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WO2001075167A1 - Strategie genetique inverse pour identifier des mutations fonctionnelles dans des genes de sequence connue - Google Patents

Strategie genetique inverse pour identifier des mutations fonctionnelles dans des genes de sequence connue Download PDF

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Publication number
WO2001075167A1
WO2001075167A1 PCT/US2001/010545 US0110545W WO0175167A1 WO 2001075167 A1 WO2001075167 A1 WO 2001075167A1 US 0110545 W US0110545 W US 0110545W WO 0175167 A1 WO0175167 A1 WO 0175167A1
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mutations
mutation
endonuclease
gene
organism
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PCT/US2001/010545
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Claire M. Mc Callum
Steven Henikoff
Trenton Colbert
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Fred Hutchinson Cancer Research Center
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Priority to AU2001253063A priority Critical patent/AU2001253063A1/en
Priority to US10/240,456 priority patent/US20040053236A1/en
Publication of WO2001075167A1 publication Critical patent/WO2001075167A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • sequence comparison tools that detect protein sequence similarity to previously studied genes often allow a related function to be inferred.
  • the present invention provides a reverse genetic strategy that combines the high density of mutations offered by traditional mutagenesis methods with rapid mutational screening to discover induced lesions.
  • the method designated TILLING (Targeting Induced Local Lesions In Genomes)
  • TILLING Targeting Induced Local Lesions In Genomes
  • mutagenesis methods e.g., chemical-induced (for example, using ethyl methanesulfonate (EMS)(Koornneef et al., Mutat. Res. 93:109-123 (1982))
  • EMS ethyl methanesulfonate
  • mutation analysis tools such as the detection of single base pair changes by heteroduplex analysis (Underhill et al., Genome Res.
  • the TILLING method generates a wide range of mutant alleles, is fast and automatable, and is applicable to any organism that can be mutagenized, stored and propagated.
  • the present invention provides a reverse genetic method for identifying functional mutations in a gene of known sequence comprising treating an organism or cell with mutagen which induces mutations in the DNA of an organism or cell; preparing isolated genomic DNA from the mutagenized organism or cell; amplifying a region of a gene of known sequence; and screening for mutations in the mutagenized DNA sequence in the gene as compared to the same sequence of the gene in the wild type parent organism or cell.
  • the method designated TILLING for Targeted Induced Local Lesion in Genomes, combines the high density of mutations provided by traditional mutagenesis methods with rapid mutational analysis methods to identify mutations of interest in genes of known sequence without inserting heterologous nucleic acids into an organism or cell.
  • Methods for mutagenizing the genome of an organism or a cell to induce mutations can include treating an organism or cell with chemical agents or radiation.
  • a traditional chemical mutagen such as ethyl methanesulfonate, methylmethane sulfonate, N-ethyl-N-nitrosourea, triethylmelamine, diepoxyalkanes (diepoxyoctane, diepoxybutane, and the like), 2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl) aminopropylamino] acridine dihydrochloride, formaldehyde, and the like can be used.
  • Nucleic acid amplification methods suitable for use in the methods of the present invention include, but are not limited to PCR methods such as RT-PCR. Primers are selected to amplify a region of the genome comprising the gene of interest. The PCR product is then analyzed for the presence of mutations. Mutations can be detected, for example, by single-stranded conformational polymorphism or by heteroduplex analysis, and the like.
  • Methods for heteroduplex analysis compatible with the methods of the present invention include constant denaturant capillary electrophoresis, denaturing high pressure liquid chromatography, and enzyme or chemical digestion of nucleotide mismatches followed by separation and detection of the digested DNA.
  • Each of these methods have been used previously to identify naturally occurring polymorphisms consisting of single base changes in genes of interest (Cotton et al., Mutation Detection: A Practical Approach, IRL Press, Oxford, England (1998)).
  • TILLING the mutations detected are either missense or nonsense mutations which result in altered or truncated protein products.
  • the organisms or cells analyzed by the disclosed methods can be either homozygous or heterozygous for the mutation of interest.
  • the methods of the present invention are applicable to any organism which can be heavily mutagenized, including both plants and animals.
  • TILLING has been applied to two Arabidopsis thaliana chromomethylase genes related to CMT 1, a DNA methyltransferase homologue with a chromodomain (Henikoff and Comai, Genetics 149:307-318 (1998)).
  • the methods are also applicable to other plants, particularly crop plants such as maize, alfalfa, wheat, barley, soy beans, cotton, pine, rice, legumes, i.e., Medicago truncatula, and the like.
  • Using the methods of the present invention it is possible to select for plants with phenotypic variations of commercial interest without introducing foreign DNA of any type into the plant genome.
  • Figure 1 depicts in the form of a cartoon the TILLING strategy applied to a plant such as Arabidopsis thaliana.
  • Figure 2 depicts the structure of Arabidopsis thaliana chromomethylase genes. Exons are shown as boxes with cytosine DNA methyltransferase blocks (black rectangles) and chromodomain blocks (gray rectangles) indicated. Fragments used for TILLING analysis are indicated as horizontal lines above CMT2 and below CMT3.
  • Figure 3 provides a depiction of dHPLC chromatograms showing typical sensitivity for detection of a transition mutation on a PCR fragment, where Ler and Col templates, which differ by a single C/G to T/A change, have been mixed in the indicated ratios and amplified. Retention time on the dHPLC column is plotted against intensity of the signal in millivolts (mV).
  • Figure 4 depicts the sites that are most susceptible to base transition mutations after treatment with EMS for the CMT3B fragment (the nucleotide sequence of the fragment is depicted as SEQ ID NO: 11, and the amino acids encoded by the wild-type sequence and the mutations detected for each amino acid position for this fragment are depicted as SEQ ID NO: 12 and SEQ ID NO: 13).
  • the position of Q479 to stop obtained in the screen is depicted as ⁇ (See Table 1).
  • Figure 5 depicts in the form of a cartoon the high throughput TILLING strategy applied to a plant such as Arabidopsis thaliana as demonstrated in Example 3.
  • the subject invention relates to methods for finding multiple mutations in genes of known sequence by combining mutagenesis with methods for finding point mutations.
  • the present invention provides a method for the creation and subsequent detection of mutations within a selected (desired) DNA region.
  • the mutations created provide a range of allele types, including knockouts and missense mutations, which will be useful in a variety of gene function and interaction studies. This method is particularly useful for studies in organisms that do not have extensive genetic tools or genomic DNA sequence available.
  • Arabidopsis is mutagenized to produce a plurality of different point mutations and screened with semi-automated nucleic acid amplification-based methods, i.e., PCR, within a gene region of interest.
  • mutations in any gene contained in the genome of Arabidopsis can be screened by the methods of the present invention in as few as a set of approximately 5-10,000 reference plants. It is expected that most phenotypes can be scored in the F2 progeny of reference plants, and therefore functional analysis can be easily performed.
  • TILLING has been specifically applied to Arabidopsis and Drosophila
  • the methods as described are of general use. Therefore, any organism that can be mutagenized can be TILLed, although plants are especially suitable.
  • the general applicability of the methods of the present invention means that organisms lacking well-developed genetic tools can be TILLed. For example, but not by way of limitation, plants such as maize, alfalfa, barley, rice, soy beans, cotton, pine, melons, and other commercially important crop plants can be analyzed with the methods of the present invention. Additionally, model plant systems, such as the legume Medicago truncatula, can be examined using the methods of the present invention.
  • seeds, pollen, germ cells and cells cultured from plants are suitable subjects for TILLING.
  • animals are also suitable subject for TILLING.
  • germ cells of animals such as nematodes, fruit flies, mice, chickens, turkeys, dogs, cats, cows, sheep, horses, pigs and other commercially important agricultural and companion animals can be analyzed with the methods of the instant invention.
  • TILLING is related to a method whereby chemically mutagenized Caenorhabditis elegans cultured in microtiter plates were screened by PCR for deletions (Liu et al., Genome Res. 9:859-867 (1999)).
  • this method requires screening of approximately 10 6 genomes (about 100 times more than that required for TILLING) to obtain a knock-out mutation, it is not likely to be generally applicable.
  • several advantages over previously disclosed reverse genetic methods are realized including, for example:
  • genomic DNAs are prepared and arrayed, the process is almost fully automated. All subsequent steps, including, for example, detection of mutations, e.g., by PCR and dHPLC analysis, can typically be performed in, for example, microtiter plates, which can be handled robotically.
  • Chemical mutagens in particular, mutagens which result in primarily point mutations and short deletions, insertions, transversions, and or transitions (about 1 to about 5 nucleotides), such as ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N-ethyl-N-nitrosourea (ENU), triethylmelamine (TEM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitosamine, N-methyl-N'- nitro-Nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7, 12 dimethyl- benz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan
  • missense mutations not just knock-outs.
  • temperature-sensitive missense alleles expected to be especially useful for gene interaction studies (Bowman et al, Plant Cell 1:37-52 (1989)), can be obtained.
  • a PCR product useful for detection by, for example, dHPLC or gel electrophoresis following cleavage of an oligonucleotide strand at the position of a base mismatch, for example, by endonuclease digestion is the size of a small gene, small targets that are likely to be missed by other methods, such as, high density transposon tagging strategies or large DNA detection strategies (Wisman et al., Plant Mol. Biol. 37:989-999 (1998)); Bevan et al., Bioessays 21:110-120 (1999) each incorporated herein by reference) present no special problem for TILLING.
  • Any gene can be targeted, whether essential or not, because mutations are detectable in both homozygotes, or heterozygotes.
  • the methods of the present invention permits one to find mutations in a gene of interest in the absence of an assayable phenotype which may later be discerned, for example mutations that are present as a heterozygous mutation may not be detected in a heterozygote, but may be detectable when the identified mutations crossed and a homozygous individual is obtained.
  • the concentration of the mutagen selected will be that which will induce a plurality of different mutations in the genome of the organism of interest.
  • Mutagenesis refers to methods for inducing a plurality of mutations in the DNA of a cell.
  • the mutations typically useful in the methods of the present invention are those which induce changes that alter or eliminate the function if the gene product (i.e., a nucleotide substitution, deletion, or insertion).
  • the methods of the present invention are especially useful in detecting point mutations.
  • Point mutations include single base transitions, base tranversions, insertions and deletions.
  • the mutagenesis comprises exposing a germ cell of an organism, a cell, or a seed with a chemical mutagen, i.e., but not limited to those listed above.
  • the cells can also be treated with, for example, radiation, i.e., x-rays and gamma-radiation, which induce primarily larger lesions, ultra-violet light, and the like.
  • radiation i.e., x-rays and gamma-radiation
  • a polynucleotide sequence encoding certain heterologous enzymes which can induce mutations i.e., a phyophosphohydrolase, such as the bacterial MutT gene which makes AT to CG transversions, and the like, can be introduced into the cell or seed.
  • Appropriate mutation rates for mutagens will typically be in the range of about 1 mutation per 500 kilobase pairs (kbp) to about 1 mutation per 10 kbp.
  • Genomic DNA is obtained from the selected F2 individuals and pooled. The pooled DNA is subjected to TILLING as described below using primers designed for one, or typically multiple (usually up to at least 12) marker regions or other regions of interest. The mutation rate at a particular region or gene locus can thus be determined by dividing the number of mutations found in all regions by the total number of base pairs screened.
  • EMS was used to induce primarily point mutations in Arabidopsis.
  • Other agents are well known to the skilled artisan which provide similar results in various other organisms as provided hereinabove.
  • PCR polymerase chain reaction
  • SSCP single-stranded conformational polymorphism
  • Primers used for amplification are designed to specific regions of genes of interest.
  • primers were designed with melting temperatures of 60°-70°C, and final annealing temperatures of T m -5°C are chosen.
  • Amplification products are denatured and reannealed under conditions permitting heteroduplexes to form. Such denaturation and annealing can be carried out in a separate step from the amplification or can be incorporated into the amplification protocol.
  • heteroduplexes can be fragmented by chemical cleavage.
  • Chemical cleavage can be carried out by, for example, hydroxylamine and osmium tetroxide to react with the mismatch in a DNA heteroduplex.
  • subsequent treatment with piperidine cleaves the mismatched strand at the point of the mismatch. Mutations are detected by the separation of the fragments and the identification of fragments smaller than the untreated heteroduplex.
  • Heteroduplexes are also detectable by electrophoresis, for example by constant denaturant capillary electrophoresis (CDCE), or by denaturing high pressure liquid chromatography (dHPLC).
  • DCE constant denaturant capillary electrophoresis
  • dHPLC denaturing high pressure liquid chromatography
  • dHPLC was chosen for the screening of mutation because it combines automation, speed of analysis and high overall detection sensitivity for unknown single base changes in a commercially available instrument.
  • endonuclease cleavage was used to identify mutations because it is a reliable and inexpensive point mutation discovery method that can be performed even more rapidly than dHPLC and in a robust manner.
  • Running time for dHPLC may limit throughput for the methods of the present invention, requiring about a week of screening for each mutation detected.
  • the use of fluorescence detection rather than UV absorbance is expected to allow an order-of-magnitude increase in the number of genomes in a pool; smaller sample loads should minimize band broadening, allowing for better separation of heteroduplexes from homoduplexes.
  • Fluorescence detection also may allow for multiplexing based on different fluorochromes, further increasing dHPLC throughput.
  • fluorescence multiplexing is not yet commercially available for dHPLC, multiplexing has been performed by co-amplifying fragments that are differentially retained on the column but have similar melting temperatures.
  • Heteroduplexes can be detected enzymatically, for example using an endonuclease that recognizes and cleaves at mismatches in a heteroduplex.
  • Suitable endonucleases for use in the instant methods include resolvases, RNases, bacteriophage T4 endonuclease VII, bacteriophage T7, endonuclease I, Saccharomyces cerevisiae endonuclease XI, Saccharomyces cerevisiae endonuclease X2, Saccharomyces cerevisiae endonuclease X3, SI nuclease, CEL I, PI nuclease, or mung bean nuclease.
  • the CEL I endonuclease (Oleykowski et al., Nucl. Acids Res. 26:4597-4602 (1998)) is used to cleave heteroduplex mismatches.
  • CEL I a plant-specific extracellular glycoprotein that belongs to the SI nuclease family (Oleykowski et al., ibid.), has been shown to be suitable for genotyping applications because it preferentially cleaves mismatches of all types (Oleykowski et al., ibid.) and has been used to detect heterozygous polymorphisms in DNA pools (Kulinski et al., Biotechniques 29: 44-46 (2000)).
  • mutations are identified using a high-throughput TILLING method that utilizes an endonuclease to cleave heteroduplex mismatches.
  • mutagenized DNA is first amplified using primers specific for a gene region of interest.
  • the primers are preferably labeled with different independently detectable labels. This differential double-end labeling of amplification products allows for rapid visual confirmation, because mutations are detected on complementary strands, and so can be easily distinguished from amplification artifacts.
  • the choice of labels useful in the methods of the present invention will be evident to the skilled artisan.
  • Independent detection can be accomplished by, for example, using fluorochrome labels, i.e., fiuorescein isothiocyanate (FITC), terachlorofluorescem, hexachlorofluoroscem, Cy3, Cy5, Texas Red, infrared dyes (IRDYE 700, IRDYE 770, IRDYE 800), or APC, and the like, that fluoresce at different wavelengths permitting clear identification of each label by its particular wavelength, or by selecting radioactive labels that are detectable using different filters.
  • FITC fiuorescein isothiocyanate
  • terachlorofluorescem terachlorofluorescem
  • hexachlorofluoroscem Cy3, Cy5, Texas Red
  • infrared dyes IRDYE 700, IRDYE 770, IRDYE 800
  • APC and the like
  • Heteroduplex analysis is then carried out by cleaving the heteroduplexes with an endonuclease under conditions and for a time sufficient to permit endonuclease cleavage at mismatches between wild-type and mutant.
  • Cleavage products are physically separated by, for example, gel electrophoresis or other means which exploits a change in size or mass.
  • Slab gel electrophoresis is well suited for large-scale mutation detection. The two-dimensional readout facilitates the detection of rare events, such as mutations, because a new band will stand out above the wild-type background and can be easily spotted.
  • each new band is also obtained, an advantage over other methods based on detection of mismatches or conformational changes (Nataraj et al., Electrophoresis 20:1177-1185 (1999)), which do not indicate where in the molecule a mutation resides.
  • the separated cleavage fragments are differentially detected using methods suitable for the labels.
  • IRDYE-labeled cleavage products separated in a polyacrylamide gel are detected by the measuring the absorbance at each wavelength characteristic of the label used, i.e., 700 or 800 nm, as the fragments pass through a detector.
  • Images of the gel are obtained by for example, direct scanning or photography followed by scanning and the images are visually analyzed using graphic display software, such as Adobe PHOTOSHOP (Adobe, San Jose, CA), QUICKTIME (Apple Computer, Cupertino, CA), NETSCAPE NAVIGATOR (Netscape, Mountain View, CA), or the like.
  • the images are analyzed with the aid of a standard commercial image processing program to identify the presence of change in fragment size indicating cleavage by the endonuclease at a mutation induced mismatch which give information on the presence of a mutation as well as its location.
  • mutations detected in a pool can be further investigated by screening the individual DNAs in the positive pools to identify the individual, e.g., plant, harboring the mutation. This rapid screening procedure determines the location of a mutation, or to within a few base pairs, for a PCR product up to 1 kb in size.
  • Differential double-end labeling of amplification products allows for rapid visual confirmation, because mutations are detected on complementary strands, and so can be easily distinguished from amplification artifacts.
  • An additional important advantage of double end-labeling for detecting both cleavage products is avoidance of false positive bands.
  • False positive bands which might result from the practice of the disclosed methods are of two types: those that appear in multiple lanes for a single detected label and those that appear in a single lane but in the same position for both detected labels.
  • IRDYE detection is carried out by viewing the gel in each of two channels which detect a different infrared (IRDYE) label. Because it is highly unlikely that the same mutation will appear in two different individuals, it is assumed that certain homoduplex sites are especially sensitive to variability in endonuclease digestion, causing bands to appear in multiple lanes above the background pattern.
  • PCR product yield was determined to typically provide a low and inconsistent signal using both IR Dye 700 and IR Dye 800 dyes on opposing primers; however, consistent results have been obtained using a mixture of IRDye-labeled and unlabeled primers.
  • DNA molecules within mixtures are improved using capillary technology.
  • capillary electrophoresis has been successfully exploited for high throughput DNA sequencing (Kheterpal et al., Anal. Chem. 71:31A-37A (1999)) and for rapid heteroduplex (CDCE) and SSCP detection applications (Larsen et al., Hum. Mutat. 13:318-327 (1999); Li-Sucholeiki et al., Electrophoresis 20:1224-1232 (1999); Nataraj et al., Electrophoresis 20: 1177-1185 (1999)). It is expected that dHPLC will also be accelerated by the development of capillary columns.
  • TILLING minimizes the effort required to find mutations
  • ascertainment of a resulting phenotype requires additional characterization.
  • Chemical mutagenesis introduces background mutations that can make phenotypic analysis uncertain, and multiple generations of outcrossing may be desirable.
  • a rapid strategy is available if two independent severe lesions are found. Briefly, the two individuals can be crossed and their progeny typed. A phenotype attributable to the two non-complementing mutations will be found in every individual carrying both lesions, whereas non-complementing background mutations will sort independently.
  • missense mutations should be identified as a by-product of screening for two severe lesions. Because it is estimated that 5-10% of EMS-induced mutations are temperature-sensitive (Ashburner, Drosophila, A Laboratory Handbook, Cold Spring Harbor Press, Cold Spring Harbor (1990)), the method of the present invention is likely to provide conditional mutants that can be used for epistasis and interaction analyses. Furthermore, by choosing evolutionarily conserved regions of proteins for TILLING, the probability of obtaining severe and conditional lesions is not only increased, but also mutations are provided in regions that are most useful for protein structure and function studies. The "Blocks" system, for example, is designed to find conserved regions amenable to the methods provided herein (Henikoff et al., Nucl. Acids Res. 27:226-228 (1999)).
  • TILLING can be performed at a genomic scale to provide gene knockouts and conditional mutations for general study. For example, a collection of approximately 10,000 mutagenized reference M2 plants in an Arabidopsis race that is most suitable for TILLING has been partially established.
  • the Columbia ecotype is a particularly suitable choice because it has been used for sequencing and EST analyses.
  • Columbia erecta a Columbia derivative that carries an induced erecta allele so that it has favorable compact growth characteristics (Yokoyama et al., Plant J. 15:301-3 10 (1998)) has been used to establish the library.
  • This line has been back-crossed to wild- type Columbia three times and self fertilized subsequent to EMS-mutagenesis, and so it is expected to be homozygous for about 90% of its genome. There should be only about 20 heterozygous mutations in the genome which could complicate the screening method described herein. However, even these heterozygotes can be eliminated by prescreening the unmutagenized parental genome.
  • Columbia erecta seeds are being mutagenized with EMS using the same protocol as described herein for plants.
  • each reference plant of the M2 generation can be grown from a separate Ml plant. It is important that DNA samples from different plants are nearly identical in concentration in order to maximize sensitivity to a mutation in any one sample plant.
  • CMT2 and CMT3 Two Arabidopsis chromomethylase genes (CMT2 and CMT3) related to CMT1 were selected. Primers were chosen based primarily on the probability of introducing a severe lesion. Mutations in the CMT2 and CMT3 genes were detected with denaturing HPLC (dHPLC), followed by sequencing to determine the mutation. Additionally, in another embodiment TILLING was used to examine functional mutations in a gene of known sequence in Drosophila. Within another embodiment, two Arabidopsis genes, hdal and Sir2B were selected and subjected to high-throughput TILLING. Primers were selected to flank the gene region of interest and to a specific Tm to facilitate amplification.
  • dHPLC denaturing HPLC
  • genes of known sequence can be chosen for TILLING using methods for analyzing the DNA sequence for regions which would have a high probability for mutation depending on the mutagen used. By assigning a score to defined regions of a target gene based on the likelihood of obtaining a desirable mutation, genes can be placed in a rank order. The ranks can be used both to pick regions of the selected gene for primers and to choose the order in which genes will be TLLLed. Preliminary data with Arabidopsis suggests that approximately 5-10,000 reference plants will suffice for obtaining the desired mutations from just a single primer pair per gene that encompasses the most favorable region for TILLING. A computer program for choosing primers can output a list for oligonucleotide synthesis.
  • Plants are especially well suited to the methods of the present invention, because they can be self-fertilized and seeds can be easily stored.
  • Arabidopsis thaliana which has been described herein as a specific example of high throughput TILLING
  • other crop plants can also benefit from TILLING.
  • the same genes discovered in Arabidopsis can be studied in crop plants as listed above.
  • genes in other plants which are the same or similar to those discovered in Arabidopsis can be selected.
  • the identification of a similar gene can be accomplished, for example, using CODEHOP PCR primer design (Rose et al., Nucl. Acids Res. 26:1628-1635 (1998)). This is a PCR primer design method for amplification of distantly related sequences.
  • each database entry can be, for example, a FASTA-formatted sequence, containing the mutation that was determined from the individual plant PCR products. Searching the TILLING database of mutant sequences (typically supplemented with a database providing a set of non-mutant controls) will return single entries for each mutation aligned with the query. The mutation itself can be easily pinpointed as (presumably) the only non-matching alignment pair.
  • a user would search an amino acid sequence database to find an amino acid mutation or a nucleotide sequence database to identify a base mismatch. Each mutation can be confirmed by sequencing both strands. Confirmation of a heterozygous mutation by sequencing can be challenging even when both strands have been sequenced, however, computational methods exist for interpreting sequence trace data to identify heterozygous mutations.
  • reverse genetics methods may have been an impediment to organizing genomics of some plant species, and the methods of the present invention will likely spur genomics in neglected but important plants. Further, the generality of TILLING means that screening for mutations by these methods is applicable to animals.
  • reverse genetic techniques in zebrafish are both labor and resource intensive and are not suitable for genome-scale analysis.
  • Other potentially suitable systems include cultured cells, for example, mutagenized mouse embryonic stem cells, which can be stored frozen and implanted when needed to obtain mice for phenotypic analysis.
  • chromomethylase a DNA methyltransferase homologue with a chromodomain
  • chromomethylase a DNA methyltransferase homologue with a chromodomain
  • CMTl is found to be homozygous null. This non-essentiality of CMTl could be explained as redundant function if other chromomethylases exist in Arabidopsis.
  • CODEHOP PCR primer design method Rose et al., Nucleic Acids Res. 26: 1628-1635 (1998) was employed and two different nucleic acid sequences evidently related to CMTl from A. thaliana genomic DNA were isolated. Using these PCR products to probe an A. thaliana genomic library, two new chromomethylase genes were identified, CMT2 and CMT3.
  • RT-PCR and isolation and sequencing of the full coding regions of CMT2 and CMT3 cDNAs revealed that their intron/exon boundaries are similar to those of CMTl (Fig. 1).
  • Quantitative RT-PCR expression studies showed that CMT2 and CMT3 are ubiquitously expressed at moderate levels, as might be expected for genes involved in silencing.
  • Arabidopsis thaliana has been mutagenized with ethyl methanesulfonate (EMS) and the chromomethylase 2 (CMT2) and chromomethylase 3 (CMT3) genes have been examined for mutations.
  • EMS ethyl methanesulfonate
  • CMT2 chromomethylase 2
  • CMT3 chromomethylase 3
  • the two PCR products were TA-cloned (Invitrogen) and sequenced. Two unique sequences related to CMTl were identified and used to probe an A. thaliana genomic library (Clontech). The cDNA sample preparation and RT-PCR conditions used were previously described (Henikoff et al., Genetics 149:307-318 (1998)).
  • EMS mutagenesis tissue collection and DNA extraction. Seeds from A. thaliana ecotype No-0 were mutagenized with 20 mM EMS for 18 hours (Koornneef et al., Mutat. Res. 93:109-123 (1982)). Seeds from these Ml plants were collected in batch for the M2 generation.
  • Leaf samples from five M2 individuals were pooled prior to DNA extraction. To ensure that approximately equal amounts of tissue were collected from every individual, leaf samples were collected as punches using a #4 (9.5 mm diameter) cork borer and stored at -80°C. A modification of a quick DNA preparation protocol (Edwards et al, Nucleic Acids Res. 19:1349 (1991)) was used. DNAs from individual plants were prepared when a pool containing a mutation was identified.
  • Samples for mutational screening and sequencing were generated in 20 ⁇ l reaction volumes containing approximately 1 ng pooled genomic DNA, 2.5 mM MgCl 2 , 100 ⁇ M dNTPs, 0.2 ⁇ M of forward and reverse primers, IX Pfu buffer and 2.5 U of Pfu polymerase (Stratagene). TOUCHDOWN PCR amplifications were performed as recommended by the manufacturer (Transgenomic Inc., San Jose, CA) (Kuklin et al., Genetic Testing 1 :201-206 (1997)). Cycle sequencing protocols were used with ABI Model 373 sequencers.
  • Mutation detection was performed using the WAVE system (Transgenomic Inc., San Jose, CA). Following PCR amplification, the Pfu polymerase was inactivated while the DNA samples were heated and cooled to form heteroduplexes. For most fragments, the predicted WAVE (v.3.5) melting temperatures and separation gradients were used (Jones et al., Clin. Chem. 45:1133-1140 (1999)). For CMT2B and CMT3B, the software predicted two melting domains, and so the corresponding samples were analyzed at each of the predicted melting temperatures. After EMS mutagenesis (Redei et al., in, Methods in Arabidopsis Research, pp. 16-82.
  • Base changes were detectable as extra peaks owing to melting of duplex regions around mismatches and reduced retention on the heated reverse phase HPLC column.
  • DNAs from individual plants were amplified and typed, and the PCR sample carrying the alteration was sequenced using an amplification primer.
  • nonsense mutations result from single base changes that convert an amino acid codon into a stop codon.
  • missense mutations result when single base changes alter the amino acid encoded by a particular codon; these can be further categorized as those resulting in conservative and nonconservative substitutions.
  • silent mutations result when a single base change to a codon does not alter the encoded amino acid. These changes are usually, but not exclusively, the result of mutations that alter the third base of a codon. Because nonsense and missense mutations that result in nonconservative substitutions are most likely to result in deleterious mutations, it is important to know the expected frequency of each class of mutation.
  • EMS produces primarily C to T changes resulting in C/G to T/A transition mutations.
  • LEAFY EMS-generated alleles http:// www.salk.edu/LABS/pbio-w/lfyseq.html
  • 20/23 are C/G to T/A
  • 2/23 are C/G to A/T
  • 1/23 is A T to T/A.
  • all changes are C/G to T/A transitions and using the standard Arabidopsis codon usage table (http://www.kazusa.
  • coding regions that are evolutionarily highly conserved, the likelihood of recovering missense mutations with detrimental effects on gene function can be maximized.
  • transition mutations in splice junctions are deleterious, and so for every intron in a chosen region there are at least two positions at which C/G to T/A mutations lead to loss of gene product. It can be calculated that overall 1% of the mutations in coding regions will be disruptions of splice junctions.
  • a screen for mutations in a cell of interest demonstrated as an example the methods of the present invention analysis of the CMT2 and CMT3 using 835 M2 plants.
  • transition mutations in splice junctions are deleterious, and so for every intron in a chosen region there are at least two positions at which C/G to T/A mutations lead to loss of gene product. It can be calculated that overall 1% of the mutations in coding regions will be disruptions of splice junctions.
  • a screen for mutations in a cell of interest demonstrated as an example the methods of the present invention analysis of the CMT2 and CMT3 using 835 M2 plants. Seven different PCR fragments ranging in size from 345-970 bp were examined, for a total of approximately 2 Mb of DNA sequence screened by dHPLC. Thirteen chromatographic alterations were detected and confirmed to be mutations by amplification of multiple samples (Table 1); no PCR errors were found. Analysis of the isolated DNA demonstrated an error rate of ⁇ 10 "6 . All detected mutations were base transitions in either homozygotes or heterozygotes, as expected for EMS-mutagenized M2 plants.
  • CMT2 In CMT2, one mutation resulted in an Asp to Asn amino acid change and another was detected within an intron.
  • Two different changes in nucleotide sequence were identified in CMT3.
  • One mutation changed a Glu codon to Lys, and the other changed a CAG Glu codon to a TAG stop codon.
  • the stop codon resulted in truncation of CMT3 (Fig. 4), which lacked four conserved blocks that are known to be crucial for enzymatic function (Posfai et al., Nucleic Acids Res. 17:2421-2435 (1989)). Transition mutations were discovered in nine other plants, but each of these is identical to one of the mutations described above.
  • This example provides a method for the examination of functional mutations in a gene of known sequence in fruit flies.
  • Male flies will be fed EMS to induce point mutation in the genome.
  • the males will be crossed enmass to Balancer 4* (Bal + ) females (where the balancer chromosome is used to suppress recombination and maintain heterozygous lines).
  • Bal/ x Bal/ wherein * means mutagenized chromosome matings are set up, removing the parent flies after a sufficient egg-laying period.
  • the number of males and females collected in each vial from the mating will depend on the sensitivity of the method of detection and the dose of mutagen used. It is expected that about 10 females and 10 males would result in about 10 * genomes represented among the resulting brood.
  • Matings for the M2 will be carried out selecting about 20 aged non-Bal ( / ) and allowing egg laying.
  • the vial will contain a sampling of mutagenized genomes and Bal chromosomes. Flies in the vial will be allowed to develop at low temperature (about 14°C) to hold the M3 generation as long as possible.
  • DNA will be prepared from the / M2 parents in 8 x 12 arrays. It is expected that each independently mutagenized chromosome might be as rare as 1/40, and would presumably be missed. However, the representation of the average chromosome should be 1/10. Therefore, about half of the mutations will be detected on average, depending on the fragment size for screening.
  • This Example describes a high-throughput TILLING method which was used to analyze the Arabidopsis hdal gene.
  • a region of interest was amplified using a "left" primer labeled with a first label and a "right” primer labeled with a second label and wherein the labels are independently detectable.
  • Heteroduplexes of the amplified and labeled DNA was nicked at a mismatch with the endonuclease CEL I.
  • the use of label at both ends of the amplification products permitted identification of mutations and sequencing at single base resolution from no farther than the middle of any fragment, thus allowing for larger segments of a gene to be analyzed.
  • Genomic DNA from each M2 plant was prepared from 0.2 g of leaf and/or stem tissue using the BIO101 FASTDNA system (Qbiogene, Carlsbad, CA) following the manufacturer's instructions. Concentrations of the DNA preparations were estimated by visualization on 1% agarose electrophoretic gels and were equalized prior to dilution (in 10 mM Tris pH 8.0, 1 mM EDTA) and pooling. The genomic DNA samples from each individual plant were pooled at either 5-fold or 8-fold representing either 5- or 8- individual plants per well, and the pools were arrayed on microtiter plates.
  • genomic DNA pools were subjected to hdal gene-specific amplification using polymerase chain reaction (PCR) using primers designed with melting temperatures of 60°-70°C, and final annealing temperatures of T m -5°C were chosen. Briefly, each PCR amplification reaction was performed in 10 ⁇ L volumes using EXTAQ polymerase (PanVera Corporation, Madison, WI) using the manufacturer's protocol with the exception that only half the manufacturer's recommended concentration of buffer was used, and MgCl 2 concentration was increased to 2 mM.
  • PCR polymerase chain reaction
  • Primers (forward primer: 5' GGTAATGGATACTGGCGGCAATTCG 3' (SEQ ID NO: 9), reverse primer: 5' ACCACCCAAGAGCAGTAGGGGAACA 3'; SEQ ID NO: 10) were obtained from MWG Biotech (MWG Biotech Inc., High Point, NC).
  • the forward primer was labeled with the infrared detectable label IRDYE 700 (IRDYE 700 (LI-COR Inc., Lincoln, NE), molecular formula: C 52 H6 7 N 4 O 5 PS) and the reverse primer was labeled with the infrared red detectable label IRDYE 800 (IRDYE 800 (LI-COR Inc.), molecular formula: C 5 H 75 N 4 O 6 PS).
  • the primers were mixed in a ratio of 3 :2 labeled to unlabeled primer (IRDYE 700-labeled primer) and 4:1 labeled to unlabeled primer (IRDYE 800-labeled primer), for final primer concentrations of 0.2 ⁇ M.
  • the reaction mixtures were subjected to amplification cycles in a MWG
  • Biotech 96-well cycler (MWG Biotech Inc.) as follows: 1) 95°C for 2 min; 2) 8 cycles of TOUCHDOWN PCR: 94°C for 20 sec (denaturation), T m + 3°cto T m - 4°C decrementing 1°C per cycle (annealing), 72°C for 45 sec to 1 min (extension for 600 to 1000 bp products); 3) 45 cycles of: 94°C for 20 sec (denaturation), T m - 5°C (annealing), 72°C for 45 sec to 1 min; 4) 72°C for 5 min; 5) 99°C for 10 min (inactivation); 6) 70 cycles of 20 sec at 70°C to 49°C, decrementing 0.3 C per cycle (reannealing).
  • the CEL I enzyme an endonuclease that preferentially cleaves mismatches in heteroduplexes between wildtype and mutant, was purified from 30 kg of celery essentially as described by Oleykowski et al. (Nucleic Acids Res.
  • Amplification products were incubated with CEL I and cleavage products were then electrophoresed using an automated sequencing gel apparatus, and gel images are analyzed with the aid of a standard commercial image processing program. Briefly, 10 ⁇ l of each amplification product was mixed with 20 ⁇ l of CEL I buffer (lOmM Hepes pH 7.5, 10 mM MgSO 4 , 0.002% Triton-X-100, 20 ng/ml of bovine serum albumin) and
  • MWG 96-well catch plates (MWG Biotech Inc.) were prepared by transferring about 1 to about 1.5 ⁇ l formamide load solution (1 mM EDTA pH 8 and 200 ⁇ g/ml bromphenol blue in deionized formamide) into each well of a fresh MWG 96-well catch plates (labeled and oriented).
  • the reactions were subjected to denaturing gel electrophoresis by first transferring the reactions to a membrane comb using a comb-loading robot and the COMBLOAD program supplied by the manufacturer (MWG Biotech).
  • An IRDYE 800- labeled 50-700 bp molecular weight marker mix (LI-COR Inc.) was applied to outside teeth.
  • LI-COR Inc. LI-COR Global IR gel scanner
  • the comb containing the CEL I-treated amplification products was inserted into a well on top of a 6.5 % acrylamide gel, electrophoresed for 1 min and removed. Electrophoresis was continued for 4 hours at 1500 V, 40 W, 40 mA limits at 50°C.
  • the DNAs were detected in two separate channels by a LI-COR scanner as generally described by Middendorf et al. (Electrophoresis 13: 487-494 (1992); incorporated by reference herein in its entirely). As described in more detail below, this method was sufficiently sensitive to detect the approximately 100 atamole of cleavage product generated by CEL I in an 8-fold pool, or one in 16 genomes for a heterozygous mutation.
  • the opposed PCR primers carried different dye labels. As there is no detectable overlap between the IRDYE 700 and IRDYE 800 dye labels, images were examined directly for the presence of novel bands in either channel, 700nm or 800nm wavelengths of excitation.
  • the image files were visually analyzed using a graphics display software program such as Adobe Photoshop (Adobe, Inc., San Jose, CA).
  • the images resulting from the gel scans showed a sequence-specific pattern of background bands resulting from endonucleolytic cleavages common to all 96 lanes.
  • images representing both channels and switching between them lanes containing a novel band in one channel and a corresponding novel band in the other channel were identified.
  • the sum of the two band sizes was equal to the full-length product visible at the top of the image.
  • This visual assay was aided by the approximate proportionality of the migration distance to molecular weight, so that a band in one channel was nearly the same distance from the leading edge as the corresponding band in the other channel was from the full-length product.
  • Image manipulation tools, rulers and guides were used for the determination of migration distances and lane numbers for the two bands.
  • the individual DNA samples were similarly screened to identify the plant carrying the mutation. This rapid screening procedure determined the location of a mutation or within a few base pairs for PCR products up to 1 kb in size. Moreover, the combination of Cell and EMS induced mutagenesis permits one to simultaneously identify and localize the mutation. Because EMS causes specific transition mutations, the use of this method permits one to determine the sequence of the mutation upon examination of the reference sequence, the wild-type sequence of the mutation and this is true of other mutagens as well. Briefly, the individual DNA samples comprising the pool containing an identified mutation were screened.
  • This screen resulted in the identification of the plant in which a point mutation had occurred and an estimated location within a few base pairs of the lesion as well as confirming the original detection event in the 8-fold pool screen.
  • Each mutation found in the arrayed plates of individual genomic DNA was re-confirmed by DNA sequencing.
  • This method provides for screening and identifying the plants which harbor detected mutations. But the method also provides, based on the size of the DNA fragments obtained on separation, i.e., gel electrophoresis, the location of the mutation.
  • the skilled artisan can determine which of the detected mutations lies in a region of interest, i.e., mutations in regions that are most likely to have a biologically functional effect, and eliminate those in other regions, such as intron and regions of low protein sequence conservation. The elimination of the need to examine all mutations in these regions saves time and allows the artisan to focus efforts on the regions of biological interest. Additionally, with the precise location of a mutation and the use of a specific mutagen, the sequence , or identity , of the mutation can be known.

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Abstract

Cette invention concerne une stratégie génétique inverse simple et générale à utiliser dans des organismes pour lesquels des outils génétiques bien développés font défaut. Ce procédé consiste en particulier à mettre un organisme ou une cellule en contact avec un mutagène qui induit principalement des mutations ponctuelles dans l'ADN génomique; à préparer un ADN à partir de cet organisme ou de cette cellule; à amplifier une région génomique d'intérêt; et à déterminer les modifications se produisant dans la séquence d'ADN mutagénisée dans la région génomique d'intérêt, par rapport à l'organisme ou la cellule parent. La détermination des mutations peut consister à détecter les hétéroduplex en vue d'identifier un organisme ou une cellule mutant. Ces procédés servent en particulier à identifier des mutations fonctionnelles dans des plantes.
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US7393996B2 (en) * 2002-10-22 2008-07-01 Arcadia Biosciences Inc. Tomatoes having reduced polygalacturonase activity caused by non-transgenic mutations in the polygalacturonase gene
EP1708559A4 (fr) * 2003-12-03 2010-01-06 Arcadia Biosciences Inc Ble comprenant une proteine cireuse reduite du fait d'alterations non transgeniques d'un gene cireux
US7786349B2 (en) 2003-04-09 2010-08-31 Bayer Bioscience N.V. Methods and means for increasing the tolerance of plants to stress conditions
WO2012004013A2 (fr) 2010-07-08 2012-01-12 Bayer Bioscience N.V. Protéine transportrice de glucosinolate et ses utilisations
US9062348B1 (en) 2005-12-22 2015-06-23 Keygene N.V. Method for high-throughput AFLP-based polymorphism detection
US9376719B2 (en) 2005-09-29 2016-06-28 Keygene N.V. High throughput screening of mutagenized populations
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US10023907B2 (en) 2006-04-04 2018-07-17 Keygene N.V. High throughput detection of molecular markers based on AFLP and high through-put sequencing
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WO2020185663A3 (fr) * 2019-03-08 2020-10-22 Board Of Trustees Of Michigan State University Maîtriser l'auto-incompatibilité chez les plantes diploïdes pour la sélection et la production d'hybrides par la modulation du ht
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Publication number Priority date Publication date Assignee Title
WO2003008589A1 (fr) * 2001-07-18 2003-01-30 Asahi Kasei Kabushiki Kaisha Elk1 gene associe a la phosphorylation
US7393996B2 (en) * 2002-10-22 2008-07-01 Arcadia Biosciences Inc. Tomatoes having reduced polygalacturonase activity caused by non-transgenic mutations in the polygalacturonase gene
US7928298B2 (en) 2002-10-22 2011-04-19 Arcadia Biosciences, Inc. Tomatoes having reduced polygalacturonase activity caused by non-transgenic mutations in the polygalacturonase gene
US7786349B2 (en) 2003-04-09 2010-08-31 Bayer Bioscience N.V. Methods and means for increasing the tolerance of plants to stress conditions
US7354715B2 (en) 2003-05-22 2008-04-08 Dow Agrosciences Llc High-throughput methods of screening DNA for deletions and other mutations
US8735649B2 (en) 2003-12-03 2014-05-27 Arcadia Biosciences, Inc. Wheat having reduced waxy protein due to non-transgenic alterations of a waxy gene
EP1708559A4 (fr) * 2003-12-03 2010-01-06 Arcadia Biosciences Inc Ble comprenant une proteine cireuse reduite du fait d'alterations non transgeniques d'un gene cireux
US10978175B2 (en) 2005-06-23 2021-04-13 Keygene N.V. Strategies for high throughput identification and detection of polymorphisms
US9898576B2 (en) 2005-06-23 2018-02-20 Keygene N.V. Strategies for high throughput identification and detection of polymorphisms
US10235494B2 (en) 2005-06-23 2019-03-19 Keygene N.V. Strategies for high throughput identification and detection of polymorphisms
US10095832B2 (en) 2005-06-23 2018-10-09 Keygene N.V. Strategies for high throughput identification and detection of polymorphisms
US9898577B2 (en) 2005-06-23 2018-02-20 Keygene N.V. Strategies for high throughput identification and detection of polymorphisms
US9896721B2 (en) 2005-06-23 2018-02-20 Keygene N.V. Strategies for high throughput identification and detection of polymorphisms
US10538806B2 (en) 2005-09-29 2020-01-21 Keygene N.V. High throughput screening of populations carrying naturally occurring mutations
US10316364B2 (en) 2005-09-29 2019-06-11 Keygene N.V. Method for identifying the source of an amplicon
US9670542B2 (en) 2005-09-29 2017-06-06 Keygene N.V. High throughput screening of populations carrying naturally occurring mutations
US9657335B2 (en) 2005-09-29 2017-05-23 Keygene N.V. High throughput screening of populations carrying naturally occurring mutations
US9574230B2 (en) 2005-09-29 2017-02-21 Keygene N.V. High throughput screening of populations carrying naturally occuring mutations
US11649494B2 (en) 2005-09-29 2023-05-16 Keygene N.V. High throughput screening of populations carrying naturally occurring mutations
US9376719B2 (en) 2005-09-29 2016-06-28 Keygene N.V. High throughput screening of mutagenized populations
US9745627B2 (en) 2005-09-29 2017-08-29 Keygene N.V. High throughput screening of populations carrying naturally occurring mutations
US10233494B2 (en) 2005-09-29 2019-03-19 Keygene N.V. High throughput screening of populations carrying naturally occurring mutations
US9062348B1 (en) 2005-12-22 2015-06-23 Keygene N.V. Method for high-throughput AFLP-based polymorphism detection
US9334536B2 (en) 2005-12-22 2016-05-10 Keygene N.V. Method for high-throughput AFLP-based polymorphism detection
US10106850B2 (en) 2005-12-22 2018-10-23 Keygene N.V. Method for high-throughput AFLP-based polymorphism detection
US11008615B2 (en) 2005-12-22 2021-05-18 Keygene N.V. Method for high-throughput AFLP-based polymorphism detection
US10023907B2 (en) 2006-04-04 2018-07-17 Keygene N.V. High throughput detection of molecular markers based on AFLP and high through-put sequencing
WO2012004013A2 (fr) 2010-07-08 2012-01-12 Bayer Bioscience N.V. Protéine transportrice de glucosinolate et ses utilisations
WO2020185663A3 (fr) * 2019-03-08 2020-10-22 Board Of Trustees Of Michigan State University Maîtriser l'auto-incompatibilité chez les plantes diploïdes pour la sélection et la production d'hybrides par la modulation du ht
WO2021069614A1 (fr) * 2019-10-10 2021-04-15 Carlsberg A/S Procédés de préparation de compositions de plantes mutantes
CN114786473A (zh) * 2019-10-10 2022-07-22 嘉士伯有限公司 制备突变植物的方法
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