Revolutionizing genomic analyses: mutation analyses using novel enzyme-based assays with laser-induced fluorescence and polymeric microfluidic devices as electrophoretic platforms

Polymer-based microelectrophoresis was investigated to analyze known (mutation detection) and unknown (mutation scanning) low-abundant mutations in genomic DNA with high diagnostic value for colorectal cancers. For our mutation detection assays, point mutations in the K-ras oncogene were identified using the ligase detection reaction (LDR). For the mutation scanning assay, which searches for sporadic mutations, an EndoV-LDR assay was utilized with mutations in the p53 tumor suppressor gene used as a model. A poly(methylmethacrylate), PMMA, microchip filled with a 4% linear polyacrylamide (LPA) gel was used to electrophoretically sort products formed from LDRs, which produced oligonucleotides \u3c65 bp in length. Using microchip electrophoresis with the LPA, a 44 bp ligation product was resolved from a 100-fold molar excess of unligated primers (25 bp) in approximately 120 s, which was ~17 times faster than conventional capillary gel electrophoresis. In order to simplify the electrophoretic process and further reduce development time, the LDR products were sorted in the absence of the sieving gel using free solution conjugate electrophoresis (FSCE). FSCE incorporated polyamide “drag-tags” onto LDR primers, which provided DNA fragment mobilities in free solution that were dependent upon their size. LDR/drag-tagged (LDR-dt) products could be formed in a multiplexed format for mutant-to-wild-type ratios as low as 1 to 100 with single base resolution. Separations were conducted using capillary array electrophoresis (CAE) and PMMA microchips filled with only a TRIS buffer. Analysis times for the LDR-dt products were less than 11 min using CAE and ~85 s for PMMA microchips with high reproducible migration times within and between microchips. PMMA-based microchips were also evaluated for the identification of sporadic mutations using an endonuclease V – LDR (Endo V/LDR) technique. Endo V cleaves heteroduplexed DNA one base 3’ of single-base mismatched sites as well as nicking DNA at some matched sites as LDR reseals miscleaved sites to reduce false positive signals. Results suggested that Endo V/LDR products from p53 mutations could be successfully separated and detected using a PMMA microfluidic chip filled with a sparsely cross-linked replaceable polyacrylamide gel in less than 6 min, which was approximately 10-fold shorter compared to CAE

EndoV/DNA ligase mutation scanning assay using microchip capillary electrophoresis and dual-color laser-induced fluorescence detection

We report the ability to detect with high sensitivity sporadic mutations using a mutation scanning assay, which employs thermostable endonuclease V (EndoV) and DNA ligase. The products of the mutation scanning assay were separated using microchip capillary electrophoresis (mu CE) and detected with a dual-color laser-induced fluorescence (LIF) detector. PCR products from mutant and wild-type DNA of p53 exon 8 were generated using Cy3-labeled forward and Cy5-labeled reverse primers to allow LIF detection with mCE. EndoV recognizes and primarily cleaves heteroduplexed DNA one base 30 to a mismatch and can nick matched sites at low levels as well. DNA ligase is used to reseal nicks generated at matched sites, which creates a highly sensitive and specific assay for analyzing sporadic mutations in genomic DNA. Heteroduplexed DNA samples were treated with EndoV alone and with both EndoV and DNA ligase and separated using a 4% (w/v) linear polyacrylamide gel constituted in 1x TTE buffer, 7 M urea, and 0.05% (w/v) methyl hydroxyethyl cellulose, which was used to suppress the EOF in the microchip. Sizing of the bands appearing in the electropherogram revealed the approximate position of the mutation. In this study, mutations present in p53 exon 8 generated Cy3-labeled cleavage products of 158 nt and Cy5-labeled cleavage products of 195 nt. The DNA fragments were simultaneously monitored at their respective color using a dual-color LIF system with the 158 and 195 nt fragments detected along with heteroduplexed fragments of 350 nt. The microchip separation was completed within 7 min, almost tenfold shorter time compared to conventional capillary gel electrophoresis.close7

Capillary and microelectrophoretic separations of ligase detection reaction products produced from low-abundant point mutations in genomic DNA

Capillary gel electrophoresis (CGE) and polymer-based microelectrophoretic platforms were investigated to analyze low-abundant point mutations in certain gene fragments with high diagnostic value for colorectal cancers. The electrophoretic separations were carried out on single-stranded DNA (ssDNA) products generated from an allele-specific ligation assay (ligase detection reaction, LDR), which was used to screen for a single base mutation at codon 12 in the K-ras oncogene. The presence of the mutation generated a ssDNA fragment that was \u3e40 base pairs (bp) in length, while the primers used for the ligation assay were \u3c30 bp in length. Various separation matrices were investigated, with the success of the matrix assessed by its ability to resolve the ligation product from the large molar excess of unligated primers when the mutant allele was lower in copy number compared to the wild-type allele. Using CGE, LDR product models (44 and 51 bp) could be analyzed in a cross-linked polyacrylamide gel with a 1000-fold molar excess of LDR primers (25 bp) in approximately 45 min. However, when using linear polyacrylamide gels, these same fragments could not be detected due to significant electrokinetic biasing during injection. A poly(methylmethacrylate) (PMMA) microchip of 3.5 cm effective column length was used with a 4% linear polyacrylamide gel to analyze the products generated from an LDR. When the reaction contained a 100-fold molar excess of wild-type DNA compared to a G12.2D mutant allele, the 44 bp ligation product could be effectively resolved from unligated primers in under 120 s, nearly 17 times faster than the CGE format. In addition, sample cleanup was simplified using the microchip format by not requiring desalting of the LDR prior to loading

Assay validation using the LOPAC ± detergent.

<p>(A) Z' factors for replicate LOPAC experiments ± detergent. (B–C) Comparison of duplicate runs of the LOPAC ± detergent. Each point represents the activity of a discrete compound from the LOPAC. (D) Comparison of the LOPAC results ± detergent. Percent inhibitions represent the means of the replicate LOPAC experiments. Trend line (solid, red), ideal correlation line (dashed, blue). (E) Percent inhibition distribution of the averaged LOPAC results ± detergent, binned in 5% intervals.</p

A Cell-Free Fluorometric High-Throughput Screen for Inhibitors of Rtt109-Catalyzed Histone Acetylation

<div><p>The lysine acetyltransferase (KAT) Rtt109 forms a complex with Vps75 and catalyzes the acetylation of histone H3 lysine 56 (H3K56ac) in the Asf1-H3-H4 complex. Rtt109 and H3K56ac are vital for replication-coupled nucleosome assembly and genotoxic resistance in yeast and pathogenic fungal species such as <i>Candida albicans</i>. Remarkably, sequence homologs of Rtt109 are absent in humans. Therefore, inhibitors of Rtt109 are hypothesized as potential and minimally toxic antifungal agents. Herein, we report the development and optimization of a cell-free fluorometric high-throughput screen (HTS) for small-molecule inhibitors of Rtt109-catalyzed histone acetylation. The KAT component of the assay consists of the yeast Rtt109-Vps75 complex, while the histone substrate complex consists of full-length <i>Drosophila</i> histone H3-H4 bound to yeast Asf1. Duplicated assay runs of the LOPAC demonstrated day-to-day and plate-to-plate reproducibility. Approximately 225,000 compounds were assayed in a 384-well plate format with an average Z' factor of 0.71. Based on a 3σ cut-off criterion, 1,587 actives (0.7%) were identified in the primary screen. The assay method is capable of identifying previously reported KAT inhibitors such as garcinol. We also observed several prominent active classes of pan-assay interference compounds such as Mannich bases, catechols and p-hydroxyarylsulfonamides. The majority of the primary active compounds showed assay signal interference, though most assay artifacts can be efficiently removed by a series of straightforward counter-screens and orthogonal assays. Post-HTS triage demonstrated a comparatively small number of confirmed actives with IC₅₀ values in the low micromolar range. This assay, which utilizes five label-free proteins involved in H3K56 acetylation <i>in vivo</i>, can in principle identify compounds that inhibit Rtt109-catalyzed H3K56 acetylation via different mechanisms. Compounds discovered via this assay or adaptations thereof could serve as chemical probes or leads for a new class of antifungals targeting an epigenetic enzyme.</p></div

Assay design and optimization.

<p>(A) Titration matrix of CoA and CPM in buffer-only conditions to determine the optimal assay levels of acetyl-CoA and CPM. (B) Titration matrix of CPM and acetyl-CoA in buffer-only conditions to verify acetyl-CoA and CPM do not form fluorescent adducts under HTS conditions. (C) Time-course study of CoA titrations with 20 µM CPM in buffer-only conditions to determine the optimal time for the reaction involving CoA and CPM. (D) HTS plate template. Arrows denote chosen HTS conditions.</p

Noteworthy PAINS substructures in the primary Rtt109-Vps75 HTS.

<p><i>Italics</i> denote the original names of published PAINS substructures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078877#pone.0078877-Baell1" target="_blank">[51]</a>. For individual substructures, the ratios denote the number of primary active compounds divided by the number of compounds tested for each HTS production run.</p

Rtt109-Vps75 HTS protocol steps.

<p>Rtt109-Vps75 HTS protocol steps.</p

Rtt109-Vps75 HTS summary.

<p>Rtt109-Vps75 HTS summary.</p