Jochen
Hoffmann
*a,
Sebastian
Hin
a,
Felix von
Stetten
ab,
Roland
Zengerle
abc and
Günter
Roth
abc
aRoland Zengerle, Laboratory for MEMS Applications, Department of Microsystems Engineering - IMTEK, University of Freiburg, Georges-Koehler-Allee, 103, 79110, Freiburg, Germany. E-mail: jochen.hoffmann@imtek.de; Fax: +49 761 203 73299; Tel: +49 761 203 73234
bHSG-IMIT, Wilhelm-Schickard-Straße 10, D-78052, Villingen-Schwenningen, Germany
cBIOSS-Centre for Biological Signalling Studies, University of Freiburg, 79110, Freiburg, Germany
First published on 13th March 2012
A universal protocol for grafting PCR primers onto glass, PDMS, COP, COC, and PP is developed and evaluated by solid-phase PCR (SP-PCR). Primers are immobilized in a PCR compatible way featuring spots with high homogeneity and integrity. Furthermore, we show a protocol for binding a PCR product to immobilized PCR primers via solid-phase PCR (SP-PCR). Previously reported “enhanced SP-PCR” (Z. Kahn et al. Anal. Biochem., 2008, 375, 391–393) is improved in terms of factorial signal increase from 9.9 to 86.8 and specificity from 11.7 to 45.9. The presented immobilization- and SP-PCR protocols may enable integration of DNA microarrays directly into microfluidic lab-on-a-chip cartridges of various materials for analysis via SP-PCR. Beside planar microarrays, another interesting application could be to coat the inner surfaces of a chip with PCR primers to recover generated PCR products in digital PCR systems.
A key requirement for grafting PCR primers onto substrates in a SP-PCR compatible manner is that the chemical bond between oligonucleotide and array-substrate withstands thermocycling conditions such as incubation at temperatures around 95 °C. In addition, the binding chemistry must ensure a free 3′-OH end to be accessible and extendable by a DNA polymerase. To realize such thermally stable and oriented bonds, a number of different immobilization protocols have been developed for glass (Fig. 1) but rarely for polymers.7,8
Fig. 1 Various chemistries reported for grafting oligonucleotides to specific materials. Numbers indicate reference. indicates validation of temperature stability complying with SP-PCR, indicates validation for DNA immobilization, not tested for SP-PCR. |
Today, several immobilization protocols have proven their suitability for array based SP-PCR5,6,9–11 whereas others have not yet been tested12–16 (Fig. 1). A comprehensive overview over different immobilization strategies is given by Todt et al.17 and Sassolas et al.18Direct immobilization methods covalently bind oligonucleotides to the substrate. An example for such a method is the EDC chemistry, where 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC) mediates the linkage of 5′-NH2 modified DNA to hydroxylated substrates.4 Another way is to UV-crosslink poly-dT modified DNA directly to glass10 or embedded in a 3-D hydrogel matrix to plastic substrates.3 Besides that, acrydite-modified oligonucleotides in an acrylamide gel can be spotted onto a substrate for oriented co-polymerization.5Indirect immobilization methods utilize homobifunctional linking molecules like glutaraldehyde11 and 1,4-phenylene diisothiocyanate (PDITC)6 for attaching oligonucleotides to activated or modified (mostly aminosilanized) surfaces. Fig. 1 summarizes immobilization chemistries that have been developed for different materials whereof only some have been applied to microarray based solid-phase PCR. From these data and current literature19,20 it becomes clear that there is a great technical need for a simple, robust, and versatile immobilization strategy that is applicable to a variety of different substrates and compatible to SP-PCR.
This work aims to identify and verify a universal protocol for grafting oligonucleotides to a variety of different substrates like glass and polymers fulfilling the requirements for SP-PCR. To address this issue, the PDITC chemistry was investigated in more detail. The chemistry is based on the homobifunctional linking molecule PDITC6,17,21 and has already been successfully demonstrated for SP-PCR,6 and the fabrication of DNA microarrays on glass6 and polypropylene (PP).13 The objective of this study is to graft arrays of solid-phase primers with a spot density of >400 spots cm−2 onto polydimethylsiloxane (PDMS), PP, cyclic olefin polymer (COP), cyclic olefin copolymer (COC) as well as on glass, and to investigate the SP-PCR performance on these substrates.
Fig. 2 Fluorescent scans of dilution series printed onto COP, PP, COC, and PDMS. Eighty replicas of a dilution series per printing-block are immobilized onto the different materials, distributed over 20 prints (row A) with 4 replicas per print (row B). Each droplet has a volume of 1 nL, depositing on each sub-array (from left to right) 50 amol, 100 amol, 200 amol, 400 amol, 800 amol, and 1600 amol of primer. Scanning is done in the Cy5 channel with exposure times of 500 ms. Intensities can be qualitatively assessed by using the colour scale on the right. (*) misalignment of individual images due to the scanning software FIPS. |
Scheme 1 DNA immobilization using PDITC chemistry. On the surface of an unmodified substrate (A), hydroxyl groups are generated using oxygen plasma (B); next, the aminosilane APTES reacts with the hydroxyl groups, leaving an amine terminated surface (C); the homobifunctional PDITC binds to the amine groups, terminating the surface with thiocyanate groups (D); finally, an oligonucleotide with 5′- amine modification is covalently bonded to the surface (E). |
Scheme 2 Schematics of solid-phase PCR used for evaluation of the immobilization protocol. Initially, a reaction compartment comprises solid-phase primers as well as forward (fwd) and reverse (rev) primers in an asymmetric ratio (A); in the beginning, PCR proceeds preferably in the liquid phase, until the fwd primer is depleted (B); then, solid-phase PCR dominates, where the immobilized primer is extended by polymerase activity. Biotin-dUTPs are incorporated into the reaction for labeling (C, D) and subsequent visualization of the SP-PCR product by staining with streptavidin-Cy5 (E, F). |
Fig. 3 Measured fluorescence intensities of the Cy5 primer dilution series bound to the investigated substrates. The signal of the 0.05 μM Cy5 primer is the lowest amount which can be distinguished from the background signal (spotting buffer only). From 0.05 μM to 0.80 μM, signals increase linearly, only 1.60 μM spots shows lower intensities than expected, indicating a saturation limit. Numerical values represent the integral over the whole area of each spot. |
Fig. 4 Cy5 scans of the arrays containing the Cy5 primer as not extendable spotting control (lane a), the extendable primer as extension control (lane b), and the not extendable primer as negative control (lane c) in rows of four spots per substrate. Scanning is done before (row A) and after SP-PCR and staining (row B). Highly specific extension of the extendable primer is observed on all polymers and also on glass, although a remarkable amount of Cy5 primers is lost on all substrates. |
Fig. 5 Measured fluorescence intensities for each substrate before (crossed bars) and after (bold bars) solid-phase PCR. On all substrates, signals from the extendable primer significantly increase after SP-PCR, whereas signals remain close to the background for the not extendable primer, indicating an excellent SP-PCR system. All solid-phase primers are spotted in end-concentrations of 2.00 μM, for which reason, intensities of the Cy5 primer before PCR are higher than intensities from Fig. 2 (end concentration of Cy5 primer: 1.60 μM). Gaussian standard deviations include slide to slide variations, n = 16. |
A prerequisite in SP-PCR is the extendibility of solid-phase primers. We determined the factorial signal increases of each type of solid-phase primer as a ratio of the intensities after and before SP-PCR. Measured signal increases of extendable primers are between 43.9 and 86.8, compared to 1.4–10.0 in the paper “enhanced SP-PCR” published in 2008.1 Signal increases of the not extendable primers (negative controls) are between 1.1 and 2.5 (Fig. 5, Table 1). When calculating specificity, in accordance to1 as the ratio between the extendable and not extendable primer after SP-PCR, we also obtain a higher specificity of 31.7 (± 4.3) (glass), 45.9 (± 20.9) (COP), 21.6 (± 3.3) (PP), 21.7 (± 2.8) (COC), and 34.2 (± 6.7) (PDMS) compared to published values of 7.6–11.7.1 The background increased only slightly after SP-PCR with factors between 1.5 (± 0.6) (COP) and 2.6 (± 0.3) (PP), which is due to the addition of 0.1% BSA (w/v) to the PCR amplification mix, preventing the absorption of PCR-components to the substrates.26 We obtained highly specific and significant signal increases of solid-phase primers on all substrates, indicating a well-balanced SP-PCR system. Details of the SP-PCR system as well as the improvements in treatment of the surfaces are described in the ESI.†
Glass | COP | PP | COC | PDMS | |
---|---|---|---|---|---|
a The factorial signal increase is calculated by dividing the measured intensity signals before and after SP-PCR, n = 16. | |||||
Extendable primer | 72.5 ± 6.7 | 45.7 ± 6.9 | 53.6 ± 5.4 | 43.9 ± 4.8 | 86.8 ± 10.2 |
Not extendable primer | 2.2 ± 0.4 | 1.1 ± 0.6 | 2.2 ± 0.6 | 2.2 ± 0.2 | 2.5 ± 0.6 |
Background | 1.6 ± 0.5 | 1.5 ± 0.6 | 2.6 ± 0.3 | 2.2 ± 0.3 | 2.4 ± 0.7 |
As an outlook, our SP-PCR protocol can be an interesting approach for detecting simultaneously multiple targets in microarrays analysis.27 In the field of lab-on-a-chip systems,28,29 comprehensive sample preparation capabilities can be combined by the multiplexing capabilities of microarrays based SP-PCR by directly grafting microarrays into lab-on-a-chip substrates. Beside microarrays, another interesting application could be to apply this immobilization protocol to the inner surface of a chip for digital PCR (dPCR) as it has been recently shown by our group.30 Thereby, generated PCR products can be recovered, which is an interesting novelty compared to currently published systems for dPCR.31–33
Footnote |
† Electronic Supplementary Information (ESI) available: See DOI: 10.1039/c2ra01250b/ |
This journal is © The Royal Society of Chemistry 2012 |