Immobilization of DNA primers
Primers synthesized on OAS supports linked to PS or CPG beads were synthesized in the 5′→3′ direction and remained attached to the PS or CPG support at the 5′-end. Thus, primers synthesized using OAS supports can be used directly as immobilized primers in PCR reactions without further manipulation. A potential disadvantage to the use of OAS supports is an inability to remove failure sequences generated during DNA synthesis. All other primers were purified after synthesis before coupling to beads, to remove failure sequences.
In order to be useful for a PCR reaction, bead-bound primers must remain intact during the thermal cycling process. We initially tested 5′-thiolated primers attached via heterobifunctional crosslinkers to amine-functionalized CPG, and, as expected (8
), this chemistry proved to be heat labile (data not shown). Thus, a soluble 5′-disulfide-modified primer was used to generate thiolated PCR product and immobilization took place as a separate step, subsequent to amplification. The remainder of the immobilized primer chemistries tested were heat stable and thus suitable for solid phase PCR experiments. Table summarizes the various combinations of primers, attachment chemistries and beads or polymeric supports which were used in this study.
Several amplification conditions were analyzed for the generation of a bead-bound DNA template that was functional for transcription by bacteriophage T7 polymerase. The primers used in the amplification reactions were as follows (see Table ): (i) soluble T7Prom and soluble T7Term; (ii) soluble T7Prom and soluble 5′-thiolated T7Term; (iii) soluble T7Prom and immobilized T7Term (PC-E, PC-H, T-PS or T-CPG); (iv) immobilized T7Prom (Pr-PS or Pr-CPG) and soluble T7Term. All PCR and transcription reactions yielded soluble DNA or RNA fragments of the expected size, as verified by electrophoretic analysis and exemplified in Figure A and B.
Figure 1 Soluble PCR amplification (A) and in vitro transcription (B) products from reactions containing indicated amounts of PC-E and PC-H immobilized primers. (A) Lane 1, 1 µM each soluble primers T7Prom and T7Term; lane 2, 1 µM and 50 nM primers (more ...)
The amount of DNA covalently bound to the CPG (PC-E or PC-H), OAS-CPG (T-CPG or Pr-CPG) or PS (T-PS or Pr-PS) beads was determined by 5′-end-radiolabeling DNA amplicons obtained following PCR with or without an immobilized primer. A 5′-biotin-labeled primer was used as one of two primers in the soluble control reaction to limit radiolabeling to just one of the 5′-ends of the amplicon and, thus, to more closely simulate the situation in which one primer is bead bound. Briefly, the gene for GFP was amplified from pETGFP using one of the following conditions: (i) two soluble primers (T7Prom and 5′-biotinylated T7Term); (ii) one soluble and one 5′-thiol-modified primer (T7Prom and 5′-SH-modified T7Term); (iii) one soluble and one immobilized primer [(T7Term and PC-E, PC-H, T-PS or T-CPG) or (Pr-PS or Pr-CPG and T7Term)]. The thiolated PCR amplicon generated under condition (ii) above was then immobilized to heterobifunctional crosslinker-modified CPG beads. All bead-immobilized templates and soluble PCR fractions were 5′-end-labeled as described in Materials and Methods. Non-specific DNA attachment was determined by amplifying a DNA template without any complementarity to the above soluble or immobilized primers and attempting to radiolabel the bead-associated product. Specific covalent attachment to the immobilized primers was determined by the amount of 32P incorporation associated with the beads minus any non-specific attachment. The specific activity of the [γ-32P]dATP was used to determine nmol DNA bound/g beads. Table compares the yield of DNA PCR amplicons on CPG, OAS-CPG or OAS-PS beads using each type of immobilized primer, or post-PCR attachment of thiolated amplicons. The results indicate a wide range of DNA attachment in the nmol DNA/g bead range for the immobilization chemistries surveyed. It is interesting to note that HDA silanized beads (H-SIAB, H-PEG and PC-H) consistently yielded higher quantities of covalently attached DNA than EDA silanized beads for all three chemical attachment methods tested. HDA (six carbons) is a longer silane than EDA (three carbons) and may be less densely packed on the surface than the EDA. A lower density of silane may provide a greater proportion of silanes that react with the heterobifunctional crosslinkers. DNA template attachment is most successful on beads to which primers were coupled prior to PCR via carbodiimide chemistry (PC-E and PC-H) and those which underwent post-PCR immobilization onto HDA crosslinker-modified CPG (see PC-E, PC-H, H-SIAB and H-PEG, Table ). The percentage of total PCR product that was bound to the immobilized beads during solid phase PCR is shown in Table . The mean bead-bound amplicon generated by PC-E and PC-H immobilized primers (expressed as a percentage of total amplicon) was 30.9 ± 0.73 and 60.4 ± 2.2%, respectively. Amplification using T-PS and T-CPG solid phase primers resulted in 51.8 ± 2.7 and 15.6 ± 3.5% of total amplicon bound to PS and CPG beads, respectively. Analysis of the four OAS supports indicated a comparatively low mean percentage of total amplicon bound to P-PS and P-CPG immobilized primers (3.2 ± 0.54 and 3.0 ± 0.53%, respectively). This result may be sequence-dependent as these primer–bead combinations used the T7Prom primer which contains a potential hairpin loop sequence that may have interfered with annealing of the primer to the target molecule.
Quantitation of bead-immobilized DNA
Percentage of total PCR amplicon immobilized
Solid phase PCR and in vitro transcription of bead-bound amplicons
In designing the solid phase PCR experiments, we selected the bacteriophage T7 transcriptional promoter and termination sequences as primers since the T7 promoter allows for high levels of tightly regulated transcription and, thus, would be useful for recombinant cloning and protein production applications (30
). We wished to create a solid phase PCR system that offered widespread utility for amplification and immobilization of a wide array of gene targets. With design of appropriate primers, however, we anticipate that this method would work equally well for other bacterial, viral or mammalian expression systems.
The solid phase PCR protocol was a modification of the procedure established by Saiki et al
). In the present study, solid phase PCR was performed using minimal, or ‘spike’, concentrations of the soluble equivalent to the immobilized primer. This was critical in the initial stages of PCR amplification to increase the ability of the DNA template to interact with bead-immobilized primers that settled in the bottom of the PCR reaction tube. Rassmussen and co-workers similarly used asymmetric PCR to immobilize PCR amplicons using PCR primers attached to microtiter plate wells (20
). Figure shows a representative result for solid phase PCR and in vitro
transcription with bead-immobilized PCR products using pET23 as the template DNA, which yields a 1.56 kb PCR product (gene encoding T4 MCP). For PCR and subsequent in vitro
transcription reactions, 10, 62 and 124 µg of beads attached to the indicated primer (PC-E or PC-H) were used. From our radiolabeling experiments, we have calculated the bead-bound primer concentrations in each PCR reaction to be 30, 175 and 352 nM (PC-E, Fig. A, lanes 3–5) and 77, 462 and 930 nM (PC-H, Fig. A, lanes 6–8). Figure A, lane 1, shows the soluble PCR product produced from two soluble primers T7Prom and T7Term (1 µM each). Lane 2 shows the soluble amplicon produced from two soluble primers T7Prom and T7Term (at 1 µM and 50 nM, respectively), under conditions which mimic the solid phase PCR conditions except that the immobilized primer is not present. The soluble PCR product from solid phase PCR reactions using 1 µM T7Prom, 50 nM soluble T7Term and the indicated amounts of PC-E and PC-H immobilized primers is shown in Figure A, lanes 3–8. The PCR reactions yielded a single soluble DNA band of the expected molecular weight (1.56 kb) for the gene encoding T4 MCP. The band intensity of the soluble PCR product shown in Figure A, lanes 3–8 (the same reaction conditions as used in lane 2 plus the indicated amount of immobilized T7Term primer), is of equal or greater intensity than the PCR product shown in lane 2. This is an expected result because the enzymatic extension of the immobilized primer creates additional template (antisense) strand that can be used to create more soluble product by the soluble primer present in the reaction. PCR reactions that contained soluble T7Prom and immobilized T7Term primers, but no soluble ‘spike’ of T7Term primer, resulted in undetectable soluble PCR product formation (data not shown). These results indicate that the small increase in soluble PCR product observed in lanes 3–8 is not due to dissociation of primer from the bead during amplification. The addition of a ‘spike’ of soluble T7Term primer was necessary to increase the likelihood of an interaction between the soluble coding strand and the bead-bound T7Term primer, which settles to the bottom of the PCR tube during amplification. To determine whether settlement of the beads during PCR was inhibiting efficient coding strand capture, we repeated select reactions but altered the protocol such that the reaction tubes were vortexed vigorously during the denaturation stage of several cycles (2, 4, 6, 8, 12 and 24). Contrary to our expectations, this resulted in a diminished yield of lower quality transcript (which reached a maximum of 66% of the non-vortexed reaction).
All bead-immobilized DNAs tested were functional as templates for in vitro transcription (representative data are shown in Fig. B). Following the PCR reactions, the 1.56 kb gene encoding T4 MCP attached to PC-E and PC-H beads was subjected to in vitro transcription. Electrophoretic analysis of the mRNA transcripts produced from these reactions is shown in Figure B. Lanes 1 and 2 show mRNA transcript production from two soluble DNA templates. As negative controls, the PCR reaction tubes that originally contained the soluble PCR products shown in Figure A, lanes 1 and 2, were washed (in parallel with the bead-containing PCR reactions, Fig. A, lanes 3–8) and used for in vitro transcription reactions. The lack of mRNA product in Figure B, lanes 3 and 4, demonstrated that the observed mRNA transcription observed in lanes 5–10 was not due to carry-over of soluble DNA non-specifically bound to the reaction tube. A comparison of Figure B, lanes 5–7 and 8–9 shows that although mRNA is produced with each bead type, the PC-E-immobilized PCR amplicon is more readily transcribed than DNA attached through PC-H primers. This result was also consistent with in vitro transcription results for amplicons produced from 5′-thiol-modified primers and subsequently attached to EDA- or HDA-modified CPG beads (E-SIAB, H-SIAB, E-PEG and H-PEG) in that more mRNA was produced when EDA-silanized beads were used. Paradoxically, the observed higher levels of amplicon attachment to HDA silanized beads may be detrimental to efficient in vitro transcription of the immobilized template DNA. This may be due to the inability of T7 polymerase and other required transcriptional components to efficiently access and transcribe because of tight DNA packing on and inside the CPG bead pores.
We examined a range of concentrations of bead-bound DNA template to establish whether or not a linear relationship exists between the quantity of bead-bound DNA template added to transcription reactions and the mRNA produced. A comparison of band intensities of mRNA transcribed from bead-immobilized DNA versus soluble DNA template showed a negligible difference in transcript production above 62 µg beads. The results relative to the Xenopus control (1.00) (Fig. B, lane 1) were 0.65, 0.80 and 0.84 for 10, 62 and 124 µg PC-E-immobilized DNA, respectively. The results for PC-H relative to the same control were 0.32, 0.28 and 0.32 for 10, 62 and 124 µg beads. These observations indicated a near saturated state with little change with increasing bead-bound amplicon concentration. For nearly all of the transcription experiments, reactions were periodically gently agitated during incubation. To determine whether the observed limitation in product formation was due to the restricted accessibility to the immobilized DNA of the transcription reactants as the beads settled to the bottom of the reaction tube, we repeated select experiments with continuous agitation during the incubation period. This resulted in an average increase of 19.5% product mRNA (when >50 µg beads were used per transcription reaction), verifying that exposure of the beads to fresh transcription reagents through mixing improved yield.
It is important to note that the relative intensities of the mRNA bands (Fig. B, lanes 5–7) were ~80% of the soluble Xenopus DNA transcriptional control (Fig. B, lane 1), demonstrating the efficiency of the solid phase templates in in vitro transcription reactions.
Our primary interest in developing a solid phase PCR amplification system using bead-bound primers was their potential use in repetitive in vitro transcription reactions. In vivo protein synthesis of polymeric proteins and certain mammalian proteins can be problematic due to the rearrangement or modification of highly repetitive DNA sequences, improper protein folding and cellular toxicity. Although in vitro transcription/translation systems could provide an alternative to in vivo protein synthesis, these methodologies can be impractical because of the laborious and often expensive necessity to resynthesize DNA templates after each transcription reaction. Conversely, automated solid phase peptide synthesis can be quite economical but may be limited by the length and sometimes the sequences of the desired protein. Bead-immobilized DNA templates would provide a convenient way to circumvent these problems by enabling the bead-bound DNA templates to be collected and recycled after each transcription reaction. This approach permits the synthesis of large proteins not available by automated synthesis and is more economical than conventional in vitro transcription/translation reactions. Figure shows the results of an experiment designed to determine whether DNA templates, coding for GFP (1.0 kb), immobilized via PC-E and E-PEG crosslinked supports, could be efficient templates for multiple sequential in vitro transcription reactions. In these experiments, bead-immobilized PCR amplicons were used as templates for in vitro transcription, then the immobilized DNA primers were collected by brief centrifugation, rewashed and exposed to fresh in vitro transcription reagents. Significant differences in sequential mRNA production were observed for each type of immobilized DNA template. Although E-PEG yielded high levels of transcript initially, these templates were unable to survive the recycling process beyond two or three cycles of repetitive rounds of in vitro transcription. This result proved to be the case for most immobilized DNA templates tested with the exception of PC-E-immobilized DNA. In this case, our findings indicated that PC-E-bound templates can be collected and recycled effectively for use in at least seven repetitive rounds of in vitro transcription.
Figure 2 Repetitive in vitro transcription using solid phase DNA templates. mRNA electrophoresed on a 1% agarose–formaldehyde gel. Lane 1, mRNA markers in kb as indicated; lanes 2–4 and 5–7, mRNA from the first, fifth and seventh (more ...)
Protein production from solid phase DNA templates
To determine whether mRNA transcripts produced from solid phase DNA templates were able to initiate translation, reagents for non-radioactive in vitro translation were added to the supernatants from in vitro transcription reactions. The biotinylated protein products were detected using a streptavidin–HRP conjugate with TMB as substrate. Figure A shows a western blot of biotinylated products produced from solid phase templates through a coupled in vitro transcription/translation system. All solid phase DNA templates produced the desired protein product (57 kDa T4 MCP). A useful feature of the bead immobilization methodology is the ability to amplify and to immobilize either single or multiple DNA target sequences. The results of such a mixing experiment are shown in Figure B. In this experiment, the genes for GFP and T4 MCP were amplified via solid phase PCR using the immobilized PC-E primer and plasmids pETGFP and pET23 as templates (either separately or mixed together). The bead-immobilized amplicons from each solid phase PCR reaction (GFP, MCP and GFP + MCP) were collected, washed and used as templates for in vitro transcription/translation reactions. Figure B, lanes 3 and 4, shows the separate production of GFP and T4 MCP and lane 5 shows in vitro translation of both proteins simultaneously from a mixture of immobilized templates. Thus, the addition of multiple DNA gene templates to solid phase PCR reactions yields efficient immobilization of both genes and the subsequent production of multiple protein products.
Figure 3 Western blot showing production of T4 MCP from immobilized DNA templates. (A) DNA templates used for in vitro transcription product production and subsequent protein translation are as follows: lane 1, soluble DNA encoding MCP; lane 2, no DNA control; (more ...)