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- Bogas, Diana, et al.
(author)
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Applications of optical DNA mapping in microbiology
- 2017
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In: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 62:6, s. 255-267
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Research review (peer-reviewed)abstract
- Optical mapping (OM) has been used in microbiology for the past 20 years, initially as a technique to facilitate DNA sequence-based studies; however, with decreases in DNA sequencing costs and increases in sequence output from automated sequencing platforms, OM has grown into an important auxiliary tool for genome assembly and comparison. Currently, there are a number of new and exciting applications for OM in the field of microbiology, including investigation of disease outbreaks, identification of specific genes of clinical and/or epidemiological relevance, and the possibility of single-cell analysis when combined with cell-sorting approaches. In addition, designing lab-on-a-chip systems based on OM is now feasible and will allow the integrated and automated microbiological analysis of biological fluids. Here, we review the basic technology of OM, detail the current state of the art of the field, and look ahead to possible future developments in OM technology for microbiological applications.
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| 3. |
- Persson, Helena, et al.
(author)
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Preparation of highly multiplexed small RNA sequencing libraries
- 2017
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In: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 63:2, s. 57-64
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Journal article (peer-reviewed)abstract
- MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Here, we develop a scalable protocol for the rapid and economical preparation of a large number of small RNA sequencing libraries using dual indexing for multiplexing. Combined with the use of off-the-shelf reagents, more samples can be sequenced simultaneously on large-scale sequencing platforms at a considerably lower cost per sample. Sample preparation is simplified by pooling libraries prior to gel purification, which allows for the selection of a narrow size range while minimizing sample variation. A comparison with publicly available data from benchmarking of miRNA analysis platforms showed that this method captures absolute and differential expression as effectively as commercially available alternatives.
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| 4. |
- Xia, Hongyan, et al.
(author)
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Development of single-tube nested real-time PCR assays with long internally quenched probes for detection of norovirus genogroup II
- 2016
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In: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 60:1, s. 28-34
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Journal article (peer-reviewed)abstract
- The high sequence variation of RNA viruses necessitates use of degenerate primers and probes or multiple primers and probes in molecular diagnostic assays. We showed previously that PCR amplification in two rounds, first with long target-specific primers and then with short generic primers, followed by detection using long probes, can tolerate sequence variation. Here we demonstrate that long primers and probes of up to 56 nucleotides can also be applied in real-time PCR for the detection of norovirus genogroup II with improved sensitivity. Probe design (method of incorporating quenchers, use of Zen internal quencher or traditional quenchers) greatly affects the sensitivity of the real-time PCR assays.
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