国际植物学墨尔本大会上命名法规的变化

2011年7月在第18届国际植物学大会命名法分会上通过了命名法规的一系列重大改动,并在全体大会上得到了接受批准。文中就命名法规的变化进行了简单概述,同时也讨论了这些变化的意义,特别是对菌物研究的意义,以期引起广大研究人员的关注。     

DNA sequence and analysis of the bet genes encoding the osmoregulatory choline—glycine betaine pathway of Escherichia coli

The sequence was determined of 6493 nucleotides encompassing the bet genes of Escherichia coli which encode the osmoregulatory choline-glycine betaine pathway. Four open reading frames were identified: betA encoding choline dehydrogenase, a flavoprotein of 61.9kDa; betB encoding betaine aldehyde dehydrogenase (52.8kDa); betT encoding a proton-motive-force-driven, high-affinity transport system for choline (75.8kDa); and betl, capable of encoding a protein of 21.8kDa, implicated as a repressor involved in choline regulation of the bet genes. Identification of the genes was supported by subcloning, physical mapping of lambda placMu53 insertions, amino acid sequence similarity, or N-terminal amino acid sequencing. The bet genes are tightly spaced, with betT located upstream of, and transcribed divergently to, the tandemly linked betIBA genes.     

Improved Multiple Displacement Amplification (iMDA) and Ultraclean Reagents

Background

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA).

Results

A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome.

Conclusions

The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.