Michael W. Reed

Multiple pH measurement during storage may detect bacterially contaminated platelet concentrates.

BACKGROUND: Bacterial contamination or platelet (PLT) metabolism can change the pH of stored PLT concentrates (PCs). Measurement of pH for quality control is currently done on a limited basis. An easy noninvasive method was developed to obtain sequential pH measurements over time, without risking contamination and/or consuming PCs.

Chemistry of Minor Groove Binder–Oligonucleotide Conjugates

Various types of minor groove binders have been attached to synthetic oligodeoxynucleotides, and the interactions of these conjugates (MB‐ODNs) with DNA are reviewed here. MB‐ODNs have enhanced DNA affinity and have improved the hybridization properties of sequence‐specific DNA probes. Short MB‐ODNs hybridize with ssDNA to give more stable DNA duplexes than unmodified ODNs with similar lengths. Mismatch discrimination of short MB‐ODNs is enhanced in comparison to longer unmodified ODNs. The stronger binding of MB‐ODNs allows for more stringent hybridization conditions to be used in DNA probe‐based assays. MB‐ODNs are especially useful in quantitative “real‐time” PCR assays since they bind efficiently during the high‐temperature primer extension cycle. The synthesis and biophysical chemistry of MB‐ODN conjugates are reviewed here. Four published structural classes of MB‐ODNs and their various dsDNA binding modes are discussed, and the well‐characterized DPI3‐type MB‐ODNs and their interactions with ssDNA target strands are described in detail.

Extraction of MS2 Phage RNA from Upper Respiratory Tract Specimens by Use of Flat Glass Devices

ABSTRACT The isolation of pure nucleic acids from clinical samples is a crucial step in the molecular diagnosis of viral infections by nucleic acid testing (NAT). In this study, novel flat glass devices (cards) were demonstrated to support the rapid and efficient extraction of nucleic acids from upper respiratory tract specimens (nasal washes and swabs). The performance of the nucleic acid extraction cards was directly compared to an existing standardized and automated platform for viral extraction from these types of specimens. The flowthrough card method improved the speed of nucleic acid purification and accommodated larger sample volumes in extraction of bacteriophage MS2 RNA from the various specimen matrices. The dynamic range and estimated sensitivity of the card extraction method for reverse transcriptase quantitative real-time PCR (RT-qPCR)-based detection approximate those of the standardized magnetic glass bead extraction method used in this study.

Synthesis, characterization, and application of substituted pyrazolopyrimidine nucleosides.

This unit describes, in detail, the preparation of 3-aminopropyl-substituted pyrazolo[3,4-d]pyrimidine analogs of the purines deoxyadenosine (dA) and deoxyguanosine (dG). Phosphoramidite reagents of these so-called aminopropyl-PPA and -PPG nucleosides (AP-PPA and AP-PPG, respectively) allow introduction of amino linkers into internal positions of synthetic DNA strands. Synthesis of suitably protected AP-PPA and AP-PPG phosphoramidites are described. The stepwise alkynylation, hydrogenation, selective protection, and phosphoramidite synthesis is similar for both the PPA and PPG analogs. To demonstrate the application of these reagents, a protocol is given in which a simple DNA strand is synthesized and conjugated to a lipophilic activated ester (dabcyl-SE) to form a stable amide linkage. Utility of this chemistry for preparing internally modified DNA conjugates is discussed.