Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries

Abstract

Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.

Spatially resolved, highly multiplexed RNA profiling in single cells

“…Denaturing poly-acrylamide gel electrophoresis and absorption spectroscopy were used to confirm the quality of the probes and revealed that this probe synthesis protocol converts 90–100% of the reverse-transcription primer into full length probe and of the probe that is constructed, 70–80% is recovered during the purification step. This protocol is similar to another recently published protocol (23) but provides a substantially larger yield. …”

“…To generate the massive number of encoding probes required, we amplified them from array-derived oligonucleotide pools containing tens of thousands of custom sequences using a modified form of the oligopaint protocol comprising in vitro transcription followed by reverse transcription (Fig. S3; Materials and Methods, “Probe Synthesis” section) (22, 23). This two-step labeling approach significantly diminished the total hybridization time for an experiment: we found that efficient hybridization to the readout sequences took only 15 minutes whereas efficient direct hybridization to cellular RNA required more than 10 hours.…”

Chromosome-Specific Painting in Cucumis Species Using Bulked Oligonucleotides

“…Each synthesized library contained 400 ng DNA. The libraries were first amplified using an emulsion PCR protocol (Murgha et al 2014). Briefly, the PCR mixture (aqueous phase,100 ml) consisted of 0.2 ng DNA from the library, 0.5 mM each of F (T7 RNA polymerase promoter plus 6 additional nucleotides) and R (CGTGGTCGCGTCTCA) primers ( Figure 1B), 0.2 mM dNTPs, 0.5 mg/ml bovine serum albumin, 4 units of Phusion High-Fidelity polymerase (New England Biolabs, catalog no.…”

Combined in Vitro Transcription and Reverse Transcription to Amplify and Label Complex Synthetic Oligonucleotide Probe Libraries

“…Finally, 288 (6.92%) of the 4161 RNA molecules were not detected across the 9 arrays for the 3 detection experiments, namely: RNA library composition, unlabeled RNA library by reporter DNA library, and post-labeling of unlabeled RNA to confirm reporter DNA calls. The missing oligonucleotides in each of the respective custom libraries may be attributed to any of the following: synthesis failure, drop-out during in vitro transcription or reverse transcription, hybridization failure (probe synthesis failure, secondary structure, steric hindrance), or a concentration of individual oligonucleotides that is below the detection threshold (17). …”

“…For example, the process is modified to generate multiple fluorescent sub-libraries for FISH from a single de novo synthesized library. Finally, the process can be scaled up to produce several hundred micrograms of labeled probe libraries (17). …”