Clonal rolling circle amplification for on-chip DNA cluster generation

Abstract

Generation of monoclonal DNA clusters on a surface is a useful method for digital nucleic acid detection applications (e.g. microarray or next-generation sequencing). To obtain sufficient copies per cluster for digital detection, the single molecule bound to the surface must be amplified. Here we describe ClonalRCA, a rolling-circle amplification (RCA) method for the generation of monoclonal DNA clusters based on forward and reverse primers immobilized on the surface. No primer in the reaction buffer is needed. Clusters formed by ClonalRCA comprise forward and reverse strands in multiple copies tethered to the surface within a cluster of micrometer size. Single stranded circular molecules are used as a target to create a cluster with about 10 000 forward and reverse strands. The DNA strands are available for oligonucleotide hybridization, primer extension and sequencing.

Keywords: forming DNA clusters; isothermal amplification; solid surface.

Figure 1:

Illustration of the ClonalRCA mechanism. (A) Linear template molecules with left and right adaptor arms (dotted line) are used for (B) ligase reaction to form circular template molecules. (C) After denaturation of circular template DNA, DNA is hybridized to primers immobilized to the solid support (horizontal bar in gray). (+) DNA strand and (−) DNA strand binds to the primers because, forward (black vertical lines on surface) and reverse primer (red vertical lines on surface) are immobilized to the surface. In addition to forward and reverse primer, spacer oligonucleotides (dotted vertical lines on surface) are immobilized to the solid support. The spacer oligonucleotides are used to regulate the DNA copy number and the DNA crowding within the cluster. After hybridization all non-hybridized circles are eliminated by an intense washing step. Thereafter an amplification reaction mixture is added and the surface is incubated at 38 °C for 2 h. During this time, the first strand is synthesized from the target circle (D) which re-hybridized to the complementary primers immobilized on the solid support and primer extension occurs (F). During the reaction progress, less primers are available for re-hybridization. Therefore, more and more single-stranded DNA cannot re-hybridize and remains single-stranded (F).

Figure 2:

Demonstration of ClonalRCA. (A) In order to show the hybridization efficiency independent of GC content, a circular library from a PCR tumor panel was hybridized to the primers immobilized on a surface. A real-time PCR was performed from hybridized circles of 4 of the ∼600 target circles (DDR-2, KRAS, DDR2-3, and IDH2). The four targets span approximately the GC content of the whole library. The relative hybridization efficiency was very similar and showed no significant difference between targets of different GC content ranging from 30% to > 60%. (B) The average DNA copy number of the ClonalRCA cluster (amplification factor) was determined after ClonalRCA by real-time PCR. ClonalRCA resulted in an amplification factor of ∼10 000-fold in average independent of the GC content of the target circles. (C) The surface was coated with forward primer, reverse primer, and the short spacer oligonucleotide in a ration of 1:1:4. In order to increase DNA copy number per cluster and the DNA density within a spot, the fraction of spacer oligonucleotides bound to the surface was reduced to zero. The amplification factor increased from 13 000-fold to > 40 000-fold indicating that the ratio of the spacer oligonucleotide compared to the specific primers binding the target circles regulated the copy number per cluster. (D) ClonalRCA clusters were analyzed on a flow cell surface using YOYO-1 as DNA specific dye. In order to make sure that no monoclonal clusters are fused, a limited amount of DNA was used for hybridization. The spots were homogeneously distributed, were of a round shape and showed a size of ∼1.2–2 µm.

Figure 3:

Presence of both strands after ClonalRCA (A) The (+) strand and the (−) strand were hybridized with strand specific primers tagged by different sequences (red sequence for reverse primer binding to the (+) strand and blue sequence for the forward primer). (B) After primer extension both extension products could be quantified individually during real-time PCR using the tags as primer binding site (while primer 810 bound to reverse primer extension product, primer 800 hybridized to forward primer extension product). In addition the total amount of ClonalRCA products and primer extension products could be determined by primers (Primer 80, 81) binding to the target circle sequence. (C) Both strand-specific real-time PCR reactions resulted in very similar Cq values indicating that both strands are present in ClonalRCA clusters. The Cq value of the primer pair binding to the target circle sequence was ∼ 2.5–3 cycles lower than the Cq value of the strand specific real-time PCR reactions indicating a reduced primer hybridization efficiency. (D) Primer hybridization efficiency was determined in quantitative PCR experiments comparing strand-specific PCRs and strand-unspecific PCR. A primer binding efficiency of 22–28% [hybridization to (+) strand] and of 50–69% [hybridization to (−) strand] was determined.

Figure 4:

Forward and reverse sequencing of ClonalRCA clusters. Circular target-1 DNA was used for ClonalRCA. After forward sequencing the newly synthesized strand was removed by denaturation and reverse sequencing was initiated. Four representative images of the same position were shown with the relevant bases in bold letters: (A) Image of forward sequencing cycle 3 (base T, dUTP-R6G), (B) image of reverse sequencing cycle 4 (base T, dUTP-R6G), (C) image of forward sequencing cycle 17 (base A, dATP-ROX), (D) image of reverse sequencing cycle 18 (base A, dATP-ROX).