Cartridge Methods for Oligonucleotide Purification

Protocols are given for purification of oligonucleotides by dimethoxytrityl‐sensitive and affinity desalting methods. The protocols are applicable for many of the convenient disposable products available for rapid oligonucleotide purification, clean‐up by selective adsorption, and elution on solid‐phase media. Many of these products are prepackaged, single‐use cartridges or columns filled with affinity or size‐exclusion media.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152627/1/cpnc1007.pd

Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip

The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. We developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. We detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. We also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing

Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip

The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. We developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. We detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. We also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing

Base Composition Analysis of Nucleosides Using HPLC

In this protocol, nuclease digestion of an oligonucleotide is followed by dephosphorylation and HPLC analysis of the monomers on a reversed‐phase C18 column. This method can be used to detect and quantitate a wide variety of nucleobase modifications in oligonucleotides. Integrated areas of the nucleoside chromatogram give precise quantitation of nucleoside composition when the relative extinction coefficient cofactors are applied to the sum of the areas of the four bases. The protocol is also useful for analysis of oligonucleotides containing conjugated moieties and carbohydrate modifications.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152749/1/cpnc1006.pd

λ Integrase Complementation at the Level of DNA Binding and Complex Formation

Site-specific recombinases of the λ Int family carry out two single-strand exchanges by binding as head-to-head dimers on inverted core-type DNA sites. Each protomer may cleave its own site as a monomer in cis (as for Cre recombinase), or it may recruit the tyrosine from its partner in trans to form a composite active site (as for Flp recombinase). The crystal structure of the λ Int catalytic domain is compatible with both cleavage mechanisms, but two previous biochemical studies on λ integrase (Int) generated data that were not in agreement. Support for cis and trans cleavage came from assays with bispecific DNA substrates for λ and HK022 Ints and from functional complementation between recombination-deficient mutants, respectively. The data presented here do not provide new evidence for cis cleavage, but they strongly suggest that the previously described complementation results cannot be used in support of a trans-cleavage mechanism. We show here that IntR212Q retains some residual catalytic function but is impaired in binding to core-type DNA on linear substrates and in forming higher-order attL intasome structures. The binding-proficient mutant IntY342F can stabilize IntR212Q binding to core-type DNA through protein-protein interactions. Similarly, the formation of higher-order Int complexes with arm- and core-type DNA is boosted with both mutants present. This complementation precedes cleavage and thus precludes any conclusions about the mechanism of catalysis. Cross-core stimulation of wild-type HK022-Int cleavage on its cognate site (in cis) by mutant λ Ints on bispecific core DNA suicide substrates is shown to be independent of the catalytic tyrosine but appears to be proportional to the respective core-binding affinities of the λ Int mutants