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1.
Br J Haematol ; 194(1): 53-60, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34114218

RESUMO

Measurement of BCR activator of RhoGEF and GTPase -ABL proto-oncogene 1, non-receptor tyrosine kinase (BCR-ABL1) mRNA levels by reverse transcription quantitative polymerase chain reaction (RTqPCR) has been critical to treatment protocols and clinical trials in chronic myeloid leukaemia; however, interlaboratory variation remains a significant issue. Reverse transcriptase droplet digital PCR (RTddPCR) has shown potential to improve testing but a large-scale interlaboratory study is required to definitively establish this. In the present study, 10 BCR-ABL1-positive samples with levels ranging from molecular response (MR)1·0 -MR5·0 were tested by 23 laboratories using RTddPCR with the QXDX BCR-ABL %IS kit. A subset of participants tested the samples using RTqPCR. All 23 participants using RTddPCR detected BCR-ABL1 in all samples to MR4·0 . Detection rates for deep-response samples were 95·7% at MR4·5 , 78·3% at MR4·7 and 87·0% at MR5·0 . Interlaboratory coefficient of variation was indirectly proportional to BCR-ABL1 level ranging from 29·3% to 69·0%. Linearity ranged from 0·9330 to 1·000 (average 0·9936). When results were compared for the 11 participants who performed both RTddPCR and RTqPCR, RTddPCR showed a similar limit of detection to RTqPCR with reduced interlaboratory variation and better assay linearity. The ability to detect deep responses with RTddPCR, matched with an improved linearity and reduced interlaboratory variation will allow improved patient management, and is of particular importance for future clinical trials focussed on achieving and maintaining treatment-free remission.


Assuntos
Proteínas de Fusão bcr-abl/sangue , Ensaio de Proficiência Laboratorial , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ásia , Biomarcadores Tumorais/sangue , Europa (Continente) , Células HL-60/química , Humanos , Células K562/química , Laboratórios Clínicos , Modelos Lineares , América do Norte , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes
2.
Nucleic Acids Res ; 40(20): 10567-75, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22904087

RESUMO

The biocompatibility of a triazole mimic of the DNA phosphodiester linkage in Escherichia coli has been evaluated. The requirement for selective pressure on the click-containing gene was probed via a plasmid containing click DNA backbone linkages in each strand of the gene encoding the fluorescent protein mCherry. The effect of proximity of the click linkers on their biocompatibility was also probed by placing two click DNA linkers 4-bp apart at the region encoding the fluorophore of the fluorescent protein. The resulting click-containing plasmid was found to encode mCherry in E. coli at a similar level to the canonical equivalent. The ability of the cellular machinery to read through click-linked DNA was further probed by using the above click-linked plasmid to express mCherry using an in vitro transcription/translation system, and found to also be similar to that from canonical DNA. The yield and fluorescence of recombinant mCherry expressed from the click-linked plasmid was also compared to that from the canonical equivalent, and found to be the same. The biocompatibility of click DNA ligation sites at close proximity in a non-essential gene demonstrated in E. coli suggests the possibility of using click DNA ligation for the enzyme-free assembly of chemically modified genes and genomes.


Assuntos
DNA/química , Escherichia coli/genética , Química Click , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Mutagênese Sítio-Dirigida , Plasmídeos/genética , Triazóis/química , Proteína Vermelha Fluorescente
3.
Proc Natl Acad Sci U S A ; 108(28): 11338-43, 2011 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-21709264

RESUMO

A triazole mimic of a DNA phosphodiester linkage has been produced by templated chemical ligation of oligonucleotides functionalized with 5'-azide and 3'-alkyne. The individual azide and alkyne oligonucleotides were synthesized by standard phosphoramidite methods and assembled using a straightforward ligation procedure. This highly efficient chemical equivalent of enzymatic DNA ligation has been used to assemble a 300-mer from three 100-mer oligonucleotides, demonstrating the total chemical synthesis of very long oligonucleotides. The base sequences of the DNA strands containing this artificial linkage were copied during PCR with high fidelity and a gene containing the triazole linker was functional in Escherichia coli.


Assuntos
DNA/síntese química , Sequência de Bases , DNA/química , DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mimetismo Molecular , Dados de Sequência Molecular , Estrutura Molecular , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido Nucleico , Triazóis/química
4.
Angew Chem Int Ed Engl ; 53(9): 2362-5, 2014 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-24452865

RESUMO

Click DNA ligation promises an alternative to the current enzymatic approaches for DNA assembly, with the ultimate goal of using efficient chemical reactions for the total chemical synthesis and assembly of genes and genomes. Such an approach would enable the incorporation of various chemically modified bases throughout long stretches of DNA, a feat not possible with current polymerase-based methods. An unequivocal requirement for this approach is the biocompatibility of the resulting triazole-linked DNA. The correct function of this unnatural DNA linker in human cells is demonstrated here by using a click-linked gene encoding the fluorescent protein mCherry. Reverse transcription of mRNA isolated from these cells and subsequent sequencing of the mCherry cDNA shows error-free transcription. Nucleotide excision repair (NER) is shown to not play a role in the observed biocompatibility by using a NER-deficient human cell line. This is the first example of a non-natural DNA linker being functional in a eukaryotic cell.


Assuntos
DNA/síntese química , DNA/genética , Transcrição Gênica , Linhagem Celular , Química Click , DNA/química , Reparo do DNA , Humanos , Proteínas Luminescentes/genética , Biologia Sintética , Triazóis/química , Proteína Vermelha Fluorescente
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