15 a treatment of a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected. An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers 20 sufficiently complementary to each strand of the specific sequence to hybridize therewith. The extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers. Following a sufficient number of rounds of synthesis of extension products, the sample is analysed to assess 25 whether the sequence or sequences to be detected are present.
Detection of the amplified sequence may be carried out by visualization 1 1229-79.DRF 1 9 Sep. 1 997 -1 6h1 2 CA 0221~870 1997-09-19 following EtBr staining of the DNA ~ol'ow;"g gel electrophoresis, or using a detectable label in accordance with known techniques, and the like. For a review on PCR techniques (see PCR Protocols, A Guide to Methods and Amplifications, Michael et al., Eds, Acad. Press, 1990).
Ligase chain reaction (LCR) is carried out in accordance with known techniques (Weiss,1991, Science 254:1292). Adaptation of the protocol to meet the desired needs can be carried out by a person of ordinary skill. Strand displacement amplification (SDA) is also carried out in accordance with known techniques or adaptations thereof to meet the particular needs (Walker et al., 1992, Proc. Natl. Acad. Sci. USA
89:392-396; and ibid., 1992, NucleicAcids Res. 20:1691-1696.
As used herein, the term "gene" is well known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide. A "structural gene" defines a DNA sequence which is transcribed into RNA and translated into a protein having a specific amino acid sequence thereby giving rise the a specific polypeptide or protein. It will be readily recognized by the person of ordinary skill, that the nucleic acid sequences of the present invention can be incorporated into anyone of numerous established kit formats which are well known in the art.
The term "vector" is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned. Numerous types of vectors exist and are well known in the art.
The term "expression" defines the process by which a structural gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more.
11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 The terminology "expression vector" defines a vector or vehicle, as described above, but designed to enable the expression of an inserted sequence following transformation into a host. The cloned gene (inserted sequence) is usually placed under the control of control element 5 sequences such as promoter sequences. The placing of a cloned gene under such control sequences is often referred to as being "operably linked" to control elements or sequences.
Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a 10 prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
As used herein, the designation "functional derivative"
15 denotes, in the context of a functional derivative of a sequence, whether nucleic acid or amino acid sequence, a molecule that retains a biological activity (either functional or structural) that is substantially similar to thatof the original sequence. This functional derivative or equivalent may be a natural derivative or may be prepared synthetically. Such derivatives 20 include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The same applies to derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the 25 sequence is generally maintained. When relating to a protein sequence, the substituting amino acid has chemico-physical properties which are 11229-79.DRF 19 Sep. 1997 - 16h12 CA 0221~870 1997-09-19 similar to that of the substituted amino acid. The similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylicity and the like. The term "functional derivatives" is intended to include "fragments", "segments", "variants", "analogs" or"chemical 5 derivatives" of the subject matter of the present invention.
Thus, the term "variant" refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention.
The functional derivatives of the present invention can 10 be synthesized chemically or produced through recombinant DNA
technology. All these methods are well known in the art.
As used herein, "chemical derivatives" is meant to cover additional chemical moieties not normally part of the subject matter of the invention. Such moieties could affect the physico-chemical characteristic 15 of the derivative (i.e. solubility, absorption, half life and the like, decrease of toxicity). Such moieties are exemplified in Remington's Pharmaceutical Sciences (1980). Methods of coupling these chemical-physical moieties to a polypeptide are well known in the art.
The term "allele" defines an alternative form of a gene 20 which occupies a given locus on a chromosome.
As commonly known, a "mutation" is a detectable change in the genetic material which can be transmitted to a daughter cell. As well known, a mutation can be, for example, a detectable change in one or more deoxyribonucleotide. For example, nucleotides can be 25 added, deleted, substituted for, inverted, or transposed to a new position.
Spontaneous mutations and experimentally induced mutations exist. The 11229-79.DRF 19 Sep. 1997 - 16h12 CA 0221~870 1997-09-19 result of a mutations of nucleic acid molecule is a mutant nucleic acid molecule. A mutant polypeptide can be encoded from this mutant nucleic acid molecule.
As used herein, the term "purified" refers to a molecule 5 having been separated from a cellular component. Thus, for example, a "purified protein" has been purified to a level not found in nature. A
"substantially pure" molecule is a molecule that is lacking in all other cellular components.
The mutagenesis of the DNA or of the cells is carried out 10 in accordance with well-known methods (Sambrook et al., 1989, supra), such that the total DNA population or cell population has statistically at least an insertion mutation in each and every gene of the genome.
Essentially, the one tube collection of mutants obtained by mutagenesis covers the complete genome. A typical mutagenesis experiment can yield mutants at frequencies varying from 10,000 clones to more than 1,000,000 clones. Such mutants can be recovered in a single tube.This mutagenesis scheme is based on the premise that the genome size is known, that mutagenesis is a random event and that a typical gene has an average size of 1 kilobase. For example and on a statistical basis, the 20 5.9 Mb Pseudomonas aeruginosa genome would require a minimum of 5,900 mutants to cover the genome at least once. This is herein defined as a 1 X genome coverage. Thus, a collection of 17,500 mutants (3 X), 29,500 mutants (5 X) or 59,000 mutants (10X) could be utilized for screening in a typical EGT assay for this particular microorganism. Of 25 course, the person of ordinary skill could also screen more than 10X.
11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 As used herein, the designation "therapeutic target"
refers to any gene or product thereof that when blocked by known or novel molecules will affect the growth of the organism coding for the target.
As used herein, the designation "Non-selective conditions" refers to in vitro and/or in vivo growth conditions wherein all the paramaters and factors which are required for optimal growth are present. Non-limiting examples of such parameters/factors include growth media nutrients, temperature, pH, cell line, and the like. Under such conditions, one would expect the organism to be maintained prior to the mutagenesis step.
As used herein, the designation "Selective conditions"
refers to conditions which are defined by the nature of the experiment done in vitro and/or in vivo and in which one specific parameter or factor or set of conditions are modified (in comparison to non-selective conditions) to determine if essentials genes or gene products can be identified in that particular condition. A non-limiting example of a selective condition includes growth at a restrictive temperature.
It will be clear to the person of ordinary skill, that insertional mutagenesis of an essential gene, within the context of a cell, will result in the death of that cell. Consequently, the genome of this particular cell will not be available as a substrate for the amplification process in accordance with the EGT method of the present invention.
The DNA molecule analysed may be a gene, a fragment thereof cloned into a vector or preferably a genome.
11229-79.DRF 19 Sep. 1997 - 1 6h12 CA 0221~870 1997-09-19 As used herein, the terminology "target region" defines a DNA region for which preliminary sequence data is sufficiently available to enable the design of a first primer pair which will, under appropriate conditions, give rise to a recognizable extension product. The target region is determined and defined by the available sequence data available for the particular genome analysed, and by the limits in the amplification method used. For PCR, for example, the conditions permit extension products to reach about 2000 nucleotides. The target region should thus be between about 50 to about 2000 nucleotides. Preferably between about 200 and about 1000. Since sequence information can be clustered, some genes might have several target regions. In any event, the mutagenesis conditions should be adapted so as to enable an insertional mutagenesis of all targeted regions. In essence, a person of ordinary skill will adapt the mutagenesis scheme so as to permit saturation mutagenesis of the DNA to be analysed.
Although in a preferred embodiment, the present invention is adapted for use with a whole genome, a DNA molecule inserted into a vector can also be used in accordance with the present invention. In such an embodiment, the vector should permit an expression of the DNA molecule in order to permit an ~ssessment of the essentiality of the gene product. In such a scheme, it will be understood that only dominant insertional mutation can provoke the lethality since, presumably, a copy of a wild type or homologous copy of the gene which is present on the vector, is present in the host cell. Consequently, it will be clear to the person of ordinary skill that although the present invention is not limited to haploid genomes, the method of the present invention is 11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 favorably used in a context of a haploid organism, and more preferably a haploid microorganism. Organisms in which conversion to homozygocity is efficient and/or complete are also covered by the scope of the present invention. In a preferred embodiment therefore, prokaryotic genomes and lower eukaryotic genomes such as the haploid genomes of parasites and protista are used. Non-limiting examples of such lower embryotic genomes include that of tachyzoite form of Toxoplasma gondii, of Plasmodia, Schistosoma and Leishmania species, as well as those of fungi such as that of Candida, Aspergillus, Neospora and other disease causing (in plants, in animals and in humans) relevant fungi are especially preferred genomes. In addition, all disease causing agents such as Influenzae, Hl\/, Herpes and other viruses may also be used in the context of the present invention.
It shall be understood that although the saturation insertional mutagenesis of the present invention is carried out by a shotgun approach (without specifically directing the insertion to specific sequences), a rational design of insertion mutation could also be carried out, especially with DNA molecules inserted into vectors.
Since the design of the first pair of primers depends on known sequence data from the genome to be analysed, it follows that minimum stretches of sequence data must be available in order to enable the EGT method of the present invention. Preferably, contiguous nucleic acid sequence data of approximately twelve nucleotides, to approximately twenty-four nucleotides in the targeted region must be available.
Although in a preferred embodiment, the method of the present invention relates particularly to genomes of organisms which do 11 229-79.DRF 19 Sep. 1997 - 1 6h12 CA 0221~870 1997-09-19 not contain or contain few introns, the present invention could be adapted by a person of ordinary skill for intron-containing genomes. Briefly, the level of mutagenesis would have to be increased in order to enable saturation to occur. Saccharomyces cerevisiae is one non-limiting 5 example of an organism which contains introns.
Numerous insertional mutagenesis method are known in the art. It will be clear to the person of ordinary skill that the method should be adapted to enable the insertion of the sequence which is complementary to that of a primer binding thereto (generally described 10 herein as primer 3).
The term "saturation mutagenesis" as used herein with reference to a genome, refers to an insertion mutagenesis in substantially every gene thereof and/or every target region thereof. Based upon statistical analysis and well known methods, at least 90%, preferably, 95% and more preferably 100% of the genes and/or target regions will have been mutagenised. Briefly, to esli,nate the conditions to permit the aiming of a complete population of mutagenised genes, the statistical analysis utilised is based on a number of criterions: 1) a completely random insertion of the insertion element (i.e. a mobile element); 2) an average size of 1 Kb for a typical gene in a prokaryote genome; 3) knowledge a priori of the genome size (Megabases). For example, a complete 1 X coverage of the P. aeruginosa 5.9 Mb genome would require a minimum of 6000 clones after the mutagenesis experiment.
Preferably, a minimum of 5 X coverage of the genome should be used by using 60,000 clones. When relating to DNA molecules present on a 11229-79.DRF 19 Sep. 1997 - 16h12 CA 0221~870 1997-09-19 vector, saturation mutagenesis refers preferably to the insertion element being present at every nucleotide position thereof.
Mutational methods include, without being limited thereto, insertional mutations in which a DNA molecule is inserted without 5 loss of native sequences, or substitutional mutations in which the DNA
molecule inserted replaces native DNA molecule of the targeted region.
It shall be understood that the choice of a particular insertional element can be adapted to particular needs, provided that it is absent from the genome which is to be analysed, that it is sufficiently long 10 to permit the generation of a primer which binds thereto (hence the need for known sequence data of about 12 contiguous nucleotides for the primer target on the genome, and disrupts the gene or target region it is inserted into. In a preferred embodiment, the insertional mutagenesis is provided by a insertional element such as transposons (i.e. Tn5, Tn10, 15 Tn916, Ty). In such cases, the insertional mutagenesis will be carried out with the insertional elements in accordance with known methods.
Insertional mutagenesis of DNA can also be carried out by using the integrases protein of retroviruses to mediate the insertion of a selected primer into a target region. Following amplification, the 20 amplified product or extension product can be detected. In a preferred embodiment they can be sized-fractionated by gel electrophoresis as well known in the art. In another embodiment the extension products can be detected after separation on columns and the like. Hybridization capture and the triplex DNA technology are non-limiting examples of technologies 25 which could be used to detect the amplified products (Lanbiewicz et al., 11229-79.DRF 19 Sep. 1997 - 16h12 CA 0221~870 1997-09-19 1997, Nucl. Acids Res.25; 2037-38; and Ito et al.,1992, Proc. Natl, Acad.
Sci 89: 495-8).
A kit for identifying essential genes in a genome contains at least three oligonucleotide primers, constituting at least two 5 primer pairs, a mutated genome, and solutions for enabling hybridization between the mutated genome sequences and the oligonucleotide primers and for enabling amplification of the extension product. Oligonucleotide primers can be suspended in solution or provided separately in Iyophilized form. The components of the kit can be packaged together in 10 a common container, the kit typically including an instruction sheet for carrying out a specific embodiment of the method of the present invention. Additional optional components of the kit include detection probes, and means for carrying out a detection step (for example, a probe or primer is labelled with a detectable marker).
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawings:
Figure 1 shows a sumll,a, i~ed schematic representation of the essential gene test (EGT) according to the present invention; and Figure 2 shows a more detailed view of the EGT shown 25 in Figure 1.
11229-79.DRF 19 Sep. 1997 - 16h12 CA 0221~870 1997-09-19 Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments with reference to the accompanying drawing which is exemplary and should not be interpreted 5 as limiting the scope of the present invention.
DETAILED DESCRIPTION
Insertional Mutagenesis of the Targeted Genome First, insertional mutagenesis must be performed so as 10 to cover most if not all genes of a particular genome in a population of cells. Under these conditions, one would expect the one tube mutagenized population to cover the spectrum of each and every gene coded by a particular organism.
Insertional Mutagen In one embodiment in which a bacterial genome is targeted, a bacterial population is mutagenized using for example a mobile element having a high frequency of transposition (Tn5, Tn10, Tn916, IS elements or any other known mobile genetic element) creating insertional mutations at diverse sites. Depending on the conditions and 20 mobile element utilized, one may produce a single tube population containing cells having an insertion in essentially all the genes. Any particular type of mutagenesis scheme including insertion elements, PCR
mutagenesis, random insertion of DNA by synthetic or biological methods would be amenable to genetic analysis by the EGT test or assay.
1 1229-79.DRF 19 Sep. 1 997 -16h12 CA 0221~870 1997-09-19 The assay can also be applied to any simple organisms such as viruses. The EGT has excellent potential in disease causing viruses from plants, from animals and from humans. Non-limiting examples include the potato blight virus in plants, the equine encephalitis 5 virus in animals and the cytomegalovirus in humans. Additional examples include single eukaryotic cells of fungi and of yeasts causing diseases such as mycoses and include Candida, Cryptococcus, Histoplasma, Blastomyces, Coccioides, Aspergillus, Fusarium, and Trychophyton, and the like. Thus, the EGT assay could be applied to all disease causing 10 organisms (See the listing of the Manual of Clinical Microbiology, 1995, ASM Press). The person of ordinary skill will adapt the EGT accordingly.
For the targeting of the yeast genome the insertional element Ty is a representative example of an insertional mutagen which can be used in accordance with the present invention. In addition, the EGT assay can be 15 utilized to dissect metabolic and genetic pathways by assessing mutagenized populations in different in vitro and in vivo conditions.
Amplification A sample of the mutagenized population is then submitted to nucleic acid amplification. In a preferred embodiment, the 20 amplification is carried out by PCR using either cells directly or by preparing an aliquot of DNA. A collection of two primers specific to the sequence under investigation (from a genomic database and assumed to encode an essential or dispensable gene where only part of the ORF is known) and defining a first primer pair, gives rise to an amplification 25 product of a defined size. A third primer specific to the insertional mutagen is also used. This three primer assay will give specific 11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 amplification products defining a sequence as essential or dispensable.
The EGT assay is performed as summarized in Figure 1 using a wild-type and a mutagenized population. The role of a particular sequence as essential or dispensable is visu~ ed as the presence (non-essential) or 5 depletion of defined satellite amplification products (essential) (Fig. 1).
A more detailed representation is shown in Fig. 2.
Intcr~,ret~lion of the results of EGT assay The primer pairs selected from the sequence of interest defines an amplification product that will be present both in essential 10 genes and in dispensable genes irrespective of the growth conditions since in the context of a population of cells, individual cells having no insertions in the targeted sequence of interest will always be present.
Thus, the first primer pair serves as an internal control for the assay conditions. If the insertion occurs in a dispensable gene, the second 15 primer pair, constituted by a primer specific to the targeted sequence and one specific to the insertional mutagen, gives rise to a specific extension product and a series of additional band products. Thus, in addition to the expected product originating from the first primer pair, additional amplification products will be visible. The difference in the size of the 20 additional product will reflect the distance between the target region of thethird primer (the insertion "point") and that of the first primer (or second primer). In contrast, insertion of an element in an essential gene will not yield an amplification product (lethal phenotype) and the only visualized amplification product will be generated by the amplification of 25 mutagenized cells containing no insertions in the essential sequence of interest (originating from the first primer pair).
11229-79.DRF 19Sep. 1997-16h12 CA 0221~870 1997-09-19 As alluded to above, the EGT assay enables automation. For example, by using fluorescent primers (labelled with distinct fluorochromes) the EGT assay could be used in conjunction with the ABI GENESCAN.
The following examples are offered by way of illustration and not by way of limitation.
E~CAMPLE 1 EGT assay on two Pseudomonas aeruginosa genes The EGT assay was applied to the Pseudomonas aeruginosa strain PAO1 5.9 Mb genome in the following way. First, a library of insertion mutants was constructed with the miniTn5 Km insertion element using standard methods. A collection of 60,000 clones (10 X
genome coverage) obtained were pooled into a single tube.
A first primer pair of 21-mers specific and internal to the ftsZ gene sequence (ftsZ1 :5'-ATC ACC ATC CCG MC GAG MG-3') and (ftsZ2:5'-TAT CCA GGT MT CCA GGT CAT-3') give a 669 bps amplified PCR product.The PCR conditions for DNA amplification were carried out in accordance with the manufacturer's recommendations (Perkin Elmer Cetus and Applied Biosystems). In a typical EGT assay, one would expect the 669 bps to be present irrespective of the mutagenesis or growth conditions.
The EGT assay was performed for ffsZ by using the following primers :(KanaputR1: 5'-GCG GCC TCG AGC MG ACG
TTT-3') and (KanaputF4: 5'-TTG GTT GTA ACA CTG GCA GAG-3') in combination with one and\or the two above-mentioned primers (ftsZ1 and 1 1229-79.DRF 1 9 Sep. 1 997 -1 6h12 CA 0221~870 1997-09-19 ftsZ2). The result of the EGT assay showed a product of 669 bps and no satellite bands, irrespective of the mutagenesis scheme. Thus, only the first primer pair gave rise to an extension product. Thus, ftsZ is therefore defined as an essential gene by the EGT method.
The EGT assay was tested with the ampC gene using primers (ampcF1: 5'- CAT CGC TTC CAC ACT GCT-3') and (ampcR1:
5'-TGC CGG GM CAC TTG CTG CTC-3') constituting a first primer pair giving rise to a PCR product of 592 bps irrespective of the mutagenesis.
When used in conjunction with the KanaputR1 and KanaputF1 primers, a PCR product of 592 bps (positive control) and additional DNA bands (due to insertions in the ampC gene) could be visualized in the agarose ethidium bromide stained gel. Thus, the EGT assay would define the ampC gene as non-essential.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
11229-79.DRF 19Sep. 1997-16h12
Claims (12)
1. A method for identifying essential and non-essential genes in a genome of a cell grown in non-selective conditions, said method comprising:
saturation mutagenesis of said genome by insertion mutagenesis, whereby an oligonucleotide sequence is inserted in the target regions of said genome such that a population of cells having at least 90% of said target regions insertionally mutated is obtained;
growing said population of cells under non-selective conditions to provide a non-selected sub-population of cells;
amplifying a target region from said non-selected sub-population of cells, using a first primer which hybridizes to a known first end of said target region, and a second primer which hybridizes to another known end of said target region, said first and second primers thereby constituting a first primer pair, giving rise to a first extension product, and a third primer which hybridizes to said oligonucleotide sequence, said third primer constituting a second primer pair with one said first or second primer, said second primer pair enabling the amplification of a second extension product; and assessing for the presence or absence of said first and second extension product, whereby the presence of the first and second extension products is indicative of a non-essential gene, whereas the presence of the first extension product and the absence of the second extension product is indicative of an essential gene.
resolving by gel electrophoresis said amplified DNA from said at least one selected and one non-selected aliquots into individual bands differing by size to identify the position of individual sequence tag insertions within said target region, whereby differences in the presence or intensity of bands between said at least one selected and one non-selected aliquots are indicative that said sequence tag insertion causes a difference in response to said selective condition employed with said at least one aliquot, resulting in the functional analysis of said target region.
saturation mutagenesis of said genome by insertion mutagenesis, whereby an oligonucleotide sequence is inserted in the target regions of said genome such that a population of cells having at least 90% of said target regions insertionally mutated is obtained;
growing said population of cells under non-selective conditions to provide a non-selected sub-population of cells;
amplifying a target region from said non-selected sub-population of cells, using a first primer which hybridizes to a known first end of said target region, and a second primer which hybridizes to another known end of said target region, said first and second primers thereby constituting a first primer pair, giving rise to a first extension product, and a third primer which hybridizes to said oligonucleotide sequence, said third primer constituting a second primer pair with one said first or second primer, said second primer pair enabling the amplification of a second extension product; and assessing for the presence or absence of said first and second extension product, whereby the presence of the first and second extension products is indicative of a non-essential gene, whereas the presence of the first extension product and the absence of the second extension product is indicative of an essential gene.
resolving by gel electrophoresis said amplified DNA from said at least one selected and one non-selected aliquots into individual bands differing by size to identify the position of individual sequence tag insertions within said target region, whereby differences in the presence or intensity of bands between said at least one selected and one non-selected aliquots are indicative that said sequence tag insertion causes a difference in response to said selective condition employed with said at least one aliquot, resulting in the functional analysis of said target region.
2. A method according to claim 1, wherein mutagenizing is performed with a transposable element.
3. A method according to claim 2, wherein said target DNA comprises a gene encoding a protein.
4. A method according to claim 1, wherein said selective condition is growth of cells in media lacking a nutrient that is an intermediate in a metabolic pathway.
5. A method for functional analysis of a target region in a sequence of interest, said method comprising:
mutagenizing said target region by insertion of a sequence tag to provide a population of DNA molecules containing a sequence tag insertion in at least 90% of nucleotide positions in said target region;
introducing said population of mutagenized DNA
molecules into host cells that express said sequence of interest;
subjecting a first aliquot of said host cells to at least one selective condition and a second aliquot to a non-selective condition to provide at least one selected and one non-selected aliquot;
amplifying target region DNA from said at least one selected and one non-selected aliquots, wherein said amplification is by polymerase chain reaction using a first primer hybridizing to said sequence tag and a second primer hybridizing to a known endpoint, said endpoint being characterized as an arbitrary unique sequence in said target DNA, to provide amplified DNA; and resolving by gel electrophoresis said amplified DNA from said at least one selected and one non-selected aliquots into individual bands differing by size to identify the position of individual sequence tag insertions within said target region, whereby differences between the presence or intensity of bands between said at least one selected and one non-selected aliquots are indicative that said sequence tag insertion causes a difference in response to said selective condition employed with said at least one selected aliquot resulting in the functional analysis of said target region.
mutagenizing said target region by insertion of a sequence tag to provide a population of DNA molecules containing a sequence tag insertion in at least 90% of nucleotide positions in said target region;
introducing said population of mutagenized DNA
molecules into host cells that express said sequence of interest;
subjecting a first aliquot of said host cells to at least one selective condition and a second aliquot to a non-selective condition to provide at least one selected and one non-selected aliquot;
amplifying target region DNA from said at least one selected and one non-selected aliquots, wherein said amplification is by polymerase chain reaction using a first primer hybridizing to said sequence tag and a second primer hybridizing to a known endpoint, said endpoint being characterized as an arbitrary unique sequence in said target DNA, to provide amplified DNA; and resolving by gel electrophoresis said amplified DNA from said at least one selected and one non-selected aliquots into individual bands differing by size to identify the position of individual sequence tag insertions within said target region, whereby differences between the presence or intensity of bands between said at least one selected and one non-selected aliquots are indicative that said sequence tag insertion causes a difference in response to said selective condition employed with said at least one selected aliquot resulting in the functional analysis of said target region.
6. A method according to claim 5, wherein mutagenizing comprises the steps of:
combining DNA comprising said target region with retroviral integrase and a first set of complementary oligonucleotide primers, said primers comprising (a) a recognition sequence for said retroviral integrase and (b) a sequence tag, wherein said retroviral integrase mediates the insertion of said first set of complementary oligonucleotide primers to provide a population of mutagenized DNA
molecules.
combining DNA comprising said target region with retroviral integrase and a first set of complementary oligonucleotide primers, said primers comprising (a) a recognition sequence for said retroviral integrase and (b) a sequence tag, wherein said retroviral integrase mediates the insertion of said first set of complementary oligonucleotide primers to provide a population of mutagenized DNA
molecules.
7. A method according to claim 5, wherein mutagenizing comprises the steps of:
combining DNA comprising said target region with retroviral integrase and a first set of complementary oligonucleotide primers, said primers comprising (a) a recognition sequence for said retroviral integrase and (b) a recognition site for a type lls restriction endonuclease, wherein said retroviral integrase mediates the insertion of said first set of complementary oligonucleotide primers to provide a population of mutagenized DNA molecules cutting said population of mutagenized DNA molecules with said type lls restriction endonuclease to provide cut DNA; and ligating to said cut DNA a second set of complementary oligonucleotide primers comprising a sequence tag.
combining DNA comprising said target region with retroviral integrase and a first set of complementary oligonucleotide primers, said primers comprising (a) a recognition sequence for said retroviral integrase and (b) a recognition site for a type lls restriction endonuclease, wherein said retroviral integrase mediates the insertion of said first set of complementary oligonucleotide primers to provide a population of mutagenized DNA molecules cutting said population of mutagenized DNA molecules with said type lls restriction endonuclease to provide cut DNA; and ligating to said cut DNA a second set of complementary oligonucleotide primers comprising a sequence tag.
8. A method according to claim 5, wherein said sequence of interest comprises a gene encoding a protein.
9. A method according to claim 8, wherein said population of mutagenized DNA molecules are cloned into a filamentous bacteriophage vector with regulatory sequences for expression of said sequence of interest.
10. A method according to claim 5, wherein said sequence of interest comprises a regulatory gene.
11. A method according to claim 10, wherein said selective condition is growth in media containing a cytotoxic agent, and said regulatory gene controls expression of a gene conferring resistance to said cytotoxic agent.
11. A method according to one of claims 1-10, wherein said genome is a haploid genome.
11. A method according to one of claims 1-10, wherein said genome is a haploid genome.
12. A method according to claim 11, wherein said haploid genome is a bacterial genome.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2215870 CA2215870A1 (en) | 1997-09-19 | 1997-09-19 | Method for the identification of essential genes and therapeutic targets |
| CA002304369A CA2304369A1 (en) | 1997-09-19 | 1998-09-21 | Method for the identification of essential genes and therapeutic targets |
| AU92476/98A AU9247698A (en) | 1997-09-19 | 1998-09-21 | Method for the identification of essential genes and therapeutic targets |
| PCT/CA1998/000893 WO1999015644A2 (en) | 1997-09-19 | 1998-09-21 | Method for the identification of essential genes and therapeutic targets |
| US10/647,566 US20040081990A1 (en) | 1997-09-19 | 2003-08-25 | Method for the identification of essential genes and therapeutic targets |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2215870 CA2215870A1 (en) | 1997-09-19 | 1997-09-19 | Method for the identification of essential genes and therapeutic targets |
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| CA2215870A1 true CA2215870A1 (en) | 1999-03-19 |
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| AU (1) | AU9247698A (en) |
| CA (1) | CA2215870A1 (en) |
| WO (1) | WO1999015644A2 (en) |
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| AU4186600A (en) * | 1999-03-31 | 2000-10-16 | Rosetta Inpharmatics, Inc. | Essential genes of yeast as targets for antifungal agents, herbicides, insecticides and anti-proliferation drugs |
| GB0008748D0 (en) * | 2000-04-11 | 2000-05-31 | Univ Manchester | Mutant bank |
| CN1329034A (en) * | 2000-06-19 | 2002-01-02 | 上海博德基因开发有限公司 | A novel polypeptide-human L1 components ORF2 protein 21 and polynucleotide for coding this polypeptide |
| WO2002083940A2 (en) * | 2001-04-06 | 2002-10-24 | Creatogen Aktiengesellschaft | Screening method for anti-microbial drug targets by genome-saturing mutagenesis (gsm) |
| GB0321232D0 (en) * | 2003-09-10 | 2003-10-08 | Arrow Therapeutics Ltd | Method for the identification of attenuating lesions in pathogens |
| EP3170902A1 (en) * | 2015-11-19 | 2017-05-24 | ETH Zurich | Parallel functional testing of synthetic dna parts, pathways, and genomes |
| CN105400876B (en) * | 2015-12-03 | 2019-06-11 | 天津科技大学 | Pseudomonas aeruginosa and phage-infect related gene and application |
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| WO1994026933A1 (en) * | 1993-05-13 | 1994-11-24 | The Board Of Trustees Of The Leland Stanford Junior University | Genetic footprinting: insertional mutagenesis and genetic selection |
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- 1998-09-21 AU AU92476/98A patent/AU9247698A/en not_active Abandoned
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| WO1999015644A2 (en) | 1999-04-01 |
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