The Universal DNA microarray is a technology platform that provides an alternate strategy in microarray design. It differs from the conventional approaches to microarray technology in that mutation detection and hybridization to the array surface are completely separate events. Since the specificity for the Universal DNA microarray is determined by LDR, it avoids the false negatives and false positives associated with direct DNA hybridization arrays. For high-throughput detection of specific multiplexed LDR products, unique zip-code sequences are
Fig. 1. (Color Plate 8 following p. 18). Overview of target amplification, probe amplification, signal amplification, and arrays used in molecular diagnostics (see ref. 1 for original references). Target amplification: PCR/TaqMan—The 5'-3' exonuclease activity of Taq polymerase releases a fluorescent group from its quencher during primer extension, allowing real-time monitoring of the PCR reaction. WGA—random oligonucleotide primers are used in conjunction with a processive polymerase with strand displacement
Fig. 1. (Color Plate 8 following p. 18). Overview of target amplification, probe amplification, signal amplification, and arrays used in molecular diagnostics (see ref. 1 for original references). Target amplification: PCR/TaqMan—The 5'-3' exonuclease activity of Taq polymerase releases a fluorescent group from its quencher during primer extension, allowing real-time monitoring of the PCR reaction. WGA—random oligonucleotide primers are used in conjunction with a processive polymerase with strand displacement attached to each LDR product, allowing for specific capture at complementary addresses on a DNA microarray (3). The Universal DNA microarray is thus programmable and can accommodate any gene without redesigning the array. The details of the methodology provide explanations as to why this technology is sensitive, rapid, and robust.
The major components of the process are simple to perform and involve PCR followed by LDR and then hybridization of the product to the Universal DNA microarray (Fig. 2; see Color Plate 9 following p. 18). To amplify multiple genomic regions while minimizing primer-specific differences in efficiency,
Fig. 1. (Continued from previous page) activity, allowing isothermal, nonspecific amplification total genomic DNA with little amplification bias. SDA—two primers hybridize to each strand of the target DNA and are extended so that the upstream primer displaces the downstream primer. Two more primers anneal to the displaced product and extend in the same way, followed by restriction endonuclease nicking and extension of the double-stranded product. Probe amplification: LCR—two primers anneal to each strand of the target DNA region and are ligated if there is perfect complementarity at the junctions. The product molecules can act as templates for the next round of LCR, resulting in exponential amplification. LDR—a fluorescently labeled allele-specific primer and a downstream primer anneal adjacently on one strand of the target DNA. If there is perfect complementarity at the junction, a thermostable ligase joins the primers, and the resulting product is detected on an array or by electrophoresis. RCA—both sides of a long primer hybridize to the target sequence and, when ligated, produce a circular product molecule, which is then amplified by a strand-displacing DNA polymerase. Signal amplification: 3D dendrimer labeling systems—a dendrimer has a core that consists of a matrix of double-stranded DNA, and an outer surface with hundreds of single-stranded "arms," which are available for hybridization to a specific sequence or to oligonucleotides that carry signal molecules. Enzymatic cascade reporters—an event, such as hybridization of the correct DNA molecule to a position on an array, stimulates an enzyme to produce a cofactor that is specific for a second enzyme. This second enzyme can, in turn, separate a fluorescent group from its quencher. Invader assay—an allele-specific oligonucleotide and a second oligonucleotide hybridize to the target so there is a single nucleotide overlap at the base in question, and cleavase exonuclease releases the "flap" at the 5' end of the oligonucleotide only if there is perfect complementarity at the junction. This "flap" is then able to bind to a specific fluorescence resonance energy transfer (FRET) moiety, allowing cleavase to release a fluorescent signal. Array format: Hybridization array—biological oligonu-cleotides that are specific for a particular organism (e.g., cDNAs) are covalently attached to an array surface at specific locations, allowing detection of their labeled complements (e.g., RNAs). This format has difficulty distinguishing all single-base mutations. Universal array—unique oligonucleotides (zip-codes) are located at specific positions on an array surface, allowing fluorescently labeled LDR products, with a tails that are complementary to the zip-codes, to hybridize. Appropriate primer design allows a single Universal array to be used for many different assays.
1. PCR amplify all p53 exons using gene-specific/universal primers and Taq polymerase. ♦
AtoG? CtoT? AtoC?
2- PCR amplify all primary products using universal primers and Taq polymerase. ♦
3, Perform LDR using mutation-specific LDR primers, common primers containing complementary zip code sequences, and thermostable ligase. •
4. Capture fluorescent products on addressable array and score for presence of mutation
Cy3 Cy 5 Bodipy Alexa
Fig. 2. (Color Plate 9 following p.18) Schematic diagram and results for the detection of multiple mutations using PCR/PCR/LDR/Universal Array. Upper panel shows schematic. p53 exons 4-8 are PCR-amplified in a multiplex format with gene-specific primers bearing 5' universal sequences, and in a second PCR, amplified simultaneously with universal PCR primers. Multiplexed LDR is performed on all PCR products with fluores-cently labeled allele-specific LDR primers and common primers containing complementary zip-code sequences. LDR products are hybridized to universal array containing zip-codes. Address and color of the microarray spot scores are given for the mutation. Lower panelshows chip results. The p53 chip can detect 110 different mutations in exons
Fig. 2. (Color Plate 9 following p.18) Schematic diagram and results for the detection of multiple mutations using PCR/PCR/LDR/Universal Array. Upper panel shows schematic. p53 exons 4-8 are PCR-amplified in a multiplex format with gene-specific primers bearing 5' universal sequences, and in a second PCR, amplified simultaneously with universal PCR primers. Multiplexed LDR is performed on all PCR products with fluores-cently labeled allele-specific LDR primers and common primers containing complementary zip-code sequences. LDR products are hybridized to universal array containing zip-codes. Address and color of the microarray spot scores are given for the mutation. Lower panelshows chip results. The p53 chip can detect 110 different mutations in exons two rounds of PCR are used. PCR primers are constructed to have gene-specific portions connected to universal sequences on the 5' ends. The first round of PCR relies on a limited number of cycles using the gene-specific portions of the primers. The reaction is supplemented with a PCR cocktail containing primers complementary to the universal sequences and the majority of the amplification cycles are completed. Following this multiplex PCR/PCR amplification of the genomic regions of interest, each mutation is simultaneously detected using a thermostable ligase that joins adjacent pairs of oligonucleotides complementary to the sequences of interest. Attached to one of the paired oligonucleotides (referred to as the common oligo) are nongenomic 24-base sequences that are complementary to 24-base zip-code sequences present at known locations on the microarray surface. The remaining oligonucleotide of the pair (referred to as the discriminating oligo) is fluorescently labeled. Ligation occurs only when the sequence at the junction between the paired oligos is exactly complementary to the template sequence. Thus, when the variant of interest is present, lig-ation joins the oligonucleotide bearing the fluorescent label to the oligonucleotide bearing the zip-code complement. Hybridizing the LDR product to the Universal DNA microarray reveals the presence of a variant. If the variant of interest is present, a fluorescent signal will be visible on the address bearing the zip-code sequence that captures the complementary zip-code on the LDR oligo. If the variant is not present, the LDR oligo with the complementary zip-code will still hybridize to the appropriate address, but no fluorescent signal will be joined to it.
The sensitivity of this system is augmented by the use of a 3D polyacry-lamide surface. This surface permits hybridization times of 30-60 min and signal intensities 100-fold better than conventional microarrays (e.g., poly-L-lysine or amino/silane-coated slides) (3).
The most significant advantage of our technique is the ability to separate and therefore optimize mutation identification independently of array hybridization.
Fig. 2. (Continued from previous page) 5, 6, 7, and 8. A total of 216 LDR primers were required for detection. The mutation status of each sample and the zip-codes expected to capture signal are indicated at the bottom of each array; fiducials are along the top and right side of all arrays. Two reactions were performed for each sample containing LDR primers that were designed to hybridize to the upper strand or lower strand of p53 sequence. The array was imaged on a Lumonics ScanArray 5000 to visualize the Cy3, Cy5, and FAM signals. The 16-bit grayscale images for each dye were captured using the MetaMorph Imaging System (Universal Imaging), rendered in color, overlaid, and merged. R72R is a polymorphism in exon 4, and R273 C^T is the mutant signal. PCR, polymerase chain reaction; LDR, ligase detection reaction.
The background signal from each step can be minimized, and consequently, the overall sensitivity and accuracy of our method can be significantly enhanced over other strategies. Direct hybridization DNA microarrays suffer from differential hybridization efficiencies owing either to sequence variation or to the amount of target present in the sample. Consequently, hybridizations are performed at low temperatures, often for several hours to overnight, and this results in increased background noise and false signals caused by mismatch hybridization and nonspecific binding, for example, on small insertions and deletions in repeat sequences (36-39). In contrast, our approach of designing divergent zip-code sequences with similar Tms, allows for a more stringent and rapid hybridization at 65 °C.
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