Cloning and constructing transformation vector carrying IbOr from the sweetpotato (Impomea batatas L.) involved in accumulation of carotenoids

The Orange gene (Or gene) is known as a gene encoding the protein containing a cysteine-rich Zinc finger domain. The Or protein is an important component of the structure that involes in accumulation of carotenoids and enhancing the resistant ability of plant to enviromental stresses. In an attempt to improve the nutritional quality and resistance to adverse environmental conditions of maize, in this article, we present the results on cloning IbOr gene from the sweetpotato cultivar Hoang Long and constructing transformation vectors carrying cloned IbOr gene under control of Ubiquitin (Ubi) or Globulin 1 (Glo1) promoter. The cloned IbOr gene was 942 bp in size and had similarity level of 100% comparing to the IbOr gene that was deposited in GenBank with Accession number HQ828087.1. The IbOr gene from sweedpotato Hoang Long was registered in GenBank under Accession number KX792094.1. The deduced protein consisted of 313 amino acids having Zin finger domain of DnaJ and HSP40 proteins. We have also constructed two transformation vectors pCambia2300/Ubiquitin/IbOr/Nos and pCambia2300/Glo1/IbOr/Nos. These vectors were transformed successfully into Agrobacterium tumefaciens strain C58. The presence of the target gene (IbOr) and promoter (Ubi or Glo1) in the A. tumefaciens strains were confirmed by colony-PCR amplification with respective specific primer pairs and digested by suitable restriction enzymes. These vectors will provide important materials for further gene transfer research for the expression of IbOr gene and production of transgenic maize with ability to accumulate high carotenoids.

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Construction of individual ddRAD libraries v2

10.17504/protocols.io.bv4tn8wn ◽

2021 ◽

Author(s):

Claire Daguin Thiebaut ◽

Stephanie Ruault ◽

Charlotte Roby ◽

Thomas Broquet ◽

Frédérique Viard ◽

...

Keyword(s):

Magnetic Beads ◽

Sex Chromosome ◽

De Novo ◽

Pcr Amplification ◽

Restriction Enzymes ◽

Tree Frog ◽

Size Selection ◽

Hyla Arborea ◽

Sequencing Method ◽

Sample Representation

This protocol describes a double digested restriction-site associated DNA (ddRADseq) procedure, that is a variation on the original RAD sequencing method (Davey & Blaxter 2011), which is used for de novo SNP discovery and genotyping. This protocol differs from the original ddRADseq protocol (Peterson et al 2012), in which the samples are pooled just after the ligation to adaptors (i.e. before size selection and PCR). The present ddRAD protocol as been slightly adapted from Alan Brelsford's protocol published in the supplementary material of this paper: Brelsford, A., Dufresnes, C. & Perrin, N. 2016. High-density sex-specific linkage maps of a European tree frog (Hyla arborea) identify the sex chromosome without information on offspring sex. Heredity 116, 177–181 (2016). https://doi.org/10.1038/hdy.2015.83 In the present protocol, all samples are treated separately, in a microplate, until final PCR amplification performed before pooling. Despite being slightly more costly and time-consuming in the lab, it allows for fine adjustement of each sample representation in the final library pool, ensuring similar number of sequencing reads per sample in the final dataset. Briefly, genomic DNA from the samples are individually digested with 2 restriction enzymes (one rare-cutter and one more frequent cutter) then ligated to a barcoded adaptor (among 24 available) at one side, and a single adaptor at the other side, purified with magnetic beads, and PCR-amplified allowing the addition of a Illumina index (among 12 available) for multiplexing a maximum of 288 sample per library. Samples are then pooled in equimolar conditions after visualisation on an agarose gel. Purification and size selection is then performed before final quality control of the library and sequencing.

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Plasmid-Encoded Metallo-β-Lactamase (IMP-6) Conferring Resistance to Carbapenems, Especially Meropenem

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.5.1343-1348.2001 ◽

2001 ◽

Vol 45 (5) ◽

pp. 1343-1348 ◽

Cited By ~ 91

Author(s):

Hisakazu Yano ◽

Akio Kuga ◽

Ryoichi Okamoto ◽

Hidero Kitasato ◽

Toshimitsu Kobayashi ◽

...

Keyword(s):

Point Mutation ◽

Antimicrobial Agents ◽

Pcr Amplification ◽

Penicillin G ◽

Gene Encoding ◽

Mature Enzyme ◽

Dna Fragment ◽

Lactamase Gene ◽

Reduced Activity ◽

Northern Japan

ABSTRACT In 1996, Serratia marcescens KU3838 was isolated from the urine of a patient with a urinary tract infection at a hospital in northern Japan and was found to contain the plasmid pKU501. Previously, we determined that pKU501 carries bla IMP and the genes for TEM-1-type β-lactamases as well as producing both types of β-lactamases (H. Yano, A. Kuga, K. Irinoda, R. Okamoto, T. Kobayashi, and M. Inoue, J. Antibiot. 52:1135–1139, 1999). pKU502 is a recombinant plasmid that contains a 1.5-kb DNA fragment, including the metallo-β-lactamase gene, and is obtained by PCR amplification of pKU501. The sequence of the metallo-β-lactamase gene in pKU502 was determined and revealed that this metallo-β-lactamase gene differed from the gene encoding IMP-1 by one point mutation, leading to one amino acid substitution: 640-A in the base sequence of the IMP-1 gene was replaced by G, and Ser-196 was replaced by Gly in the mature enzyme. This enzyme was designated IMP-6. The strains that produced IMP-6 were resistant to carbapenems. The MICs of panipenem and especially meropenem were higher than the MIC of imipenem for these strains. The k cat/Km value of IMP-6 was about sevenfold higher against meropenem than against imipenem, although the MIC of meropenem for KU1917, which produced IMP-1, was lower than that of imipenem, and the MIC of panipenem was equal to that of imipenem. These results support the hypothesis that IMP-6 has extended substrate profiles against carbapenems. However, the activity of IMP-6 was very low against penicillin G and piperacillin. These results suggest that IMP-6 acquired high activity against carbapenems, especially meropenem, via the point mutation but in the process lost activity against penicillins. Although IMP-6 has reduced activity against penicillins due to this point mutation, pKU501 confers resistance to a variety of antimicrobial agents because it also produces TEM-1-type enzyme.

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PCR amplification and sequence analyses of reverse transcriptase-like genes in Crinipellis perniciosa isolates

Fitopatologia Brasileira ◽

10.1590/s0100-41582007000500001 ◽

2007 ◽

Vol 32 (5) ◽

pp. 373-380 ◽

Cited By ~ 3

Author(s):

Jorge F. Pereira ◽

Mariana D.C. Ignacchiti ◽

Elza F. Araújo ◽

Sérgio H. Brommonschenkel ◽

Júlio C.M. Cascardo ◽

...

Keyword(s):

Reverse Transcriptase ◽

Pathogenic Fungus ◽

Pcr Amplification ◽

Restriction Enzymes ◽

Sequence Comparisons ◽

Copy Numbers ◽

A Genome ◽

Close Relationship ◽

Pcr Products ◽

Broom Disease

Reverse transcriptase (RT) sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative RT sequences were analyzed in the genome of the pathogenic fungus C. perniciosa, the causal agent of witches' broom disease of cocoa. A 394 bp fragment was amplified from genomic DNA of different isolates of C. perniciosa belonging to C-, L-, and S-biotypes and collected from various geographical areas. The cleavage of PCR products with restriction enzymes and the sequencing of various RT fragments indicated the presence of several sequences showing transition events (G:C to A:T). Southern blot analysis revealed high copy numbers of RT signals, forming different patterns among C-, S-, and L-biotype isolates. Sequence comparisons of the predicted RT peptide indicate a close relationship with the RT protein from thegypsy family of LTR-retrotransposons. The possible role of these retrotransposons in generating genetic variability in the homothallic C. perniciosa is discussed.

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Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.10.245 ◽

2014 ◽

Vol 10 ◽

pp. 2348-2360 ◽

Cited By ~ 13

Author(s):

Kristen K Merritt ◽

Kevin M Bradley ◽

Daniel Hutter ◽

Mariko F Matsuura ◽

Diane J Rowold ◽

...

Keyword(s):

Solid Phase ◽

Pcr Amplification ◽

Kanamycin Resistance ◽

Dna Fragments ◽

Gene Encoding ◽

Synthetic Dna ◽

Information Density ◽

Letter Alphabet ◽

New Strategy ◽

Autonomous Assembly

Background: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide “letters”, the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson–Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so. Results: We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson–Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2’-deoxyisocytidine and 2’-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with optimally designed standard nucleotides lacking AEGIS components gave successful assemblies of up to 16 fragments, but generally failed when larger autonomous assemblies were attempted. Conclusion: AEGIS nucleotides, by increasing the information density of DNA, allow larger numbers of DNA fragments to autonomously self-assemble into large DNA constructs. This technology can therefore increase the size of DNA constructs that might be used in synthetic biology.

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Inactivation of the Neurospora crassa gene encoding the mitochondrial protein import receptor MOM19 by the technique of "sheltered RIP".

Genetics ◽

10.1093/genetics/136.1.107 ◽

1994 ◽

Vol 136 (1) ◽

pp. 107-118 ◽

Cited By ~ 5

Author(s):

T A Harkness ◽

R L Metzenberg ◽

H Schneider ◽

R Lill ◽

W Neupert ◽

...

Keyword(s):

Neurospora Crassa ◽

Target Gene ◽

Protein Import ◽

Selectable Marker ◽

Mitochondrial Protein ◽

Mutant Gene ◽

Mitochondrial Protein Import ◽

Gene Encoding ◽

Precursor Proteins ◽

Import Receptor

Abstract We have used a technique referred to as "sheltered RIP" (repeat induced point mutation) to create mutants of the mom-19 gene of Neurospora crassa, which encodes an import receptor for nuclear encoded mitochondrial precursor proteins. Sheltered RIP permits the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant gene in a heterokaryon with an unaffected nucleus. Furthermore, the nucleus harboring the RIPed gene contains a selectable marker so that it is possible to shift nuclear ratios in the heterokaryons to a state in which the nucleus containing the RIPed gene predominates in cultures grown under selective conditions. This results in a condition where the target gene product should be present at very suboptimal levels and allows the study of the mutant phenotype. One allele of mom-19 generated by this method contains 44 transitions resulting in 18 amino acid substitutions. When the heterokaryon containing this allele was grown under conditions favoring the RIPed nucleus, no MOM19 protein was detectable in the mitochondria of the strain. Homokaryotic strains containing the RIPed allele exhibit a complex and extremely slow growth phenotype suggesting that the product of the mom-19 gene is important in N. crassa.

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Out-Lab Therapy Approach Based on Elected A Restriction Enzyme to Transfer Target Gene

Al-Kitab Journal for Pure Sciences ◽

10.32441/kjps.02.02.p13 ◽

2018 ◽

Vol 2 (2) ◽

pp. 196-208

Author(s):

Ayad Ismaeel

Keyword(s):

Restriction Enzyme ◽

Target Gene ◽

Restriction Enzymes ◽

Tp53 Gene ◽

Restriction Enzyme Digestion ◽

Therapy Approach ◽

Software Packages ◽

Important Approach ◽

Effective Cost ◽

Target Gene Sequence

An important approach of therapy the target gene sequence causes diseases via repair/recombine the mutated gene (gene transfer) using a restriction enzymes in the laboratory. This approach will cause multiple problems happening accompany to biological laboratory if ruled out problems outside of it like the digested DNA ran as a smear on an agarose gel, incomplete restriction enzyme digestion, extra bands in the gel, etc. The paper suggested new approach of therapy via repair/replacement mutated gene caused disease by detecting primers and finding restriction enzymes using bioinformatics tools, software, packages etc. then achieving the repair/ recombine of mutations before going to the biologic lab (out-lab) to avoid the problems associated these laboratories. Implement and apply this a proposed therapy approach on TP53 gene (which caused more than 50% of human cancers) and after confirming there is mutations on P53 tumor protein shows an effective cost, friendly therapy methodology and comprehensive.

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ISOLASI FRAGMEN GEN PENYANDI PUTRESIN N-METILTRANSFERASE DAN QUINOLINAT FOSFORIBOSILTRANSFERASE ASAL TEMBAKAU LOKAL TEMANGGUNG (Nicotiana tabacum)

Jurnal Penelitian Tanaman Industri ◽

10.21082/jlittri.v17n3.2011.109-117 ◽

2020 ◽

Vol 17 (3) ◽

pp. 109 ◽

Cited By ~ 1

Author(s):

SESANTI BASUKI ◽

NURHAJATI AA MATTJIK ◽

SUWARSO SUWARSO ◽

DESTA WIRNAS ◽

SUDARSONO SUDARSONO

Keyword(s):

Dna Sequences ◽

Pcr Amplification ◽

Data Bank ◽

Degenerate Primer ◽

Gene Encoding ◽

Methyl Transferase ◽

Dna Database ◽

Genes Encoding ◽

Tobacco Cultivar ◽

Dna Template

ABSTRAKUpaya untuk menurunkan kandungan nikotin merupakan salah satuprioritas utama penelitian tembakau. Nikotin adalah senyawa alkaloidutama berpotensi dikonversi menjadi senyawa nor-nikotin yang bersifatkarsinogen. Gen PMT sebagai penyandi enzim putresin n-metiltransferase(PMT) dan gen QPT - penyandi enzim quinolinat fosforibosiltransferase(QPT) merupakan dua gen kunci yang berperan penting pada proses bio-sintesis nikotin. Penelitian ini bertujuan untuk mengisolasi potongan genPMT dan QPT asal tembakau lokal Indonesia, mengkarakterisasi danmenganalisis runutan DNA-nya. Tahapan penelitian dimulai dengan me-rancang primer degenerate berdasarkan informasi yang ada di pangkalandata Bank Gen NCBI (National Centre for Biotechnology Information),mengamplifikasi PCR menggunakan templat DNA genomik tembakaulokal cv. Sindoro1, mengklon potongan DNA hasil PCR dan menentukanrunutan DNA-nya. Hasil penelitian menunjukkan dari dua belas pasangprimer degenerate yang dirancang, hanya dua pasang primer yang meng-hasilkan potongan DNA hasil amplifikasi PCR, yaitu pasangan primerPMt-7 (F & R) untuk gen PMT dan primer QPt-3 (F & R) untuk gen QPT.Setelah dilakukan penentuan runutan DNA-nya, amplikon yang didapatdari hasil PCR dengan pasangan primer PMt-7 sebesar 1418 bp, sedangkanuntuk primer QPt-3 sebesar 205 bp. Runutan DNA gen PMT dan gen QPTasal tembakau lokal cv. Sindoro1 mempunyai tingkat kesamaan yang ting-gi dengan gen PMT dan gen QPT asal tembakau lainnya yang ada dipangkalan data Bank Gen NCBI.Kata kunci : Gen PMT, gen QPT, lintasan biosintesis nikotin, perunutanDNA, amplifikasi PCR, primer degenerateABSTRACTIsolation of Genes encoding Putrescine N-Methyl-transferase and Quinolinat Phosphoribosyl transferasederived from Temanggung Tobacco Cultivar (Nicotianatabacum)Reduction of nicotine content is one of the major objective intobacco research. Nicotine is the main alcaloid compound that potentiallycould be converted into a carcinogenic compound (nor-nicotine). The PMTgene encoding putrescine N-methyl transferase (PMT) and the QPT gene -encoding quinolinate phosphoribosyl transferase (QPT) are the two keyenzymes involved in nicotine biosynthesis. The objectives of this researchwere to isolate PMT and QPT gene fragments originated from Indonesianlocal tobacco, to characterize, and to analyze their DNA sequences. Theresearch activities included: degenerate primer design based oninformation available in the GenBank DNA Database NCBI (NationalCentre for Biotechnology Information), PCR amplification usingdegenerate primer and genomic DNA template of a local tobacco cv.Sindoro1, clone the PCR amplified products, and determine their DNAnucleotide sequences. Results of the experiment indicated that from 12degenerate primer pairs synthesized, only two were able to yield positivePCR amplified products. These primer pairs were PMt-7 (F & R primers)for PMT and QPt-3 (F & R primers) for QPT. After DNA sequencing, theamplified DNA product amplified using PMt-7 degenerate primer pairswere 1418 bp, while that using QPt-3 primer pairs were only 205 bp.Nucleotide sequences of PMT or QPT gene fragments originated fromlocal tobacco cv. Sindoro1 showed a high nucleotide sequences identity ascompared to that of the respective genes from other tobacco species thatwere available in the GenBank DNA Database NCBI.Key words: PMT gene, QPT gene, nicotine biosynthetic pathways, DNAsequencing, PCR amplification, degenerate primer

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Bioinformatics and wet laboratory analysis of rs1800562 to predict genetic aetiology of iron overload in Nigeria

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqab191.310 ◽

2021 ◽

Vol 156 (Supplement_1) ◽

pp. S145-S146

Author(s):

A B Bolarinwa ◽

F Onawoga

Keyword(s):

Iron Overload ◽

In Silico ◽

Protein Localization ◽

Restriction Enzymes ◽

Protein Product ◽

Laboratory Analysis ◽

Hfe Gene ◽

Excess Iron ◽

Stability Change ◽

Suitable Restriction

Abstract Introduction/Objective The most reported single nucleotide polymorphism (SNP) of the HFE gene is rs1800562, representing the substitution of Adenine for Guanine at position 847 of the HFE gene. This has been widely implicated in hereditary haemochromatosis and other conditions like altered cholesterol balance, Alzheimer’s disease and cutaneous photosensitivity. Abnormal HFE protein resulting from the mutant HFE gene leads to formation of excess iron which has been postulated as likely mechanism for these diseases. Although there is evidence of iron overload in Africans, only few studies have explored possible genetic causes, and prevalence of rs1800562 is not known in West African population. Hence the need to determine the prevalence of rs1800562 in Nigeria using computational and wet laboratory approach. Methods/Case Report Details of rs1800562 were retrieved from Ensembl Genome Browser version 99. Severity of the consequences of this SNP on protein product was determined using bioinformatics tools including SIFT, Polyphen, Mutation Assessor, HOPE, I-mutant and MutPred2. Genotyping of rs1800562 was done In silico using restriction fragment length polymorphism (RFLP). Primer3plus was used for primer design, NCBI BLAST and SMS were used for primer validation. We used Webcutter 2.0 to determine suitable restriction enzymes. The genotyping was simulated using USCS virtual PCR and RestrictionMapper. Whole blood samples were obtained from 200 participants selected randomly from a pool of blood donors. DNA was extracted and flanking region of rs1800562 was amplified. The amplified product was digested by RSA1and fragments examined on agarose gel electrophoresis. Results (if a Case Study enter NA) The MAF was found to be 0.01 globally and 0.02 in Africa. In the two Nigerian population examined (Yoruba and Esan population), MAF was 0.00. Mutation Assessor and SIFT Polyphen consistently predicted the mutation to be of severe consequences. Analysis on HOPE, I-mutant and Mutpred2 revealed loss of protein stability, change in net charges affecting the HFE protein localization and its interaction with other proteins. All the participants in the wet laboratory analysis were homozygous for the wild type allele of rs1800562 (MAF=0). Conclusion This study confirmed the In silico prediction of the absence of rs1800562 in Nigeria. Future studies should focus on other SNPs of the HFE gene as well as other gene involved in iron metabolism.

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Protocols for 16S rDNA Array Analyses of Microbial Communities by Sequence-Specific Labeling of DNA Probes

The Scientific World JOURNAL ◽

10.1100/tsw.2003.44 ◽

2003 ◽

Vol 3 ◽

pp. 578-584 ◽

Cited By ~ 9

Author(s):

Knut Rudi ◽

Janneke Treimo ◽

Hilde Nissen ◽

Gerd Vegarud

Keyword(s):

Microbial Communities ◽

Pcr Amplification ◽

Dna Probes ◽

Dna Array ◽

Gene Encoding ◽

Variable Regions ◽

Conserved Regions ◽

Rdna Array ◽

Array Hybridization ◽

Array Analyses

Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for DNA array-based analyses of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Protocols are presented for DNA purification, probe construction, probe labeling, and DNA array hybridizations.

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Molecular Follow-Up of Disease Progression and Interferon Therapy in Chronic Myelocytic Leukemia

Blood ◽

10.1182/blood.v90.12.4918 ◽

1997 ◽

Vol 90 (12) ◽

pp. 4918-4923 ◽

Cited By ~ 31

Author(s):

Dina Ben-Yehuda ◽

Svetlana Krichevsky ◽

Eliezer A. Rachmilewitz ◽

Ayelet Avraham ◽

Giuseppe A. Palumbo ◽

...

Keyword(s):

De Novo ◽

Methylation Status ◽

Pcr Amplification ◽

Chronic Phase ◽

Restriction Enzymes ◽

Cytogenetic Response ◽

Interferon Α ◽

Chronic Myelocytic Leukemia ◽

Myelocytic Leukemia

Abstract We previously reported that the abl promoter (Pa) undergoes de novo DNA methylation in the course of chronic myelocytic leukemia (CML). The clinical implications of this finding are the subject of the present study in which samples of CML patients, including a group treated with interferon α (IFNα) were surveyed. The methylation status of the abl promoter was monitored by polymerase chain reaction (PCR) amplification of the Pa region after digestion with several site-methylation sensitive restriction enzymes. Some 74% of the DNA samples from blood and marrow drawn in the chronic phase were nonmethylated, similar to control samples from non-CML patients. The remaining 26% were partially methylated in the abl Pa region. The latter samples were derived from patients who were indistinguishable from the others on the basis of clinical presentation. Methylated samples were mostly derived from patients known to have a disease of longer duration (26 months v 7.5 months, P = .01). Samples of 30 IFNα-treated patients were sequentially analyzed in the course of treatment. Fifteen patients with no evidence of Pa methylation before treatment remained methylation-free. The remainder, who displayed Pa methylation before treatment, reverted to the methylation-free status. The outcome is attributed to IFNα therapy, as the Pa methylation status was not reversed in any of the patients treated with hydroxyurea. Methylation of the abl promoter indicates a disease of long-standing, most likely associated with a higher probability of imminent blastic transformation. It appears to predict the outcome of IFNα therapy far better than the cytogenetic response.

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