Technical Bulletin

pdfOligoMix® is a versatile, innovative, custom product for genomics discoveries. We synthesize thousands of oligonucleotide sequences at once in massive parallel on a microarray chip and then cleave the oligos, releasing them into solution in a single microtube. Synthesis occurs via standard DMT chemistry assuring efficient stepwise yield and high quality final product. The product is delivered as a pool in a single microtube.

Users need to be aware that the amount of each individual sequence is very small and the raw OligoMix® product must be amplified prior to use. To date, many different methods have been developed for successful OligoMix® amplification (see publications). However, there are drawbacks to some amplification methods such as conventional PCR.

Recently, an international team, comprised of researchers from the Dana-Farber Cancer Institute, Harvard Medical School, and the Technical University Dresden, have developed a rolling-circle method for amplification (RCA) that overcomes the drawbacks associated with other amplification methods while also remaining economical and easy to use. This new method provides an alternative amplification strategy for selectively purifying subsets of hundreds to thousands of single-stranded oligonucleotides, which can be useful for applications such as DNA origami, multiplexed PCR, gene synthesis, fluorescence in situ hybridization (FISH), targeted sequencing and others. The team used LC Sciences’ OligoMix® to demonstrate the amplification method and subsequent application of the oligonucleotide library as Oligopaint probes for a FISH, a single-cell assay.

Rolling Circle Amplification of OligoMix®

Figure 1: Oligonucleotide amplification by circle-to-circle amplification.

(a) For selective amplification of their oligonucleotide sub-pools, the researchers used small aliquots of their total OligoMix® pool and subjected the aliquots, in parallel, to three sequential rounds of RCA. To enable amplification of the subpools, the reverse complement of each target sequence was extended with two nicking sites, a restriction site and one to two orthogonal subpool-specific barcodes of 10 nucleotides. Ligation allowed the template strands to cyclize into circular template strands.

(b) Subsequently, a polymerase with high processivity and strand displacement capacity is added which synthesizes chain-like repeated copies of the circular template. The researchers observed a 10,000X increase in transcript copy numbers from one round of RCA under optimal conditions. Two subsequent rounds of RCA were incorporated to deliver quantities suitable for the FISH application.

(c) For the second round of amplification, a second-round primer (blue dotted line) was hybridized to the intervening region of the first concatemer and the resulting double-stranded recognition site (orange) was digested with a restriction enzyme (HindIII) into monomers. A heat-inactivation step is used to inactivate the restriction enzyme for the next steps and the cut fragments of the second-round primer dissociate during this step. On cooling, an excess of the second-round primer hybridizes to the cut monomeric units and colocalizes the ends for a second ligation and RCA step. This second circular template has the reverse complementary sequence of the first circular template.

(d) These steps are repeated in the third round of amplification with the third-round primer to yield the final concatemer.

(e) Following the third round of RCA, one or two nicking primers are hybridized to this concatemer and a double-nicking reaction excises the intervening region from the target sequences (green). Because the nicking enzymes cut outside of their recognition sites, the entire intervening sequence are removed to yield the (green) sequences of interest of a given subpool.

This entire process can be performed as a one-pot reaction, without any intermediary workup steps and the excess of nicking primers, the intervening sequences, residual undigested concatemers (as well as the excess primers and enzymes) can be removed using a final anion exchange chromatography step.

For scalable production of primer-free single-stranded oligonucleotides, RCA offers numerous advantages compared to standard PCR:

  1. RCA generates only single stranded products, so the final concentration of oligonucleotide copies can be ~15 times higher than in PCR, because double-stranded products re-anneal to themselves at high concentrations after denaturation which can prevent further amplification.
  2. RCA does not require quick temperature changes which impede the scalability of PCR reactions, because it is an isothermal process.
  3. RCA produces less waste material (less reverse PCR strands) which can be a cost factor for PCR reactions.
  4. There is less sequence-dependent amplification bias with RCA than with PCR, as fewer rounds of amplification are needed.
  5. Nicking reactions are about 10 times more efficient in RCA, which results in a lower cost in the long run.

The research team applied RCA to an OligoMix® oligonucleotide library used for FISH, a single-cell assay that allows direct visualization of the in situ positioning of DNA and RNA molecules, to observe what affect RCA would have on their final data. They designed a multiplexed library which targeted a centromere-proximal portion of the right arm of Drosophila chromosome 3 and subsequently performed three-color FISH in S2R+ cells. One probe set consisted of a 679 oligonucleotides targeting a 56-kilobase region at 82A1, while another consisted of 719 oligonucleotides targeting a 50-kilobase region at 82D2-82D5 and a third, single Cy5-labelled oligonucleotide targeted the highly repetitive dodeca percentromeric satellite sequence.

The results were striking, as the researchers observed crisp, clean signals with very low background. They staining efficiency was reported from 94-100%.

Oligopaint FISH probes

Figure 2: Highly efficient Oligopaint FISH with probe sets made by barcoded c2ca

(a) Oligopaint probes strategy

(b) Two confocal images of three-color FISH performed in Drosophila S2R+ cells with a probe set of 679 oligonucleotides targeting 56 kb at 82A1 (green), a probe set of 719 oligonucleotides targeting 50 kb at 82D2-82D5 (red) and a single oligonucleotide targeting the highly repetitive dodeca percentromeric satellite sequence (white).

Conclusions

Through the data presented in this recent paper, Shih et al. have demonstrated that rolling circle amplification can be used as a robust amplification method for producing primer-free single-stranded oligonucleotides from chip-synthesized oligonucleotide libraries such as OligoMix®. The method was proven to offer a number of benefits which could be very useful to a number of DNA nanotechnology, genetics and synthetic biology applications, as it overcomes many of the limitations associated with conventional PCR amplification. These benefits could prove to be very valuable to researchers who are looking for an economical way to make the most of their OligoMix® libraries.

The ultimate strategy for OligoMix® amplification will be dependent on the specific needs for your application.


Visualizing genomes with Oligopaint FISH probes Cell Sample Preparation for Single Cell RNA Analysis Studies