Rapid incorporation kinetics and improved fidelity of a novel class of 3'-OH unblocked reversible terminators

Recent developments of unique nucleotide probes have expanded our understanding of DNA polymerase function, providing many benefits to techniques involving next-generation sequencing (NGS) technologies. The cyclic reversible termination (CRT) method depends on efficient base-selective incorporation of reversible terminators by DNA polymerases. Most terminators are designed with 3′-O-blocking groups but are incorporated with low efficiency and fidelity. We have developed a novel class of 3′-OH unblocked nucleotides, called Lightning Terminators™, which have a terminating 2-nitrobenzyl moiety attached to hydroxymethylated nucleobases. A key structural feature of this photocleavable group displays a ‘molecular tuning’ effect with respect to single-base termination and improved nucleotide fidelity. Using Therminator™ DNA polymerase, we demonstrate that these 3′-OH unblocked terminators exhibit superior enzymatic performance compared to two other reversible terminators, 3′-O-amino-TTP and 3′-O-azidomethyl-TTP. Lightning Terminators™ show maximum incorporation rates (k_pol) that range from 35 to 45 nt/s, comparable to the fastest NGS chemistries, yet with catalytic efficiencies (k_pol/K_D) comparable to natural nucleotides. Pre-steady-state kinetic studies of thymidine analogs revealed that the major determinant for improved nucleotide selectivity is a significant reduction in k_pol by >1000-fold over TTP misincorporation. These studies highlight the importance of structure–function relationships of modified nucleotides in dictating polymerase performance.     

Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates.

Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-O-methyl-dNTPs were shown to terminate DNA syntheses mediated by a number of polymerases and may be used as alternative terminators in Sanger sequencing. 3'-O-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown to be incorporated by several thermostable DNA polymerases. Base specific termination and efficient photolytic removal of the 3'-protecting group was demonstrated. Following deprotection, DNA synthesis was reinitiated by the incorporation of natural nucleotides into DNA. The identification of this labile terminator and the demonstration of a one cycle stop-start DNA synthesis are initial steps in the development of a novel sequencing strategy.     

Are there valid proxy measures of clinical behaviour? a systematic review

Background

Accurate measures of health professionals' clinical practice are critically important to guide health policy decisions, as well as for professional self-evaluation and for research-based investigation of clinical practice and process of care. It is often not feasible or ethical to measure behaviour through direct observation, and rigorous behavioural measures are difficult and costly to use. The aim of this review was to identify the current evidence relating to the relationships between proxy measures and direct measures of clinical behaviour. In particular, the accuracy of medical record review, clinician self-reported and patient-reported behaviour was assessed relative to directly observed behaviour.

Methods

We searched: PsycINFO; MEDLINE; EMBASE; CINAHL; Cochrane Central Register of Controlled Trials; science/social science citation index; Current contents (social & behavioural med/clinical med); ISI conference proceedings; and Index to Theses. Inclusion criteria: empirical, quantitative studies; and examining clinical behaviours. An independent, direct measure of behaviour (by standardised patient, other trained observer or by video/audio recording) was considered the 'gold standard' for comparison. Proxy measures of behaviour included: retrospective self-report; patient-report; or chart-review. All titles, abstracts, and full text articles retrieved by electronic searching were screened for inclusion and abstracted independently by two reviewers. Disagreements were resolved by discussion with a third reviewer where necessary.

Results

Fifteen reports originating from 11 studies met the inclusion criteria. The method of direct measurement was by standardised patient in six reports, trained observer in three reports, and audio/video recording in six reports. Multiple proxy measures of behaviour were compared in five of 15 reports. Only four of 15 reports used appropriate statistical methods to compare measures. Some direct measures failed to meet our validity criteria. The accuracy of patient report and chart review as proxy measures varied considerably across a wide range of clinical actions. The evidence for clinician self-report was inconclusive.

Conclusion

Valid measures of clinical behaviour are of fundamental importance to accurately identify gaps in care delivery, improve quality of care, and ultimately to improve patient care. However, the evidence base for three commonly used proxy measures of clinicians' behaviour is very limited. Further research is needed to better establish the methods of development, application, and analysis for a range of both direct and proxy measures of behaviour.     

Monilethrix: mutational hotspot in the helix termination motif of the human hair basic keratin 6

Monilethrix is a rare autosomal dominant disease characterized by hair fragility and follicular hyperkeratosis. Mutations in the human basic hair keratins hHb1 and hHb6 have recently been reported in this disease. Twelve families and sporadic cases were clinically diagnosed with monilethrix and were available for the study. The gene segment encoding the helix termination motif region of keratin hHb6 was PCR amplified and sequenced. Mutations were recognized in 6 families. Four families had the previously described mutations, Glu413Lys and Glu413Asp. In 2 unrelated families, a novel mutation, Glu402Lys, was identified. No clear association was found between the severity of the phenotype and the mutation carried. Furthermore, heterozygous members of the same family had variable degrees of hair and skin involvement. Homozygous patients identified in one large consanguineous family were more severely affected. Other genetic or environmental factors may also play a role in monilethrix.     

Evaluation of the Best disease gene in patients with age-related macular degeneration and other maculopathies

Vitelliform macular dystrophy (VMD2, Best disease, MIM153700) is an early onset, autosomal, dominant macular degeneration characterized by the deposition of lipofuscin-like material within and below the retinal pigment epithelium (RPE); it is associated with degeneration of the RPE and overlying photoreceptors. Recently, we cloned the gene bestrophin, which is responsible for the disease, and identified a number of causative mutations in families with VMD2. Here, we report that the analysis of bestrophin in a collection of 259 age-related macular degeneration (AMD) patients provides evidence that mutations in the Best disease gene do not play a significant role in the predisposition of individuals to AMD. However, our results suggest that, in addition to Best disease, mutations within the bestrophin gene could be responsible for other forms of maculopathy with phenotypic characteristics similar to Best disease and for other diseases not included in the VMD category. Received: 11 March 1999 / Accepted: 6 April 1999     

Friend or foe? Evolutionary history of glycoside hydrolase family 32 genes encoding for sucrolytic activity in fungi and its implications for plant-fungal symbioses

Background  

Many fungi are obligate biotrophs of plants, growing in live plant tissues, gaining direct access to recently photosynthesized carbon. Photosynthate within plants is transported from source to sink tissues as sucrose, which is hydrolyzed by plant glycosyl hydrolase family 32 enzymes (GH32) into its constituent monosaccharides to meet plant cellular demands. A number of plant pathogenic fungi also use GH32 enzymes to access plant-derived sucrose, but less is known about the sucrose utilization ability of mutualistic and commensal plant biotrophic fungi, such as mycorrhizal and endophytic fungi. The aim of this study was to explore the distribution and abundance of GH32 genes in fungi to understand how sucrose utilization is structured within and among major ecological guilds and evolutionary lineages. Using bioinformatic and PCR-based analyses, we tested for GH32 gene presence in all available fungal genomes and an additional 149 species representing a broad phylogenetic and ecological range of biotrophic fungi.     

Improved nucleotide selectivity and termination of 3'-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates

We describe a novel 3'-OH unblocked reversible terminator with the potential to improve accuracy and read-lengths in next-generation sequencing (NGS) technologies. This terminator is based on 5-hydroxymethyl-2'-deoxyuridine triphosphate (HOMedUTP), a hypermodified nucleotide found naturally in the genomes of numerous bacteriophages and lower eukaryotes. A series of 5-(2-nitrobenzyloxy)methyl-dUTP analogs (dU.I-dU.V) were synthesized based on our previous work with photochemically cleavable terminators. These 2-nitrobenzyl alkylated HOMedUTP analogs were characterized with respect to incorporation, single-base termination, nucleotide selectivity and photochemical cleavage properties. Substitution at the α-methylene carbon of 2-nitrobenzyl with alkyl groups of increasing size was discovered as a key structural feature that provided for the molecular tuning of enzymatic properties such as single-base termination and improved nucleotide selectivity over that of natural nucleotides. 5-[(S)-α-tert-Butyl-2-nitrobenzyloxy]methyl-dUTP (dU.V) was identified as an efficient reversible terminator, whereby, sequencing feasibility was demonstrated in a cyclic reversible termination (CRT) experiment using a homopolymer repeat of ten complementary template bases without detectable UV damage during photochemical cleavage steps. These results validate our overall strategy of creating 3'-OH unblocked reversible terminator reagents that, upon photochemical cleavage, transform back into a natural state. Modified nucleotides based on 5-hydroxymethyl-pyrimidines and 7-deaza-7-hydroxymethyl-purines lay the foundation for development of a complete set of four reversible terminators for application in NGS technologies.