Patent Application
20090068646
Exposure to mutagens in the environment can pose a serious health threat, particularly to workers in certain high risk occupations. Accurate methods for measuring mutations are critical to estimating potential health risks associated with exposure to radiation and other mutagens. Dosimetry systems provide information concerning the extent of exposure, information that is useful in instituting measures to reduce risk of further exposure. Biological dosimetry provides additional information concerning how radiation affects the individual receiving the radiation. Gross chromosomal changes can be detected by fluorescence in-situ hybridization (“FISH”), a biodosimetric method. However, the accuracy of long-term biodosimetry by cytogenetic means is affected by the loss of chromosomal aberrations over time.
Nearly one-third of the human genome is composed of DNA repeats. Repetitive DNA sequences have been identified as susceptible to mutation in response to mutagens. Microsatellite loci are a class of DNA repeats, each of which contains a sequence of 1-9 base pairs (bp) that is tandemly repeated. Loci having larger repeat units of 10 to 60 bp are typically referred to as minisatellites. Microsatellites and minisatellites are inherently unstable and mutate at rates several orders of magnitude higher than non-repetitive DNA sequences. Due to this instability, microsatellites and minisatellites have been evaluated for increased mutation rates after exposure to mutagens.
Ishizaki et al. (Aviat Space Environ Med 2001 72(9):p. 794-8) examined the effect of radiation exposure (0.02 Gy) on mismatch repair deficient colon cancer cells aboard a 9-day space shuttle flight using six microsatellite loci, including the mononucleotide repeat marker BAT-26. No increase in mutation rate was observed relative to controls. In view of the relatively low radiation dose, this result was not unexpected. Similarly low doses of radiation did not cause a significant increase in chromosomal aberrations in astronauts using standard cytogenetic chromosomal analysis.
Boyd et al. (Int J Radiat Biol, 2000. 76(2):2.169-176) reported a dose-response relationship for radiation-induced mutations at mini- and microsatellite loci in human somatic cells. Various sizes of minisatellite loci were analyzed; microsatellite loci analyzed were di- and tetranucleotide repeats. Boyd identified that the microsatellites were less sensitive than the minisatellites. See Boyd, FIG. 2, page 172.
Microsatellite markers were reported to be altered in A-bomb survivors with leukemia. Nakanishi et al. (Int J Radiat Biol, 2001. 77(6):p. 687-94) analyzed leukemia cells from 13 individuals with acute myelocytic leukemia and with a history of radiation exposure, and from 12 individuals with acute myelocytic leukemia and without a known history of exposure using 10 microsatellite markers, including the mononucleotide repeat marker BAT-40. Estimated radiation exposures ranged from 0.05 to over 4 Gy. Microsatellite Instability (MSI) analysis revealed a high frequency of multiple microsatellite changes in the exposed individuals (85%) compared with non-exposed individuals (8%). Those patients exposed to >1 Gy exhibited a high frequency of MSI (MSI-H), with mutations in greater than 30% of markers. However, only 3 of 13 A-bomb survivors exhibited changes in BAT-40, compared with 2 of 12 non-exposed leukemia patients, which suggests that there is no difference in the stability of BAT-40 in exposed or unexposed patients. Therefore, it appeared that BAT-40 was not sensitive enough to allow detection of radiation-induced mutation. The latter finding is consistent with an earlier report by Okuda et al. (J Radiat Res (Tokyo), 1998. 39 (4):p. 279-87) that exposure to 2 Gy X-rays did not result in increased mutations of BAT-26. Therefore, it appeared that BAT-40 and BAT-26 were not sensitive enough to allow detection of radiation-induced mutation.
Accordingly, persons in the art had come to believe that minisatellites were better able to detect radiation-induced mutations. Furthermore, it was expected that this finding applied to any mutation regardless of what mutagen was the cause of the mutation. For example, Dubrova identified minisatellites as the most unstable in the human genome. Swiss Med Wkly, 2003, volume 133 pages 474-478.
Yamada examined the mutation frequency of G17 and A17 mononucleotide repeats and (CA)17 dinucleotide repeat in human cells lines exposed to oxidative stress (Environmental and Molecular Mutagenesis, (2003) 42:75-84). No effect was observed for either mononucleotide locus, and a small increase in mutation frequency was observed for the dinucleotide locus.
A relatively high level of chromosomal alterations occur on the Y chromosome due to the presence of repetitive elements clustered along the length of the chromosome and the inability of the Y chromosome to participate in recombination repair (Kuroda-Kawaguchi et al. Nature 2001 29:279). The Y chromosome has about 60 million base pairs, of which 95% are in non-recombining regions (NRY) that do not undergo recombination due to the haploid nature of the Y chromosome (Tilford et al. Nature 2001 409:943). Radiation exposure of 1.5 Gy or more often results in persistent azoospermia or reduced sperm production, presumably due to deletions encompassing genes necessary for spermatogenesis (Birioukov, et al. Arch Androl 1993 30(2):99-104; Greiner Strahlenschutz Forsch Prax 1985 26:114-121). Germline mutation rates in short tandem repeats on the Y chromosome are similar to those observed on autosomal chromosomes (i.e., about 1.6×10−3) Bodowle, et al. (Forensic Science International 2005 150(1):1-15). Twelve short tandem repeat loci Y chromosome haplotypes: Genetic analysis on populations residing in North America. Forensic Science International).
Susceptibility to ROS-induced DNA damage is in part a function of DNA sequence, due to intrinsic secondary structural differences between DNA molecules. Lower probabilities of irradiation-induced DNA strand breakage at certain DNA sequences may be explained by reduced minor groove width that limits accessibility to the hydroxyl radical produced by ionizing radiation. Certain secondary DNA structures have been shown to be recognized by DNA repair enzymes and this may also contribute to the relative susceptibility of specific DNA sequences to mutations, particularly some types of repetitive DNA sequences. For example, a 5-bp tandem repeat satellite derived from variants of the core 5′-TTCCA-3′ has been shown to be a “hot spot” for radiation-induced single and double strand breaks (Vazquez-Gundin, F. et al. Radiation Research 2004 157:711-720). This vulnerability of specific sequences may relate to chromatin or tertiary DNA structure that could affect access of hydroxyl radicals to the DNA or exclude water molecules from the proximity of DNA, resulting in lower rate of radiation-induced hydroxyl radicals (Ljungman, M. Radiation Research 1991 126:58-64). The mutagenic potential of different DNA sequences may therefore be due to a balance between specific sensitivities of a particular DNA sequence and protection exerted by DNA structure or chromatin organization or the local sequence environment.
There is a continuing need in the art for methods of assessing exposure to mutation-inducing conditions, such as radiation or chemicals that cause mutations.
In one aspect, the present invention provides a method for monitoring an organism or cell population for exposure to a mutagen by amplifying a set of at least one microsatellite locus from a DNA sample from the organism or cell population. The set of microsatellite includes the at least one microsatellite from mononucleotide repeat loci having at least 38 repeats, Y chromosome short tandem repeats of 1-6 bp, or A-rich short tandem repeats having repeating units selected from the group consisting of AAAAG, AAAAC, and AAAAT. The size of the amplification product is compared with the expected size of the amplification product. A difference between the size of amplification product and the expected size of the amplification product is indicative of exposure of the organism or cell population exposure to a mutagen.
In another aspect, the invention provides a method for evaluating the mutagenicity of an agent by exposing an organism or cell culture to the agent and then amplifying a set of at least one microsatellite locus from a DNA sample from the organism or cell culture. The set of microsatellite includes the at least one microsatellite from mononucleotide repeat loci having at least 38 repeats, Y chromosome short tandem repeats of 1-6 bp, or A-rich short tandem repeats having repeating units selected from the group consisting of AAAAG, AAAAC, and AAAAT. The size of the amplification product is compared with the expected size of the amplification product. A difference between the size of amplification product and the expected size of the amplification product is indicative of indicative of mutagenicity.
The present invention also provides a method of detecting microsatellite instability in a human putative cancerous or precancerous cell or tumor cell. A set of at least one microsatellite locus including at least one of a mononucleotide repeat locus having at least 41 repeats and a Y chromosome short tandem repeat of 1-6 bp is amplified from a DNA sample from the putative cancerous or precancerous cell or tumor cell. The size of the first amplification product is determined and compared with the expected size of the amplification product. Microsatellite instability is indicated by a difference between the size of first amplification product and the expected size of the amplification product.
In another aspect, the invention provides a method of detecting microsatellite instability in a mouse putative cancerous or precancerous cell or tumor cell. A set of at least one microsatellite locus including at least one of a mononucleotide repeat locus having at least 48 repeats is amplified from a DNA sample from the putative cancerous or precancerous cell or tumor cell. The size of the first amplification product is determined and compared with the expected size of the amplification product. Microsatellite instability is indicated by a difference between the size of first amplification product and the expected size of the amplification product.
The invention further provides a method for detecting a mutation in a microsatellite locus by amplifying at least one microsatellite including at least one mononucleotide repeat locus having at least 41 repeats from DNA sample from a human cell line or individual to form an amplification product. The size of the amplification product is determined and compared to the expected size of the amplification product. A difference in size between the amplification product and its expected size is indicative of a mutation in the microsatellite repeat locus.
The invention also provides a method for detecting a mutation in a microsatellite locus by amplifying at least one microsatellite including at least one mononucleotide repeat locus having at least 48 repeats from DNA sample from a mouse cell line or individual organism to form an amplification product. The size of the amplification product is determined and compared to the expected size of the amplification product. A difference in size between the amplification product and its expected size is indicative of a mutation in the microsatellite repeat locus.
Also provided is a method for distinguishing between a mutation or artifact. The method involves amplifying a mono-, di- tri-, tetra-, penta-, or hexanucleotide repeat locus from a DNA sample using three different primers. The first primer hybridizes to a first sequence and the second primer hybridizes to a second sequence, the first and second sequences flanking or partially overlapping the target DNA sequence. The third primer hybridizes to a third sequence between the first and second sequences. The DNA between the first and second primers is amplified to form a first amplification product and the DNA between the first and third primers is amplified to form a second amplification product. The sizes of the amplification products are determined and compared to the expected sizes. An equivalent size difference in the first and second amplification products relative to their respective expected sizes indicates a mutation.
In another aspect, the present invention provides a construct comprising a polynucleotide encoding a detectable reporter marker linked to repeat sequence having at least 19 repeats such that a deletion of one or more base pairs of the repeat sequence alters the expression of the reporter marker.
The present invention provides methods for detecting mutations by observing allelic length variations in mononucleotide repeat tracts or in certain other short tandem repeats comprising repeating units of 1-6 base pairs that are sensitive to exposure to mutagens, such as radiation or chemical mutagens.
As used herein, “mutagen” refers to a substance or condition that causes a change in DNA including, but not limited to, chemical or biological substances, for example, free radicals, reactive oxygen species (ROS), drugs, chemicals, radiation and the normal aging process. By “exposing” it is meant contacting a cell or organism with a mutagen or treating a cell or organism under conditions that result in interaction of the cell or organism with a mutagen. It should be understood that “exposing” a cell or organism to a mutagen does not necessarily require an active step. Rather, exposure of a cell or organism to a mutagen may result from the cell or organism being present in an environment in which the mutagen occurs.
The methods allow detection and monitoring of genetic damage in individuals exposed to mutagens. Additionally, the methods may be used to measure mutagenesis in response to exposure of cultured cells or experimental animals to mutagens. In one embodiment, the methods may be used to test the mutagenicity of a particular mutagen by exposing a cell or organism to a mutagen or potential mutagen by comparing amplified microsatellite loci of exposed cells to those of a non-exposed cell or organism. In another embodiment, a cell or organism cell or organism carrying a polynucleotide encoding a detectable reporter marker linked to a microsatellite repeat locus having at least 19 repeats such that a deletion in the microsatellite repeat on exposure to a mutagen alter expression of the reporter marker.
As described in the Examples, numerous extended mononucleotide repeats (i.e., mononucleotide repeats containing from 38-200 repeats) in human or mouse DNA were identified in a search of available sequence information (Tables 1A-1D). Extended mononucleotide repeat sequences have not previously been evaluated for use in detecting an increase in instability in response to environmental insults (i.e., mutagens) or to identify conditions associated with mismatch repair deficiency because relatively long repeats were generally thought to be too highly mutable to afford meaningful results. The general suitability of extended mononucleotide repeats for use in monitoring exposure to mutagens was evaluated using select extended mononucleotide repeats, as described in the Examples. The results indicate that mutations in extended mononucleotide repeats occur with higher frequency in cells exposed to mutagens than in control cells. Extended mononucleotide repeat loci, preferably comprise at least 38 nucleotides repeats. Extended mononucleotide repeat loci suitably have repeats of between 38 and 200 nucleotides, between 41 and 200 nucleotides, between 38 and 90 nucleotides, between 41 and 90 nucleotides, between 42 and 90 nucleotides or between 42 and 60 nucleotides.
Similarly, mutations in extended mononucleotide repeats were found to occur with greater frequency in mismatch repair deficient cells than in cells having a functional mismatch repair system. Mononucleotide repeat loci having 41 or more repeats were found to be useful in detected microsatellite instability in mismatch repair in human cells. Extended mononucleotide repeat loci suitably have repeats of between 41 and 200 nucleotides, between 41 and 90 nucleotides, between 42 and 90 nucleotides, or between 42 and 60 nucleotides.
Extended mononucleotide repeat loci are named according to the species, the base contained in the mononucleotide repeat, and the number of times the base is repeated, as reported in deposited GenBank sequences. However, due to variation between individuals and alleles, the number of bases in mononucleotide repeat may be more or fewer than the number indicated in GenBank. For example, mBAT47 is used to designate a mouse sequence with a 47 base adenine repeat with reference to the GenBank sequence. However, different mouse cell lines or individual organisms may contain one or more alleles having fewer than 47 adenine repeats at that locus.
Other loci suitable for use in the methods of the invention include Y chromosome microsatellite loci comprising repeated sequences of from 1-6 bases (YSTRs or YSTR loci). As described below in the Examples, YSTRs exhibit increased mutation rates following exposure to ROS or radiation, relative to that of unexposed cells, and in MMR deficient tumor cells, relative to that of MMR proficient tumor cells. YSTRs suitable for use in evaluating exposure to a mutagen or in evaluating the microsatellite instability of a putative precancerous or cancerous cell or tumor cell include, but are not limited to, DYS438, DYS389-II, DYS390, DYS439, DYS392, DYS385b, DYS19, DYS389-I, DYS385a, DYS393, DYS437, and DYS 391. It is reasonably expected that other YSTR loci of the Y chromosome will be suitable for detecting ROS or radiation exposure, or microsatellite instability of putative precancerous or cancerous cells or tumor cells, including, but are not limited to, Y chromosome microsatellite loci shown in Table 7, which were identified in a search of available sequence information (i.e., DYS453, DYS456, DYS446, DYS455, DYS463, DYS435, DYS458, DYS449, DYS454, DYS434, DYS437, DYS435, DYS439, DYS488, DYS447, DYS436, DYS390, DYS460, DYS461, DYS462, DYS448, DYS452, DYS464a, DYS464b, DYS464c, DYS464d, DYS459a, and DYS459b). It is specifically envisioned that any other mono-, di-, tri-, tetra-, or pentanucleotide repeat on the non-recombining regions (NRY) of the Y chromosome would be suitable in the methods of the invention.
Methods that identify mutations in microsatellite loci may be used to evaluate exposure to mutagens, including those that cause oxidative stress. Mutations in microsatellite loci are generally found in non-coding regions, and are not deleterious to the cell. Thus, mutations in non-coding repetitive sequences can accumulate, providing a stable molecular record of DNA damage from past exposures.
Accumulation of reactive oxygen species (ROS), which occurs with aging or in response to exposure to certain chemicals, results in mitochondrial DNA (mtDNA) deletions and defective repair of DNA damage. Oxidative DNA damage by elevated ROS is characterized by the production of superoxide anions (O2-), hydrogen radicals (OH) and their common product hydrogen peroxide (H2O2). Accumulation of ROS causes damage to macromolecules, including lipid peroxidation, oxidation of amino acid side chains, formation of DNA-protein cross-links, oxidation of polypeptide backbones resulting in protein fragmentation, DNA damage and DNA strand breaks. Mitochondrial DNA is composed of a 16,569 bp closed circular double stranded genome, and exhibits a common 4977 bp deletion (Δ-mtDNA4977) that has been reported to increase with age and mitochondrial degeneration. Mitochondrial DNA is particularly susceptible to damage by ROS. Damage by hydrogen peroxide is more extensive in mtDNA than in nuclear DNA, and the mutation frequency of mtDNA is 10-1000 fold higher than in nuclear DNA.
As described in the Examples, the effect of accumulated ROS due to aging or exposure to paraquat was evaluated in C57BL/6 mice by examining mtDNA deletion (Δ-mtDNA4977) and genomic stability as measured by mutations in mononucleotide repeat loci. Paraquat is an herbicide that reacts with molecular oxygen in vivo to form ROS. Mutations were detected by amplifying DNA samples containing mBat-24, mBat-26, mBat-30, mBat37, mBat-59, mBat-64, or mBat-67. The results indicated that extended mononucleotide repeats are more susceptible to ROS-induced deletion mutations than are shorter mononucleotide repeats, and that amplification of the extended mononucleotide repeats provided a more sensitive test for ROS damage.
Mutational load profiling, through analysis of changes in mononucleotide repeat sequences over time, is a non-invasive and generalized approach for monitoring an individual's cumulative record of mutations. This approach is useful in predicting and minimizing health risks for individuals exposed to mutagen. The methods of the invention can be used measure genetic damage to cell cultures or whole animals caused by exposure to drugs or chemicals.
In addition, detection of mutations in extended mononucleotide repeat will facilitate detection of tumors or other conditions associated with mismatch repair deficiencies. Individuals with hereditary non-polyposis colorectal cancer (HNPCC) carry germline mutations in DNA mismatch repair genes including MLH1 and MSH2. Individuals with these mutations are predisposed to the development of cancer of the colon, as well as other tissues, especially the endometrium in females. Microsatellite loci mutations occur more frequently in colorectal tumors and other mismatch repair (MMR) deficient cancer cells, presumably because the cells are deficient in MMR. Detection of increased microsatellite instability in a tumor cell provides important diagnostic information relevant to treatment and prognosis. As illustrated in the Examples, amplification of mononucleotide repeat loci having 41 or more repeats and YSTRs provides a sensitive and specific means for evaluating microsatellite instability in mismatch repair deficient tumors. In addition, evaluation of extended mononucleotide repeat amplification products is useful in detecting mutations associated with radiation-induced acute myeloid leukemia.
Cells may be considered to be a putative precancerous or cancer cell if, for example, the cells appear atypical microscopically, in culture or are contained in a polyp or other abnormal mass. Microsatellite stability can be assessed by comparing the amplification products from these cells to amplification products from matched normal cells. Normal cells are cells that are microsatellite stable and do not exhibit any precancerous characteristics, including for example, normal blood lymphocytes or normal intestinal cells.
Briefly, methods for monitoring exposure to a mutagen or for evaluating the mutagenicity of an agent involve amplifying a microsatellite locus in a DNA sample using primers that flank or partially overlap the target sequence in an amplification reaction, suitably, a polymerase chain reaction (PCR). Suitably, the microsatellite loci include mononucleotide repeats, preferably mononucleotide repeat loci having at least 38 repeats, Y STRs, and A-rich pentanucleotide repeat loci (i.e., AAAAG, AAAAT, or AAAAC). The upper limit of the size of the target DNA to be amplified will depend on the efficiency of the amplification method. The size of the target DNA may be selected to reduce length variations due to incomplete copying of the target DNA. Preferably, the target DNA is at most about 1000 base pairs in length.
In the Examples, exposure to a mutagen, the mutagenicity of an agent, or microsatellite instability status of a putative precancerous or cancerous cell or tumor cell is evaluated by comparing the size of an amplification products to the expected size of the amplification product. The expected size of the amplification product can be established, for example, using a suitable control cell. For example, a control cell for mutagenicity studies could be cells obtained prior to exposure to an agent, or unexposed cells that are substantially identical to the exposed cells. A suitable control cell for evaluating microsatellite instability may be a normal, non-cancerous, microsatellite stable cell from the same individual. If a microsatellite locus has a predominant allele in the population (i.e., a monomorphic or quasimonomorphic allele), then the expected size of the amplification product could be the size of the predominant allele in the population. Alternatively, the expected size of the amplification product can be established by pedigree analysis.
In the Examples, the sizes of amplified products were evaluated by capillary electrophoresis. However, sizes of amplified products may be assessed by any suitable means, e.g., sequencing alleles, or by observing increased or decreased expression of reporter proteins in cells containing a DNA construct comprising a reporter gene fused to a DNA repeat such that alterations in the length of the DNA repeat result in a frame shift and loss or gain of reporter gene expression, as described in the Examples.
When performing the methods of the invention, the microsatellite loci may be amplified and analyzed individually, or in combination with other loci as part of a panel. Inclusion of multiple loci in a panel increases the sensitivity of the panel. Suitably, at least four different loci are used in a panel when assessing the microsatellite instability of a putative precancerous or cancerous cell or tumor cell. Preferably, at least five loci are evaluated for microsatellite instability. Multiple loci may be amplified separately or, conveniently, may be amplified together with other loci in a multiplex reaction.
In amplifying a repeat locus according to the methods of the invention, one may use any suitable primer pair, including, for example, those described herein below or those available commercially (e.g., PowerPlex®Y System, Promega Corporation, Madison, Wis.). Alternatively, one may design suitable primer pairs that are adjacent to or which partially overlap each end of the locus to be amplified using available sequence information and software for designing oligonucleotide primers, such as Oligo Primer Analysis Software version 6.86 (National Biosciences, Plymouth, Minn.).
Amplification of DNA containing short tandem repeat (STR) loci (i.e., tandem repeats of mono-, di-, tri-, tetra-, penta-, or hexanucleotide sequences) is associated with a high incidence of PCR products that vary in length due to slippage during amplification rather than because of mutations in those loci. This phenomenon, known as stutter artifact, can make it difficult to determine whether a variation in the size of amplification products is due to stutter or a mutation. The present invention also provides a method of amplifying STR loci that facilitates interpretation of results by allowing one to distinguish between artifactual stutter products and allelic variations. The method employs three primer PCR to generate two partially overlapping PCR products of different sizes, each of which contains the STR. If a mutation (i.e., a deletion or addition) occurred in an STR, both PCR products would show a shift in size of the same magnitude. In contrast, it is unlikely that identical stutter would occur in both amplification products. This method is particularly useful in analyzing mutations in a single cell or a small number of cells, or their DNA equivalent (e.g., small pool PCR). The methods may be used in prenatal or preimplantation diagnostic testing.
A reporter system including a microsatellite locus susceptible to mutation on exposure to mutagens will be constructed. The construct will comprise an expression vector comprising a repeat sequence comprising at least 19 repeats mono-, di-, tri-, tetra-, penta-, or hexanucleotide repeats linked to polynucleotide encoding a detectable reporter marker such that a deletion of one or more base pairs of the repeated sequence alters the expression of the reporter marker in a host cell. The system can be used to evaluate the mutagenicity of an agent by contacting the host cell with the agent and detecting a change in expression of the reporter.
A dual reporter system is described as a prophetic example in the Examples below. The dual reporter system described below includes a 5′ sequence encoding firefly luciferase linked to a 3′ sequence encoding Renilla luciferase through a repeat sequence having at least 19 repeats such that the sequence encoding Renilla luciferase is out-of-frame. A functional Renilla luciferase will be not expressed absent a mutation upstream of the Renilla luciferase coding sequence that restores the reading frame. Downstream of, and in-frame with, the Renilla luciferase coding sequence is a sequence encoding a neomycin resistance marker to permit selection of host cells in which expression of neomycin resistance has been restored through an upstream mutation. To reduce background, the repeat sequence is flanked by a 5′ out-of-frame stop codon and a 3′ in-frame stop codon.
Although the Example below describes a construct having dual detectable markers and further including a selectable marker, it is envisioned that a construct according to the invention may suitably include a sequence encoding any reporter linked to a repeat sequence such that a mutation in the repeat sequence alters (increases or decreases) the expression of the reporter. For example, the construct could include a single reporter and a repeat sequence 3′ of the initiation sequence such that a mutation in the repeat sequence alters expression of the reporter.
A reporter may include any polypeptide having a measurable phenotype. Suitable reporters include, but are not limited to, luminescent proteins (e.g., luciferases), fluorescent proteins (e.g., green fluorescent protein), enzymes that catalyze reactions that produce a detectable effect (e.g. β-galactosidase or β-lactamase). For systems employing two reporters, preferably both reporters can be readily quantified in a single sample.
Two different types of reporters can also be combined. For example, β-galactosidase and firefly luciferase could be combined, and both could be detected in a single sample (Dual-Light® Combined Reporter Gene Assay System from Applied Biosystems). Measuring luminogenic and non-luminogenic reporters has been described in US20050164321A1, which is incorporated by reference.
Reporters could be selected such that a second reporter activates or changes the activity of a first reporter (e.g., Fluorescent Resonance Energy Transfer (FRET) or Bioluminescent Resonance Energy Transfer (BRET).
To reduce false positives, a construct may be designed such that sequences encoding two reporter proteins are separated by a viral peptide insert or linker. When a frameshift mutation occurs, the second reporter is expressed as unfused to the first reporter due to a translational effect or “skip” by the ribosomal machinery.
To facilitate the manufacture or cloning of the reporter construct, selectable markers such as antibiotic resistant markers, fluorescent reporters for use in flow cytometry sorting, or an auxotrophic system (Li et al. (2003) Plant 736-747) may be used.
In a dual reporter system, such as that described in the Examples, a fusion between the second reporter (e.g., Renilla luciferase) and a sequence encoding a toxic substance (e.g., Barnase) can be included to select against anything that already includes frameshifts that would otherwise result in false positives.
The following non-limiting examples are intended to be purely illustrative.
Cell culture and irradiation. Immortalized wildtype mouse MC5 embryonic fibroblast cells derived from C57BL/6 mice were grown in standard cell culture conditions. Exponentially growing cells plated in T-25 tissue culture flasks were irradiated at room temperature with a single dose 1 Gy of 1 or 3 GeV/nucleon 56Fe ions accelerated with the Alternating Gradient Synchrotron (AGS) at the Brookhaven National Laboratory at a rate of 0.5 Gy/min. Cells were grown for 3 days post irradiation to allow recovery, trypsinized, concentrated by centrifugation, and frozen at −80 C.
SupFG1 mice (Leach et al. 1996 Mutagenesis 11(1):49-56) were irradiated with 1 or 3 Gy 56Fe high-LET ionizing radiation using the Alternating Gradient Synchrotron (AGS) at the Brookhaven National Laboratory at a rate of 0.5 Gy/min. The mice were maintained for 10 weeks under standard conditions and diet, and then sacrificed. DNA was isolated from blood using standard procedures.
PCR amplification of microsatellite repeats. Genomic DNA from irradiated or control cells was extracted by standard phenol/chloroform extraction methods and quantified by UV spectrometry and PicoGreen dsDNA Quantitative Kit (Molecular Probes, Eugene, Oreg.) following manufacturer's protocol. Mononucleotide repeats with extended poly-A tracts were identified from BLAST searches of GenBank database. Primers for PCR amplification were designed using Oligo Primer Analysis Software version 6.86 (Molecular Biology Insights, Inc., Cascade, Colo.).
Small pool PCR (SP-PCR) amplification of loci containing extended mononucleotide repeats mBat-24, mBat-26, mBat-30, mBat-37, mBat-59, mBat-64, mBat-66, and mBat-67 was performed using fluorescently labeled primer pairs for each loci (Table 2). PCR reactions were performed by using 6-15 pg of total genomic DNA in a 10 μl reaction mixture containing 1 μl Gold ST*R 10× Buffer (Promega, Madison, Wis.), 0.05 μl AmpliTaq gold DNA polymerase (5 units/μl; Perkin Elmer, Wellesley, Mass.) and 0.1-10 μM each primer. PCR was performed on a PE 9600 Thermal Cycler (Applied Biosystems, Foster City, Calif.) using the following cycling conditions: initial denaturation for 11 min at 95° C. followed by 1 cycle of 1 min at 96° C., 10 cycles of 30 sec at 94° C., ramp 68 sec to 58° C., hold for 30 sec, ramp 50 sec to 70° C., hold for 60 sec, 25 cycles of 30 sec at 90° C., ramp 60 sec to 62° C., hold for 30 sec, ramp 50 sec to 70° C., hold for 60 sec, final extension of 30 min at 60° C. and hold at 4° C. The SP-PCR products were separated and detected by capillary electrophoresis using a Applied Biosystems 3100 Genetic Analyzer and data analyzed using AB GeneScan and Genotyper Software Analysis packages to identify presence of microsatellite mutations.
Mutational Analysis. Mutations were not detected in the mBat-24, 26, 30 or 37 markers in DNA isolated from control cells or cells irradiated with 1 Gy iron ions. In contrast, mutant alleles were found with extended mononucleotide repeat marker mBat-59 in 1% (4/408) of alleles from cells irradiated with 1 Gy iron (
The mutation frequency for mBat-37, 67, 59, 64, and 66 in SupFG1 mice exposed to ionizing radiation was plotted as a function of repeat length (
Cell culture and irradiation. Male human fibroblast cell line #AG01522 from Coriell Cell Repository was grown in DMEM media with 2 mM L-glutamine, 10% fetal bovine serum, 0.5 Units/ml of penicillin, 0.5 μg/ml of streptomycin, and 0.1 mM essential and non-essential amino acids and vitamins (Invitrogen Corporation). Cell cultures were grown at 37° C. and 5% CO2 under sterile conditions. Exponentially growing cells were plated in 25 cm2 tissue culture flasks were irradiated at room temperature with a single dose 0.5, 1 or 3 Gy of 1 GeV/nucleon 56Fe ions accelerated with the Alternating Gradient Synchrotron (AGS) at the Brookhaven National Laboratory at a rate of 0.5 Gy/min. Following irradiation, media was replaced and cells grown for 3 days then collected and frozen at −70° C. until ready for DNA extraction.
Small-pool PCR amplification of microsatellite repeats. Small-pool PCR assays were conducted as described in Example A above using primer pairs (Table 7) to amplify the following microsatellite loci: (1) mononucleotide repeat markers (NR-21, NR-24, BAT-25, BAT-26 and MONO-27); (2) extended mononucleotide repeat markers (hBAT-51d, hBAT-52a, hBAT-53c, hBAT59a, and hBAT-60a); (3) tetranucleotide repeat markers on autosomal chromosomes (D7S3070, D7S3046, D7S1808, D10S1426 and D3S2432); (4) tri-, tetra- and penta-nucleotide repeats on the Y chromosome (DYS391, DYS389 I, DYS389 II, DYS438, DYS437, DYS19, DYS392, DYS393, DYS390, and DYS385); and (5) penta-nucleotide repeats (Penta C and D) (Bacher, J and Schumm, J. Profiles in DNA, 1998 2(2): 3-6; Bacher et al. Disease Markers, 2004 20:237-250.)
Mutational Analysis. Mutations were detected in the microsatellite repeats of DNA isolated from cells irradiated with 0.5 or 3 Gy iron ions. Mononucleotide repeats with polyA runs of up to 36 bp exhibited little or no increase in mutation rates over controls. Similarly, tetranucleotide repeats on autosomal chromosomes that are sensitive to MSI did not exhibit any evidence of radiation-induced mutations. In contrast, extended mononucleotide repeats with polyA runs of 38 bp or more (
Dose-response curves. A linear dose response was observed for microsatellite markers tested on the Y chromosome and extended mononucleotide repeat markers. Normal human fibroblast cells AG01522 were irradiated with 0, 0.5, 1 or 3 Gy iron ions and the combined mutation frequency of 13 microsatellite markers on the Y chromosome were determined by SP-PCR and plotted (
Identification of microsatellite loci and primers. Previously uncharacterized mouse or human mononucleotide repeats were identified through analysis of sequences found in National Center of Biotechnology Information public DNA sequence databases using BLASTN searches for repetitive sequences (Tables 1A-1D). Primers for microsatellite markers were designed with Oligo Primer Analysis software (National Biosciences, Plymouth, Minn.).
Treatment of mice used in paraquat studies. C75BL/6 mice were housed and inbred at the University of Wisconsin, with an average life span 30 months. Four groups of three mice were included in the study: 5-month old mice (young control or YC); 5-month old mice treated with paraquat; 24-month old mice (old age); and 24-month old mice treated with paraquat. Paraquat-treated animals received a single intraperitoneal injection of 50 mg/kg body weight dissolved in PBS 24 hours after their last feeding. Each mouse was sacrificed by cervical dislocation.
Tissue Preparation, DNA Extraction and Quantification. The entire liver from each mouse was dissected, washed with PBS, placed in a 1.5 ml Ependorf tube, snap frozen in liquid nitrogen, and stored at −80° C. DNA was prepared from the mouse liver tissue by using the DNA-IQ Tissue and Hair Extraction Kit (Promega Corporation, Madison, Wis.) and was quantified using the PicoGreen dsDNA Quantitative Kit (Molecular Probes, Eugene, Oreg.) following manufacturers protocols.
mtDNA Deletion Detection. Based on the mouse mtDNA sequence, four fluorescence labeled primer pairs were designed to detect both wildtype sequences and mtDNA deletions. The primer sequences, fluorescent labels, and position are shown in Table 3.
Detection of Mutations in Microsatellites using small pool PCR. Mutations were detected by amplifying loci containing mononucleotide repeats of different lengths using fluorescent labeled primers pairs (Table 2) in multiple replicates of small pool PCR (SP-PCR). The stability of four short mononucleotide repeats (mBAT-24, mBAT-26, mBat-30, mBAT-37) and three extended mononucleotide repeats (mBAT-59, mBAT-64 and mBAT-67) were evaluated.
PCR Conditions. For mtDNA deletion detection, PCR amplification was performed by using 1 ng of total genomic DNA in a 10 μl reaction mixture containing 1 μl Gold ST*R 10× Buffer (Promega, Madison, Wis.), 0.05 μl AmpliTaq gold DNA polymerase (5 units/μl; Perkin Elmer, Wellesley, Mass.) and 0.5 μM mixed primers. PCR was performed on a PE 9600 Thermal Cycler (Applied Biosystems, Foster City, Calif.) using the following cycling conditions: initial denaturation for 11 min at 95° C. followed by 1 cycle of 1 min at 96° C., 10 cycles of 30 sec at 94° C., ramp 68 sec to 62° C., hold for 30 sec, hold for 30 sec, ramp 50 sec to 70° C., hold for 60 sec, 25 cycles of 30 sec at 90° C., ramp 60 sec to 62° C., hold for 30 sec, ramp 50 sec to 70° C., hold for 60 sec, final extension of 30 min at 60° C. and hold at 4° C.
SP-PCR was performed for mutation analysis using 1-2 copies of genomic DNA (6-12 pg). PCR cycles and conditions are the same as described above except that the annealing temperature was 58° C.
Identification of PCR Products. Separation and detection of amplified fragments was performed on an ABI PRISM® 3100 Genetic Analyzer following the manufacturer's protocol (Applied Biosystems, Foster City, Calif.). Data was analyzed with the GeneScan and Genotyper computer software packages (Applied Biosystems).
Statistical Analysis. Statistical analysis was done using the statistical Package Sigma Stat version 3, where P value was determined by using one-way ANOVA for each specific group, using Holm-Sidak method did comparisons between the groups.
Δ-mtDNA4977 detected in old age or paraquat treated mice. Wild type mtDNA is detected by amplification used primers 1, 3, and 4 to yield fragments of 465 bp, 130 bp and 98 bp, respectively. Primer 2 was designed to amplify deleted mtDNA fragments, resulting in a PCR product of 620 bp. No deletion was detected in any of three mice in the young control group (5-month-old mice), whereas one of the three mice in the old age non-paraquat-treated group (25-month-old) showed Δ-mtDNA4977. All three mice in the old age paraquat-treated group showed Δ-mtDNA4977 (Table 4).
Detection of Mutations by PCR. Analysis of amplification products obtained by PCR amplification of loci mBAT-24, mBAT-26, mBat-30, or mBAT-37 detected no mutations out of 1608 alleles in old paraquat-treated group (Table 5). In contrast, analysis of amplification products obtained by SP-PCR amplification of loci mBAT-59, mBAT-64, mBAT-66 or mBAT-67 showed that the paraquat-treated old age group exhibited mutations in over 1.8% (25/1649) of mononucleotide repeat alleles assayed (Table 5,
The use of multiple SP-PCR replicates allows detection of mutant alleles that occur less frequently than wild type alleles. The results indicate that extended mononucleotide repeats are more susceptible to mutations in response to oxidative stress than are shorter mononucleotide repeats. Mice exposed to oxidative stress exhibited mutations only in mononucleotide repeats with polyA tracts of 38 bp or more (
Cell culture. Male human fibroblast cell line #AG01522 from Coriell Cell Repository were cultured in MEM Eagle-Earle BSS 2× concentration of essential and non-essential amino acids and vitamins with 2 mM L-glutamine, 10% fetal bovine serum, 0.5 Units/ml of penicillin, 0.5 μg/ml of streptomycin. Cell cultures were grown at 37° C. and 5% CO2 under sterile conditions and split at a ratio of 1:5 when cells were confluent by releasing cells with trypsin-EDTA treatment. Cells were treated with 0.0 uM (PBS), 0.1 mM, 0.4 mM, 0.8 mM or 1.2 mM hydrogen peroxide diluted in PBS for 1 hour at the same culture conditions described. After treatment, media with hydrogen peroxide was replaced with fresh media and allowed to recover for 3 days. Cells were pelleted and DNA extracted.
Mutation Detection. Mutant alleles were identified by small-pool PCR as described in Example B above using primer pairs specific for microsatellite markers (Tables 2 and 7) including: (1) mononucleotide repeat markers (NR-21, NR-24, BAT-25, BAT-26 and MONO-27); (2) extended mononucleotide repeat markers (hBAT-51d, hBAT-52a, hBAT-53c, hBAT59a, hBAT-60a and hBAT-62); (3) tetranucleotide repeat markers on autosomal chromosomes (D7S3070, D7S3046, D7S1808, D10S1426 and D3S2432); (4) tri-, tetra- and penta-nucleotide repeats on the Y chromosome (DYS391, DYS389 I, DYS389 II, DY8438, DYS437, DYS19, DYS392, DYS393, DYS390, and DYS385); and (5) penta-nucleotide repeats Penta B, C, D, and E (Bacher, et al. 1999. Proceedings from the Ninth International Symposium on Human Identification 1998; and Bacher, et al. Proceedings from the 18th International Congress on Forensic Haemogenetics. 1999).
Mutational Analysis of human cultured cells following oxidative stress. Mutations were detected in the extended mononucleotide repeats, Y-STRs and A-rich pentanucleotide repeats in DNA isolated from cells exposed to 0.1 to 1.2 mM hydrogen peroxide (
Isolation of DNA from tumor and matching normal tissue samples. C57BL/6 M1h1-deficient mice and B6 Msh2-deficient mice were sacrificed by CO2 asphyxiation. The entire intestinal tract were removed and washed with 1×PBS. Tumors and adjacent normal tissue was removed and snap frozen in liquid nitrogen. DNA was prepared from each sample using DNA IQ chemistry (Promega Corp., Madison, Wis.). In addition, DNA was extracted from leukemia cell lines from C3H mice in which acute myeloid leukemia (AML) had been induced by a whole-body dose of radiation (Pazzaglia et al. 2000 Molecular Carcinogenesis 27(3):219-228).
Detection of microsatellite instability. PCR amplification of loci containing extended mononucleotide repeats mBat-24, mBat-26, mBat-30, mBat-37, mBat-64, mBat-59, or mBat-67 was performed using primer pairs for each loci (Table 2). Amplification of mononucleotide repeats was performed using fluorescently labeled primers in 10 μl PCR reactions containing: 1 μl GoldST*R 10× Buffer (Promega, Madison, Wis.), 0.1-1 μM each primers, 0.05 μl AmpliTaq Gold DNA Polymerase (5 Units/μl; Perkin Elmer, Wellesley, Mass.) per locus and 1-2 ng DNA. PCR was performed on PE 9600 Thermal Cycler (Applied Biosystems, Foster City, Calif.) using the following cycling profile: 1 cycle 95° C. for 11 minutes; 1 cycle 96° C. for 1 minute; 10 cycles 94° C. for 30 seconds, ramp 68 seconds to 58° C., hold for 30 seconds, ramp 50 seconds to 70° C., hold for 60 seconds; 20 cycles at 90° C. for 30 seconds, ramp 60 seconds to 58° C., hold for 30 seconds, ramp 50 seconds to 70° C., hold for 60 seconds; 60° C. for 30 minutes final extension; 4° C. hold. For single template PCR, DNA was diluted to 6-12 pg (1-2 genome equivalents) based on quantification with Picogreen dsDNA Quantitative Kit (Molecular Probes, Eugene, Oreg.) following manufacturer's protocol and confirmed by serial dilution of DNA until PCR failure rates reached 30-50%. PCR amplification was the same as that outlined above except that the number of cycles was increased to a total of 35 cycles.
Separation and detection of amplified fragments was performed on an ABI PRISM® 3100 Genetic Analyzer following the manufacturer's protocol (Applied Biosystems, Foster City, Calif.). Data was analyzed with GeneScan Analysis and Genotyper Software packages from Applied Biosystems to identify predominate allele sizes for each locus. Allelic patterns or genotypes for normal and tumor pairs were compared and scored as MSI-positive if the tumor DNA samples contained one or more alleles not found in normal samples from the same mouse.
The classification of microsatellite instability was based on guidelines suggested by a National Cancer Institute workshop (Boland et al. (1998) Cancer Research 58:5248-5257; Umar et al. (2004) J Natl Cancer Inst 96:261-268, each of which is incorporated by reference herein). Using the Bethesda panel of five microsatellite repeats, tumor samples with 40% MSI were classified as MSI-high (MSI-H), less than 40% as MSI-low (MSI-L), and no alterations were classified as microsatellite-stable (MSS). If more than five markers are to be used, MSI-H group was defined as tumors having MSI at 30% or more of the markers tested, whereas the MSI-L tumors exhibit MSI in 1-29% of the markers.
DNA was isolated from numerous colon tumor samples and matching normal tissues using standard methods. The DNA was amplified using primers specific for extended mononucleotide repeat markers (Table 1C) in PCR amplification as described above. The sizes of amplification products for the colon tumor cells were determined and compared with those of the matching normal tissues. New alleles found in tumor samples that were not present in matching normal samples indicted microsatellite instability. The data for two tested extended mononucleotide repeat markers (hBAT-54 and hBAT-60) are presented in
The ability to distinguish mutations from stutter artifacts is particularly important in genotyping and/or mutational analysis with microsatellite markers (1-6 bp tandem repeats) on single cells or small pools of cells, or their DNA equivalent. The method overcomes a major problem associated with microsatellite analysis with very low amounts of template DNA. During amplification of microsatellite loci, stutter molecules, repeat slippage products formed during PCR, are generated. When formed during the first few PCR cycles, stutter molecules can outnumber the original template molecule(s). The formation of stutter molecules interferes with the ability to distinguish between stutter products and true alleles, thereby confounding interpretation of the data.
The method relies on coamplification of overlapping amplicons using three primers as illustrated schematically in
In order to facilitate analysis of amplified target DNA comprising a microsatellite loci from a single cell or using small pool PCR, primers are designed such that the a third primer hybridizes to a region between the members of a primer pair so that two partially overlapping products are formed, each of which contains the repeat locus (
DNA was obtained from mouse embryonic fibroblasts exposed to either 0 Gy or 0.5 Gy iron ions and analyzed for mutations in mBat-26 microsatellite marker using three primers: TCACCATCCATTGCACAGTT (SEQ ID NO:153) labeled with JOE; OH attCTGCGAGAAGGTACTCACCC (SEQ ID NO:167); and OH attACTAGAATCGTACATTGTCCAAAA (SEQ ID NO:168) as shown generally in
In order to provide a reporter system for detecting mutations in response to mutagens, a dual reporter construct will be developed. The construct will include a polynucleotide sequence encoding two different luciferases. Specifically, the construct will contain a first luciferase linked to a second luciferase by an intervening sequence that includes a microsatellite repeat locus having repeats of from 1-6 bases repeated at least 19 times. Preferably, the overall length of the intervening sequence is from about 19 to about 101 bases. The second luciferase will be expressed only if there is a mutation in the intervening sequence that causes the sequence of the second luciferase to be in the proper reading frame. In one embodiment, the construct is represented schematically as follows:
Luciferase (1)-repeat sequence (frame shift)-Luciferase (2)
Luciferase (1) will be constituitively expressed, and Luciferase (2) would be expressed only if a frame shift occurred in the repeat sequence. The ratio of Luciferase (1) to Luciferase (2) expressed would minimize other sources of variation in gene expression and cell viability.
The construct will ideally be designed with a sequence encoding a selectable marker such as an antibiotic resistance marker (e.g., neomycin) fused in-frame to the Luciferase (2). For example, a firefly luciferase (Ffluc) coding sequence can be linked to a sequence encoding Renilla luciferase (Rluc) and a neomycin resistance marker downstream of the Rluc coding sequence, as shown below:
5′-FFluc-repeat sequence (frame shift)-Rluc/neo-3′
To reduce background caused by frame shifts in other regions of the sequence 5′ to repeat sequence, appropriate translation stops will be placed on each side of the repeat sequences as follows:
FFluc-(out-of-frame stop) repeat sequence (in-frame stop)-(frame shift)-Rluc/neo
The construct will be ligated to a suitable vector, preferably an episomal vector having a high copy number. A high copy number vector will enhance the sensitivity of dection by amplifying any mutation that occurs through replication of the episomal vector, thus increasing the rate at which the mutation accumulates. The episomal vector will be capable of replicating in both bacteria (e.g., Eschericia coli) and in mammalian cell lines. Episomal vectors afford simplified clonal purification. Episomal vector systems for mammalian cells have been previous described (Craenenbroeck et al (2000) Eur. J. Biochem. 267:5665-5678; and Conese et al (2004) Gene Therapy 11:1735-1741, each of which is incorporate by reference).
The construct thus produced will be introduced into a cell line or organism will be used as a cellular or in vivo assay for determining the mutagenicity of chemical or biological substances in a manner similar to the Ames test (Ames et al. Science 1972 176:47-49) or Stratagene's Big Blue Mouse (Short et al. Fed. Proc. 1988 8515a; Kohler et al., Proc. Natl. Acad. Sci. USA 1991 88: 7958-7962; and Jakubczak et al., Proc. Natl. Acad. Sci. USA 1996 93: 9073-9078).
Cells containing this reporter vector will be exposed to a mutagen resulting in deletions or insertions in the repeat region and restoration of the reading frame. Subsequent expression of the luciferase coding sequence will increase light signal in a luminescence assay and will be compared to unexposed controls to determine rate of mutation induction.
Each publication or patent application cited herein is incorporated by reference in its entirety.
1Mean Shift is the average change in size in tumor alleles, excluding zeros.
2Sensitivity is the percent of tumors that displayed instability in a particular marker.
This application claims priority to U.S. Provisional Application No. 60/621,277, filed Oct. 22, 2004, to U.S. Provisional Application No. 60/661,646, filed Mar. 14, 2005, and to U.S. Provisional Application No. 60/697,778, filed Jul. 8, 2005, each of which is incorporated by reference, and is being filed simultaneously with an application entitled “Methods and Kits for Detecting Germ Cell Genomic Instability”, filed Oct. 24, 2005 under the Patent Cooperation Treaty, which is incorporated by reference.
This invention was made with Government support under grant ______ awarded by the NASA. The United States Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/38433 | 10/24/2005 | WO | 00 | 11/13/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60621277 | Oct 2004 | US | |
| 60661646 | Mar 2005 | US | |
| 60697778 | Jul 2005 | US |
