Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments

Abstract

We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.

Figure 1

Selector construct and circularization reaction principles. (A) The selector consists of two oligonucleotides, one selector probe of ∼70 nt and one general vector oligonucleotide of 34 nt. The selector probe has two target-specific ends (black) of ∼18 nt complementary to the selected target and a primer-pair motif (gray) of 34 nt. The vector oligonucleotide is complementary to the primer-pair motif (gray). (B) The circularization procedure starts with digestion of the DNA to generate targets with defined ends. The digested DNA is then combined with selectors and the mixture is denaturated, allowing the selectors to hybridize to their respective targets. This can be performed in two different ways: (i) a selector probe hybridizes to both ends of the selected target and the ends are connected to the vector by ligation, or (ii) a selector probe hybridizes with one end to the 3′ end of the selected target and the other end to an internal sequence of the target, forming a branched structure that can be cleaved by the endonucleolytic activity of Taq DNA polymerase. Both ends of the selected single-stranded target are now ready for ligation to the vector oligonucleotide, forming a closed circular molecule. The circularization mixture is then exonuclease-treated and finally PCR-amplified using a common primer pair.

Figure 2

Multiplex selector PCR. A total of 96 sequences were selected from a 7.5 k cDNA array in a pattern of a UU. Ninety-six selectors were then designed to select and amplify the corresponding targets from the human genome. The circularization reaction was performed as described in Figure 1B, where three fragments were circularized using procedure (i) and 93 using procedure (ii). A multiplexed PCR with a Cy3-labeled primer pair was then performed. (A) The PCR product from the 96-plex PCR was analyzed on a 1.5% agarose gel. The gel was loaded with a 100 bp ladder (left lane), PCR product from circularization reaction with ligase (middle lane) and PCR product from a circularization reaction without ligase. A histogram showing the expected length distribution of the 96 PCR products plotted against relative frequency (%). (B) The PCR product was also hybridized to the cDNA array. Fluorescence intensity is represented as a pseudo-color gradient ranging from green to red.

Figure 3

Reproducibility of selector PCR. The selection reaction described in Figure 2 was performed using DNA from five different individuals to investigate the reproducibility of the method. The graph demonstrates the variation in signal for each of the 78 selected fragments that were defined as positive. Signals were normalized relative to the total intensity of the spots in the UU pattern. Fragments are ordered with increasing average signal with bars representing 1 SD.

Figure 4

Design simulation demonstrating scalability of the method. Selector design was performed on five arbitrary sets of human genomic sequences. The figure shows the average design success rate for increasing numbers of fragments. Bars represent 1 SD. Line I represents one digestion reaction with one restriction enzyme, line II represents one digestion reaction with two restriction enzymes, line III represents two digestion reactions with one restriction enzyme in each reaction and line IV represents two digestion reactions with two restriction enzymes in each reaction. Arbitrarily selected 2 kb fragments containing 250 bp long sequences of interest were chosen as targets. After in silico digestion with different enzymes and combinations thereof, those target–enzyme combinations resulting in at least one fragment fulfilling the design parameters were considered successful. The parameters for the restriction fragments were set to a content of at least 50 bp of the desired sequence, a maximum flap length of 500 bp, and size of the selected fragment of 100–200 bp.