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Using Tiling Arrays to Diagnose Drug Resistance in Clinical Isolates of Gonorrhea - Essay Example

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"Using Tiling Arrays to Diagnose Drug Resistance in Clinical Isolates of Gonorrhea" paper introduces a novel method of diagnosis of drug resistance in clinical isolates of gonorrhea. The method involves the use of a Tiling array for the diagnosis of drug resistance in isolates of Neisseria gonorrhea. …
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Using Tiling Arrays to Diagnose Drug Resistance in Clinical Isolates of Gonorrhea
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APPLIED MOLECULAR BIOLOGY: OUTLINE MODULE PROJECT PROJECT USING TILING ARRAYS TO DIAGNOSE DRUG RESISTANCE IN CLINICAL ISOLATES OF GONORRHEA SUMMARY OF PROJECT One of the major global burdens of sexually transmitted diseases is gonorrhea, second only to Chlamydia.106 million gonococcal infections have been reported annually, making it a disease that demands immediate attention and treatment. However, during the recent years frequent incidences of treatment failures have been reported for this disease, causing rising concern to health care providers and researchers (Blomquist, et al., 2014). The causal organism Neisseria gonorrhoeae has been reported to exhibit resistance to first line therapeutic drugs and has even shown ability to develop resistance to high spectrum cephalosporin, ceftriaxone; making the most recent entries in the list of superbugs. (Unemo & Nicholas, 2012). The developing multi drug resistant Neisseria gonorrhoeae, the severe symptoms of the gonococcal infections, added to the socioeconomic burden and epidemiologically threatening aspects of the disease; drug resistance gonorrhea, its detection and diagnosis has acquired immense significance (Blomquist, et al., 2014). It is important to focus on preventing the spread of resistant forms as part of disease management. An essential requirement for controlling spread is enhancing surveillance through better diagnostic methods for identification and isolation of disease resistant pathogens. This project aims to introduce a novel method of diagnosis of drug resistance in clinical isolates of gonorrhea. The method presented in this paper involves the use of Tiling array for diagnosis of drug resistance in clinical isolates of Neisseria gonorrhoeae. The protocol is based on similar technology already used for development of protocols for drug resistance in other organisms. THE NOVEL ELEMENT IS TILING ARRAY Tiling array is a derivation of microarray technology developed by Kapranov and colleagues (2002) and Shoemaker and colleagues (2001) that facilitates identification of previously unidentified transcripts through genome wide annotations. ANY PROBLEMS I AM ENCOUNTERING None USING TILING ARRAYS TO DIAGNOSE DRUG RESISTANCE IN CLINICAL ISOLATES OF GONORRHEA The initial euphoria associated with the utility and significance of antibacterial and antimicrobial drugs seems to fade with the rise in the bacterial strains exhibiting resistance to single as well as multiple drugs. Hence drug resistance has become an exponentially rising global health hazard rendering world population once again vulnerable to the threats of common diseases (Levy & Marshall, 2004). Both Gram positive and Gram negative bacteria have been known to exhibit multi-drug resistance leaving health care providers with no antimicrobial therapeutic agents ensuring control and management of these diseases. The list of drug resistance bacteria includes Staphylococcus aureus, Enterococcus spp., many of the Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp etc (Magiorakos et al., 2012). Drug Resistant Gonorrhea Gonorrhea has one of the longest histories among the diseases, being first reported by Albert Neisser in 1879, and was the second bacteria to be isolated. In males the disease causes symptomatic urethritis followed by epididymitis and urethral stricture. Cervicitis is the most common symptom among women that are further complicated by pelvic inflammatory disease, ectopic pregnancies, and infertility. The causal organism Neisseria gonorrhoeae, has laso been reported to cause conjunctivitis, endocarditis, tenosynovitis, arthritis, meningitis, inflammation of the liver (Fitzhugh-Curtis syndrome) and ophthalmic infection among infants (Kent et al., 2005). Diagnosis and treatment of gonococcal infections has been possible from historical times. Diagnosis was initially done by Gram staining, and cultures (commonly on Thayer-Martin media) earlier, along with clinical signs and symptoms of cervicitis/urethritis. Later molecular techniques developed and nucleic acid tests became more prevalent. However unlike the culture techniques, the molecular methods fail to test antimicrobial susceptibility of the bacteria (Barry & Klausner, 2009). For treatment on the other hand a single dose therapeutic agent was found to exhibit efficacy in 95% of the cases. These agents included sulfonamides, penicillin, tetracyclines and fluoroquinolones. But gradually the various strains of Neisseria gonorrhoeae were found to develop resistance for each of these antimicrobial drugs. Finally the only drug the health care providers found to remain effective against the bacterial was third-generation cephalosporins (Barry & Klausner, 2009). However, as early as 2000, cases where in the third generation cephalosporins had failed to treat the disease were reported from Japan. Cefixime ceased to be used for first line therapy in Japan by the year 2006; the therapeutic agents being used now included only ceftriaxone and spectinomycin. Third generation cephalosporin resistant Neisseria gonorrhoeae became prevalent in regions of Asia and Australia by the year 2009 (Barry & Klausner, 2009). The year 2011 witnessed the first reporting of Neisseria gonorrhoeae strain (H041), a strain found to be extremely resistant extended-spectrum cephalosporin (ESC) ceftriaxone, which till then was considered to be the final successful method of first line treatment. Two mechanisms have been reported for the development of drug resistance in the bacteria. Neisseria gonorrhoeae. The first mechanism is development of altered penicillin-binding proteins (PBPs). The pathogen has three PBPs (1, 2 and 3), of which PBP2 has the forms the primary binding site for β-lactam antimicrobials such as cephalosporins. Any variations in this protein which is coded by penA gene has led to altered capacity of binding antimicrobials. The second mechanism responsible for development of resistant strains is lowering of intracellular concentration of antimicrobial agents. This is accomplished either by prohibiting the antimicrobials to enter the bacterial cells or by actively pumping out the same. The pathogen in this case has a system of efflux pumps, of which MtrC-D-E system of pumps has been reported to actively pump out penicillin, tetracycline, macrolides etc and thereby confer resistance to these antibiotics. The pump is controlled mtrR gene, hence a mutation in this gene cause a lack of repression of the pump. Whether this pump is involved in imparting resistance to cephalosporins was not known, but there could be the involvement of this or a similar pump in reducing concentration of cephalosporins in the cells of pathogen, thereby making it resistant to the drug (Lindberg et al., 2007). In a recent paper Golparian and colleagues (2014), describe the presence of three multidrug efflux pumps (MtrC-MtrD-MtrE, MacA-MacB, and NorM); that are responsible for development of multidrug resistant strains. In the light of the above information, it is obvious that strong surveillance system armed with fast and accurate diagnostic techniques for the identification and isolation of drug resistant Neisseria gonorrhoeae is essentially and urgently required. Molecular methods capable of identifying molecular markers of resistance need to be developed. This project is an initiative in this direction. Tiling Array Figure 1: Whole Genome Tiling Array, a suitable technique for genomic data (Mockler & Ecker, 2004) The science of genomics facilitates researchers with the list of genes that encode the entire living organism, thereby enabling a precise system for understanding and categorizing the organism (Lander, 1999). One of the most significant tools of genomics is DNA microarrays that enable measurement of gene expression levels, and identification of single nucleotide polymorphisms (SNPs). In very simple terms, DNA microarray is a set of microscopic components (usually DNA), which can be subjected to probes made of target molecules, and can be utilized for quantitative estimation that is gene expression, and for producing qualitative such as diagnostic data. Beginning with much simpler techniques such as Southern blot and Dot blot involving radioactive macroarrays, the technique has come a long way to two dimensional and three dimensional microarrays (Miller and Tang. 2009). The principle of the technology is based on the central dogma molecular inheritance. DNA coding all information within the cell is copied in to mRNA by the process of transcription; this mRNA transcript is then translated in to proteins. The level of mRNA in the cell at a specific time indicates the expression levels of the cellular genome and the complete set of transcripts present within the cell at a specific developmental stage is referred to as the transcriptome. Using a reference transcriptome, the level of transcription of a specific gene can be assayed at different stages of cell. The technique has enabled identification of gene expression in non coding and pseudogene areas of the genome and has even facilitated understanding of the certain aspects of metabolism in organisms such as E.coli. Further, changes in functional behavior of a gene such as repression, induction etc in response to environmental conditions, presence of specific drugs, or during certain phases of cell cycle has also been detected for example identification of genes related to oxidative stress, cell multiplication in Yersinia pestis, Salmonella enteric etc (Akama et al. 2009; Wiltgen & Tilz, 2007). During the last two decades, development in the field of bioinformatics and robotics has lead to exponential rise in the efficiency, sensitivity, reproducibility and specificity of the DNA Microarray technique (Miller & Tang, 2009). Tiling array is a derivation of microarray technology that facilitates identification of previously unidentified transcripts through genome wide annotations. Designed by Kapranov and colleagues (2002) and Shoemaker and colleagues (2001), using probes derived from sequences of human chromosomes 21 and 22; the technique enabled identification of yet unidentified active gene expression sites. Similar method was developed by Yamada and colleagues (2003) for study of gene expression of Arabidopsis genome using probes spanning the entire genome of the plant. The probes used in this technique are complementary to sequences present in complete genome, irrespective of whether they contain genes or not. Thus probes designed for tiling arrays target contiguous regions of the genome. Further, since both of the DNA strands are capable of transcription, the tiling array uses probes for both of the DNA strands; one chip with probes for one strand, and another one for that of second strand. Thus tiling arrays utilize two chips. The method has been proved to identify novel sites where active transcription is occurring but which remains concealed. The method has also found application in studies investigating alternative splice sites, transcription factor binding sites and methylation of genes, and for comparative hybridization of genomes (Yazaki et al., 2007). Figure 2: Tiling Array Protocols (Mockler & Ecker, 2004) Tilling array has been used for identification of proteins and genes responsible for resistance to specific drugs in various pathogenic microbes. Protocols have been developed for the same by researchers for the use of this technique to enable better understanding of the mechanism of disease resistance developed by pathogens. Manjunatha and colleagues (2005) describe the use of tiling array for study of mechanism of resistance to PA-824, a novel compound that was exhibiting sound prospects as an agent for treatment of tuberculosis. PA-824, a compound belonging to the class of nitoimidazole, showed antitubercular impact only after bioreductive activation of one of the aromatic nitro groups. This process of reduction is due to electrons facilitated by a specific glucose 6 phosphate dehydrogenase (FGD1) or its deazaflavin cofactor F420. Loss of any of these two molecules results in resistance to the prodrug PA-824, however, additional factors were also involved in conferring resistance; since these molecules do not exhibit direct interaction with the PA-824. Using comparative microarray of entire genomes of sensitive mutants resulted in detection of lesions in Rv3547, which is a conserved protein without any known function. Where conventional techniques of genomic studies failed to identify the gene responsible for sensitivity to the drug; the technique of comparative genome sequencing (CGS) developed by NimbleGen Systems was developed and found to be highly applicable. The method involves two phases of genetic mapping with the first phase being a mutational analysis spanning the entire genome using hybridization of reference strain DNA probe. During the second phase SNPs exhibiting higher probability of impact were reassayed through customized microarrays. The technique has been found to exhibit great efficiency for identification of SNPs and holds high potential as specific gene identification technique from entire cell genome. Tiling array has also been used for study of entire genome of Mycobacterium leprae, which is made of 3.3Mbp. More than half of its genome comprises of non coding sequences and pseudogenes, the expression of some of which changes post infection. A tiling array of the entire genome revealed expression of pseudogenes and non-coding sequences along with genes. Further the expression intensities of non coding regions is higher than that of pseudogenes. The functional significance of the RNA transcribed by pseudogenes and non coding regions is not yet known, but it definitely provides an impetus for further researches in the functional genomics of the pathogen (Akama et al. 2009). During the last few years there has been a rapid rise in applications of DNA microarray and related technologies. They are increasingly being used in the field of health and medicine for diagnosis of infectious diseases and in researches related to defense against pathogens that are increasingly becoming resistant to common therapeutic agents due to mutations in microbial genome. High density DNA probe arrays have been used for identification of gene sequences responsible for drug resistance for diseases since long. Multidrug resistant tuberculosis has become a worldwide menace during the last few years. Phenotypic antimycobacterial procedures used earlier were found to be highly time consuming. Protocols based on DNA microarrays were developed therefore, for determination of drug resistance in antituberculosis. Troesch and colleagues (1999) used the technique for identification of species of Mycobacterium resistant to rifampicin. Methods have been developed for detection of gene mutations responsible for isoniazid and rifampicin resistance in Mycobacterium tuberculosis using non-fluorescent low density DNA microarrays (Aragon et al., 2006). The novel method was cheaper and faster and involved the use of LCD chips. These chips were prestructured polymer supports that carried a non fluorescent detecting agent. The method gave results concurrent with the previously sequenced data. Methods have also been developed for detection of strains resistant to other antimicrobial drugs such as streptomycin, kanamycin, pyrazinamide, thambutol etc. A novel protocol for detection and characterization of genetic mutations credited with fluoroquinolones resistance has been developed by Antonova and colleagues (2008). Another method combining PCR amplification and suspension bead array, QIAplex system has been developed for the identification of genes conferring resistance to rifampicin, isoniazid, streptomycin, and ethambutol (Gegia et al., 2008). Figure 3: Bacterial transcriptome analyses using genomic tiling array (Aikawa et al., 2010) Clinical diagnosis of antimicrobial resistance presents several challenges that can be dealt with DNA microarrays only among the available technologies. One of the biggest challenges is continuous mutations in genomes of pathogen as a consequence of selection pressure. For instance for HIV-1; an Affymetrix microarray has been developed for profiling of antiretroviral drug resistance conferring genes. An advanced version of the protocol is now being used by the company for identification of rapidly appearing mutations in the viral genome. The method involves the use of high density DNA microarrays. It must be remembered that a foolproof and complete protocol for identifying all mutations causing resistance and the application of the developed technology to clinical diagnostic procedures would require years of focused research. Meanwhile, phenotypic identification must be relied upon as primary procedure for the purpose (Miller and Tang, 2009). Efforts are also underway to develop molecular markers for antimalarial drug resistance in falciparum malaria. An integrated approach involving in vivo trials, in vitro assays and molecular markers have been utilized for study of mechanisms of drug resistance in malaria (Abdul-Ghani et al., 2014). Protocol involving tiling array for diagnosis of drug resistance in clinical isolates of Gonorrhea Neisseria gonnorrhoeae, an obligate human pathogen exhibits multidrug resistance and hence extensive genomic studies have been conducted on the pathogen to identify and characterize genes responsible for resistance, and to understand the significance of horizontal gene transfer in the organism. The efforts lead to entire annotated gene sequence of the organism N. gonorrhoeae strain NCCP11945. It contains single circular chromosome of 2,232,025 bp; along with one plasmid of 4153 bp. Further the genome comprises of multiple repetitive sequences with the most abundant sequence (5’-GCCGTCTGAA-3’) having 1,966 copies and the next most abundant repetitive sequence element occurring 123 copies (Chung et al., 2008). Tiling array has recently been used for identifying post selection mutations in Plasmodium falciparum recently. The protocol used can be suitably applied for identification of genes conferring resistance in clinical isolates of N. gonnorrhoeae as well, as both organisms have small genomes and single chromosome thus enabling easy sequencing. The protocol for falciparum bacteria was based on rodent malarial parasite genome studies that utilized Illumina Genome Analyzer with 36bp single reads and 50bp paired end reads to sequence bacterial lines that have been subjected to various drugs. Further probable mutation sites were identified prior to sequencing using LGS or conventional method of linkage mapping. Thus point mutations could be identified in these studies (Anderson et al., 2011). The novel protocol presented in this dissertation involves the use of Tiling Microarray technology that can be used to detect NPs and CNVs by hybridization methods from the entire genome of N. gonorrohoeae. A high density microarray will first be designed with multiple probes covering approximately 90% of the bacterial genome coding genome. The high density of the probes ensures that they are overlapping and have high probability of detecting polymorphism. Sample Isolation and Preparation Determination of Susceptibility: The determination of antibiotic susceptibility of the target strain will be done by traditional agar plate dilution method followed by categorization of the available population into three categories: susceptible, incompletely susceptible and highly resistant. Sample DNA Preparation: A lytic solution containing EDTA, NaOH, proteinase, Triton X-100 will be used for DNA extraction from the 3 categories of bacterial strains. This would be followed by centrifugation to isolate DNA. Peltir Thermal Cycle 225 instrument can be used for centrifugation. The supernatant after centrifugation is thereafter isolated for amplification by PCR. The isolatd DNA will be fragmented using DNaseI and end-labelled with biotin. The samples obtained will be hybridized to microarrays and left overnight in Affymetrix buffers. Primers for PCR are designed for the entire genome of N. gonorrohoeae, on the basis of sequences obtained from the Genbank Database. PCR will be done using a thermal cycler (PCR express. Both directions of the PCR amplicons are then sequenced using ABI PRISM dye terminator cycle sequence ready reaction kit and the sequences will next be analyzed using Sequence Navigator software (both from Perkin-Elmer, Applied Biosystems). Tiling Microarray Background control probes are used for background subtraction in each microarray using Affymetrix background control probes. It is essential to normalize the tiling arrays to a synthetic baseline array obtained by calculating the mean GC content of all probes utilized in the process. Log transformed probes are then arranged in bins with the means of each of the bins correlated with that of the adjacent ones, and the outliers will be excluded. Probes selected for use in microarrays are only those that are exhibit only one perfect match to the reference genome, the rest will be excluded. A comparative analysis of the various metabolites synthesized after treatment with specific drugs would also be required to understand the impact of drug on pathogen metabolism. A variation in the intensities of hybridization of genomic sequences to the probes is indicative of appearance of SNPs. Next the copy number of genes and their transcript levels will be obtained in a comparative analysis of resistant and non resistant or wild type strains using Real time PCR and Real time RT-PCR. The technique will thus be used for the detection of both copy number variations (gene amplification) and SNPs. Position of SNPs can be detected by loss of hybridization at the probable position of SNPs in the probes. Limitations and Strengths of the Novel Protocol Tiling microarray is an efficient technology to identify non-coding and unidentified and undetected sequences in the entire genome of the organism. CNVs which are the most probable mechanisms of development of disease resistance usually remain undetected by traditional sequencing protocols. Added to statistical methods tiling array technique can yield detailed and accurate information allowing identification of SNPs, CNVs and even intergenic sequences using hundreds of probes simultaneously. This is also the weakness of the technique. Further insertions and deletions cannot be as accurately reported by tiling array as by traditional sequencing methods. Conclusion Appearance of multidrug resistant N. gonorrhoeae has made it imperative to diagnose and clinical isolate resistant strains of the pathogen prior to its spread. Traditional methods to identify and isolate resistant strains are both slow and throw no light on the mechanism of development of resistance. Further sequencing methods are not capable of identifying gene sequences responsible for resistance since most mutations responsible for resistance involve the SNPs and the CNVs; that are the non coding and intergenic sequences. Tiling Microarray on the other hand enable study of the entire genome of the organism, hence are suitable technique for identification of genes responsible for resistance. Besides being fast and efficient, this method also enables the gradual development of understanding of mechanism of resistance. References 1. Abdul-Ghani, R., Al-Maktari, M. T., Al-Shibani, L. A. & Allam, A. F., 2014. A better resolution for integrating methods for monitoring Plasmodium falciparum resistance to antimalarial drugs. Actatropica. 2. Aikawa, C., Maruyama, F. & Nakagawa, I., 2010. The dawning era of comprehensive transcriptome analysis in cellular microbiology. Frontiers in Microbiology. 3. Akama, T. et al., 2009. Whole-genome tilng array analysis of Mycobacterium leprae RNA reveals igh expression of pseudogenes and non coding regions. Journal of bacteriology, pp. 3321-7. 4. Anderson, T., Nkhoma, S., Ecker, A. & Fidock, D., 2011. How can we identify parasite genes that undrlie antimalarial drug resistance?. Pharmacogenomics, pp. 59-85. 5. Antonova, O. V. et al., 2008. Detecton of mutations in Mycobacterium tuberculosis genome determining resistance to fluoroquinolones by hybridization on biological microchips. Bulletin of experimental biology medicine, pp. 108-13. 6. Aragon, L. M. et al., 2006. Rapid detection of specific gene mutations associated with isoniazid or rifampicin reistance in Mycobaterium tuberculosis clinical isolates using non-fluorescent low density DNA micrarrays. Journal of antimicrobial chmotherapy, pp. 825-31. 7. Barry, P. M. & Klausner, J. D., 2009. The use of cephalosporins for gonorrhea: the impending problem of resistance. Expert Opinion on Pharmacotherapy, pp. 555-77. 8. Blomquist, P. B., Miari, V. F., Biddulph, J. P. & Charalambous, B. M., 2014. Is gonorrhea becoming untreatable?. Future Microbiology, pp. 189-201. 9. Dowson, C. G., Jephcott, A. E., Gough, K. R. & Spratt, B. G., 1989. Penicillin-binding protein 2 genes of non-beta-lactamase-producing, penicillin-resistant strains of Neisseria gonorrhoeae. Mol Microbiol, pp. 35-41. 10. Gegia, M. et al., 2008. Prevalence of and molecualr basis for tuberculosis drug resistance in the Republic of Georgia: validation of a QIAplex system for detection of drug resistance related mutations. Antimicrobial Agents Chemotherapy, pp. 725-9. 11. Gegia, M. et al., 2008. Prevalence of and molecular basis for tuberculosis drug resistance in the Republic of Georgia: validation of a QIAplex system for detection of drug resistance-related mutations. Antimicrob. Agents Chemother, pp. 725-9. 12. Golparian, D., Shafer, W. M., Ohnishi, M. & Unemo, M., 2014. Importance of Multidrug Efflux Pumps in the Antimicrobial Resistance Property of Clinical Multidrug-Resistant Isolates of Neisseria gonorrhoeae. Antimicrobial agents and chemotherapy, pp. 3556-9. 13. Kapranov, P. et al., 2002. Large-scale transcriptional activity in chromosomes 21 and 22. Science, pp. 916-9. 14. Kent, C. K. et al., 2005. Prevalence of rectal, urethral, and pharyngeal chlamydia and gonorrhea detected in 2 clinical settings among men who have sex with men: San Francisco, California, 2003. Clin Infect Dis., pp. 67-74. 15. Lander, E. S., 1999. Array of hope. Nature Genetics, pp. 3-4. 16. Levy, S. B. & Marshall, B., 2004. Antibacterial resistance worldwide: causes, challenges and responses. Nature Medicine, pp. S122-S129. 17. Lindberg, R., Fredlund, H., Nicholas, R. & Unemo, M., 2007. Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: association with genetic polymorphisms in penA, mtrR, porB1b, and ponA. Antimicrob Agents Chemother, pp. 2117-22. 18. Liu, X. S., 2007. Getting Started in Tiling Microarray Analysis. PLoS Computational Biology. 19. Magiorakos, A. P. et al., 2012. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology and Infection, pp. 268-81. 20. Manjunatha, U. H. et al., 2006. Identification of a nitroimidazole-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis. pnas, pp. 431-6. 21. Miller, M. B. & Tang, Y., 2009. Basic concepts of Microarrays and potential applications in clinical microbiology. Clinical Microbiology, pp. 611-33. 22. Mockler, T. C. & Ecker, J. R., 2004. Applications of NA tiling arrays for whole genome analysis. Genomics. 23. Shoemaker, D. D. et al., 2001. Experimental annotation of the human genome using microarray technology. Nature, pp. 922-7. 24. Troesch, A. et al., n.d. Mycobacterium Species Identification and Rifampin Resistance Testing with High-Density DNA Probe Arrays. 25. Unemo, M. & Nicholas, R. A., 2012. Emergence of multidrug-resistant, extensively drug-resistant and untreatable gonorrhea. Future Microbiology, pp. 1401-22. 26. Wiltgen, M. & Tilz, G. P., 2007. DNA microarray analysis: principals and clinical impact. Hematology, pp. 271-87. 27. Yamada, K. et al., 2003. Empirical analysis of transcriptional activity in the Arabidopsis genome. Science, pp. 842-6. 28. Yazaki, J., Gregory, B. D. & Ecker, J. R., 2007. Mapping the genome landscape using tiling array technology. Current opinion in plant biology, pp. 534-42. Read More
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