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Summary of the December 2015 Issue of BioTechniques

The December 2015 issues of BioTechniques will feature new methods articles for:

  • ultra sensitive ELISA detection of immunoreactive insulin
  • comparisons of electrophenograms across chips and experiments
  • multispectral karyotyping using a standard confocal microscope
  • one-pot “click” chemistry conjugations and purifications
  • single-stranded library preparation methods for ancient DNA sequencing

In addition to these articles, our Tech News feature for December will examine the tools being used to explore the human microbiome.

Article Descriptions

To decrease patient suffering in clinical settings, collection of blood samples for the diagnosis and monitoring of disease should be minimized. In this issue of BioTechniques, researchers from Japan describe a newly developed ultrasensitive ELISA coupled with a thio-NAD cycling which can be employed to measure immunoreactive insulin in blood serum using WHO international standard insulin or its equivalent products as a reference. The increased sensitivity of this new assay means less patient sampling is needed when testing.

Keywords: ELISA, immunoreactivity; insulin analysis; ultrasensitive assays

Ribosomal Intergenic Spacer Analysis (RISA) is a reproducible, high-resolution fingerprinting technique to discriminate microbial communities. Community profiles can be visualized using the Agilent 2100 Bioanalyzer where fingerprint comparisons rely on the precise estimation of all amplified DNA fragment lengths. When more than one chip is involved, electrophoregram comparisons are challenging due to the unavoidable discrepancies between chips and absence of correction software. In an article in this issue, researchers from France present RisaAligner, a software platform for analyzing and comparing electrophoregrams from Agilent chips using a non-linear ladder-alignment algorithm. The authors demonstrate the utility of RisaAligner with soil microbial profiles obtained with Agilent DNA 1000 and High Sensitivity chips.

Keywords: RISA comparisons; microbial ecology; computational biology; fingerprinting

Karyotyping detects numerical and structural abnormalities of chromosomes. Multispectral karyotyping analyzes all chromosomes in a single cell by labeling with chromosome-specific probes conjugated to unique combinations of fluorophores. Conventional laser scanning confocal microscopes that are capable of separating multiple overlapping emission spectra through spectral imaging and linear unmixing can be utilized for classifying chromosomes painted with multicolor probes. In this December article, researchers from Kansas City, MO generated multicolor chromosome paints from single-sorted chromosomes for human and mouse and developed a set of freely available open source ImageJ tools for spectral unmixing and karyotyping experiments.  Chromosome spreads painted with these multispectral probe sets can be imaged on widely available spectral laser scanning confocal microscopes and analyzed anywhere using ImageJ.

Keywords: cytogenetics; image analysis; karyotyping; confocal microscopy; chromosomes; spectral unmixing

Copper-catalyzed azide-alkyne cycloaddition (CuAAC) “click” chemistry has been demonstrated to be particular useful in bio-orthogonal conjugation reactions. In the December issue, scientists from Los Angeles, California describe a one-pot, heterogeneous bioconjugation and purification technique for selectively activated CuAAC. A Cu(II) precursor, with either the neutral ligand 1,10-phenanthroline-5,6- dione or the anionic ligand 4,7-diphenyl-1,10-phenanthroline-disulfonic acid, is converted to the active Cu(I) species within an ion-exchange matrix using zinc amalgam as the reducing agent. The Cu(I) complexes are then layered on top of a size exclusion matrix within a commercial microcentrifuge spin column; passing a mixture of an ethynyl-labeled biomolecule and an azide-bearing ligand through the column results in clean and efficient coupling. The authors demonstrate their methodology by glycosylating a DNA oligonucleotide, as well as labeling a membrane-penetrating peptide (octa-arginine) with a coumarin dye.

Keywords: bioconjugation; heterogenous click conjugation; DNA labeling; DNA glycosylation

Innovative new single-stranded DNA (ssDNA) library preparation methods have sparked great interest amongst ancient DNA (aDNA) researchers, especially after reports of endogenous DNA content increases of >20-fold in some samples. To investigate the behavior of this method, a team of researchers from Denmark generated ssDNA and conventional double-stranded DNA (dsDNA) libraries on 23 ancient and historic plant and animal specimens. The scientists report in the December 2015 issue of BioTechniques that they found ssDNA library preparation substantially increased endogenous content when dsDNA libraries contained <3% endogenous DNA, but enrichments are less pronounced when dsDNA preparations successfully recover short endogenous DNA fragments (mean size <70bp). These findings will help researchers determine when to utilize time- and resource-intensive ssDNA library preparations.

Keywords: next-generation DNA sequencing; sequencing library production; sequence analysis tools; ancient DNA; paleogenomics

 Finally, the Tech News slated for the December 2015 issue will focus on microbial and microbiome analysis techniques. We are coming to realize that the bacteria living on and within each of us creates an important community that influences both general health and susceptibility to disease. In this month’s feature, our contributing writing Jeffrey Perkel explores that latest techniques and tools being used to uncover the vast number, and diversity, of these microorganisms as scientists work to understand the complex and important interplay between our bodies and the microbial inhabitants within.

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