DNA analysis with multiplex microarray-enhanced PCR

Abstract

We have developed a highly sensitive method for DNA analysis on 3D gel element microarrays, a technique we call multiplex microarray-enhanced PCR (MME-PCR). Two amplification strategies are carried out simultaneously in the reaction chamber: on or within gel elements, and in bulk solution over the gel element array. MME-PCR is initiated by multiple complex primers containing gene-specific, forward and reverse, sequences appended to the 3' end of a universal amplification primer. The complex primer pair is covalently tethered through its 5' end to the polyacryl- amide backbone. In the bulk solution above the gel element array, a single pair of unattached universal primers simultaneously directs pseudo-monoplex PCR of all targets according to normal solution-phase PCR. The presence of a single universal PCR primer pair in solution accelerates amplification within gel elements and eliminates the problem of primer interference that is common to conventional multiplex PCR. We show 10(6)-fold amplification of targeted DNA after 50 cycles with average amplification efficiency 1.34 per cycle, and demonstrate specific on-chip amplification of six genes in Bacillus subtilis. All six genes were detected at 4.5 pg of bacterial genomic DNA (equivalent to 10(3) genomes) in 60 independent amplification reactions performed simultaneously in single reaction chamber.

Figure 1

Working model for MME-PCR. (A) Cycle 0 shows two gel elements (Pad A and Pad B) containing unique complex PCR amplification primers. Every complex primer consists of a universal primer (U1 or U2) and gene-specific forward (F_a, F_b, etc.) or reverse (R_a, R_b, etc.) primers. Free-floating U1 and U2 primers are supplied as part of the amplification cocktail in solution over the chip. (B) In Cycle 1, the DNA targets in solution anneal to their respective gene-specific PCR primer sequences within the gel elements, and the DNA polymerase extends from the gel-immobilized primers. (C) In Cycle 2, a nucleic acid ‘bridge’ is formed between the extended strand (from Cycle 1) and the complementary reverse primer. For the sake of simplicity, only one extended primer on each pad is shown in (C). Once the ‘bridge’ is formed, the polymerase can extend the second strand and synthesize a modified target containing sequences (cU1 and cU2) that are complementary to universal primers U1 and U2. (D) By Cycle 3, free-floating universal amplification primers U1 and U2 can then serve as amplification primers, not only on the gel elements themselves (D) but also in solution (E). (E) Beginning in Cycle 4, then, a pseudo-monoplex PCR amplification becomes established in the bulk solution over the gel element array. (F) Pseudo-monoplex PCR eventually dominates the reaction due to higher amplification efficiency in solution. (G) In parallel, hybridization kinetics will force more of the amplified product into the gel element arrays and therefore ‘accelerate’ the within-gel PCR amplification reaction. (H) After PCR, the microarrays were washed and the immobilized PCR products detected and confirmed by hybridizing with internal fluorescently labeled reporting probes. Thus, only the products of within-gel amplification are specifically detected. Parental and newly synthesized nascent DNA strands are shown as solid and dashed lines, respectively.

Figure 2

Modified targets containing universal sequences are produced and released into solution during MME-PCR. (A) Microarrays were manufactured by immobilizing complex primers U1-bm16S-1 and U2-bm16S-2 into 10 replicate gel elements. Universal primers U1, U2 and 10⁶ copies of target T-bm16S-723 were included in the amplification cocktail. (B) Amplification with different combinations of gene-specific and universal primers. Primers U5-bm16S-5 and U6-bm16S-4 were immobilized within gel elements, and universal primers U5, U6 and 10⁶ copies of target T-bm16S-726 were supplied in the amplification cocktail. In both experiments, MME-PCR proceeded for 50 cycles. The solution-phase reaction mixture was analyzed on 1.2% agarose gels. M is low weight marker (A) and a 100 bp DNA marker (B). Numbers 1 and 2 shown to the left of figures indicate amplified fragment and primer dimers amplification artifacts, respectively.

Figure 3

MME-PCR with template DNA of different complexity and sizes. (A) Microarrays were manufactured by immobilizing complex primers U5-bm16S-5 and U6-bm16S-4 into 10 gel elements on replicate arrays. The amplification of the 152 bp fragment was supplied with universal primers U5, U6 and either 10⁶ copies of target T-bm16S-157 (157 bp DNA fragment), T-bm16S-726 (726 DNA fragment), B.mycoides genomic DNA or no DNA. After 50 cycles of MME-PCR, the microarrays were hybridized with internal probe P-bm16S. (B) The images were quantified, and the data plotted in arbitrary fluorescent units as the mean ± confidence interval, as described in Materials and Methods.

Figure 4

MME-PCR is specific. (A) Microarrays were manufactured by immobilizing six different pairs of complex primers, designed for the amplification of six different functional genes of B.subtilis: dnaK, ebrA, fruR, grpE, spo0A and yisY (see Table 1 for the primer list). Each pair of primers was immobilized into the 10 replicate gel elements on each array. The reaction mixture was supplied with universal primers U5, U6 and 4.5 ng of B.subtilis genomic DNA (10⁶ copies). After PCR, the microarrays were hybridized with a mixture of six reporting probes, P-bsDnaK, P-bsEbrA, P-bsFruR, P-bsGrpE, P-bsSpo0A and P-bsYisY (Table 1), to verify the identity of the resulting amplicons. Intensities of hybridization signals are shown in arbitrary units. (B–G) The experiment described in (A) was repeated, except that the genomic DNA was replaced with 10⁶ copies of one of the six amplicons, T-bsDnaK, T-bsEbrA, T-bsFruR, T-bsGrpE, T-bsSpo0A or T-bsYisY (Table 1). Labels above each panel (A–G) identify a target supplied with amplification cocktail. Numbers 1–6 below each bar in the bar graphs represent complex immobilized primers for B.subtilis genes dnaK, ebrA, fruR, grpE, spo0A and yisY, respectively. Bar number 7 represents average background signal from empty gel elements on the chip.

Figure 5

Detection limit of the MME-PCR. (A) Serial dilutions of total genomic DNA of B.subtilis were prepared. Eight separate conventional PCR reactions with bsGrpE-F/bsGrpE-R primers (Table 1) were run for 50 cycles. The products of the reactions were separated on 1.2% agarose gel. The labels above the lanes denote the amount of B.subtilis genomic DNA used in the reaction, expressed in equivalents of the bacterial genomes. ‘No DNA’ lane contains the product of mock PCR reaction, performed without the addition of DNA. M, 100 bp DNA ladder. (B) Microarrays, identical to those described in Figure 4 were used for MME-PCR with different amounts of input B.subtilis genomic DNA (10⁵, 10⁴, 10³, 10², 10¹ or zero copies). After PCR, the microarrays were hybridized with the set of six B.subtilis reporting probes used in the experiment shown on Figure 4 to verify the identity of the amplicons. The numbers below each bar in the bar graph panels denote the name of targeted genes, or the background as shown in Figure 4. Labels below each panel denote the amount of the bacterial genomic DNA, expressed in the number of copies of the genome.