Universal Probe Library: A Comprehensive Overview

The Universal Probe Library (UPL) is a powerful tool in molecular biology, offering a versatile approach to gene detection and quantification. This article will delve into the various aspects of UPL, from its underlying principles and applications to its design and validation. It also covers unique molecular identifiers (UMIs) and their role in error correction in NGS libraries.

Introduction

Understanding gene function requires efficient and accurate methods for detecting and quantifying specific nucleic acid sequences. The Universal Probe Library provides a comprehensive solution for researchers, enabling them to study gene expression, detect pathogens, and perform various other molecular analyses.

Understanding Universal Probe Library (UPL)

Probe Chemistry: Locked Nucleic Acid (LNA) Technology

At the heart of the Universal ProbeLibrary lies a unique nucleotide chemistry known as LNA (Locked Nucleic Acid). LNA technology allows the use of very short (8-9 bases) oligonucleotides as effective hybridization probes in real-time PCR assays. LNA modification introduces a methylene bridge that "locks" the ribose sugar in a specific conformation, increasing the binding affinity of the oligonucleotide to its target sequence. This results in an unusually high melting temperature (Tm) for these short probes, making them fully compatible with commonly used PCR conditions and the hydrolysis-probe detection format. The enhanced binding affinity also makes these probes sensitive enough to detect single-base mismatches.

Assay Design with ProbeFinder Software

Designing effective assays is crucial for successful real-time PCR experiments. The free online ProbeFinder software, available at the Universal ProbeLibrary Assay Design Center, simplifies this process. The software designs one or more intron-spanning assays for a target gene based on user-submitted information such as gene name, accession number, or sequence. For each assay, ProbeFinder specifies a set of specific primers and a UPL probe that will provide optimal results. The combination of primers and probe ensures specific amplification and detection of the target sequence in a standard real-time PCR assay.

Labeling and Instrumentation

UPL probes are pre-labeled with a reporter fluorophore (FAM) and a dark quencher dye. This eliminates the need for additional labeling steps, saving time and resources. Standard real-time PCR instrumentation is all that is required to use UPL probes.

Available Sets and Coverage

In addition to being available individually, Universal ProbeLibrary probes are available in sets (90 probes/set), each of which covers virtually all the transcripts from a single organism. Together, the sets cover approximately 99% of the human, primate, mouse, rat, C. elegans, Drosophila, and Arabidopsis gene transcripts listed in the NCBI Reference Sequence Database.

Applications of Universal Probe Library

Gene Expression Analysis

UPL is commonly used for gene expression analysis, allowing researchers to quantify mRNA levels for specific genes of interest. This information can be used to study the effects of various treatments, identify biomarkers for disease, and gain insights into fundamental biological processes.

Pathogen Detection

UPL can be used to detect the presence of specific pathogens in a sample. By designing probes that target unique sequences in the pathogen's genome, researchers can quickly and accurately identify infections.

SNP Genotyping

The sensitivity of UPL probes to single-base mismatches makes them useful for SNP genotyping. By designing probes that specifically target each allele of a SNP, researchers can determine the genotype of an individual at that locus.

Advantages of Universal Probe Library

Versatility

UPL can be used to detect virtually any gene in a wide range of organisms.

Simplicity

The ProbeFinder software makes assay design easy, even for researchers with limited experience in real-time PCR.

Sensitivity

LNA technology enhances the sensitivity of UPL probes, allowing for the detection of low-abundance targets.

Specificity

The combination of specific primers and probes ensures that only the target sequence is amplified and detected.

Convenience

Pre-labeled probes eliminate the need for additional labeling steps.

Universal Molecular Identifiers (UMIs)

Unique molecular identifiers (UMIs) are short, random sequences used to uniquely tag individual molecules in a sample library. These molecular barcodes are incorporated onto each molecule within a given sample library. UMIs are particularly useful in sequencing applications, especially for addressing PCR duplicates in DNA and cDNA libraries.

Error Correction with UMIs

Sequencing with UMIs can significantly reduce false-positive variant calls and increase the sensitivity of variant detection. This is because UMIs allow for the identification and removal of PCR duplicates, which can introduce errors into sequencing data. TruSight Oncology UMI Regents, for example, provide error correction with unique molecular identifiers (UMIs) for NGS libraries.

Unique Dual Indexes (UDIs)

Unique dual indexes (UDIs) ensure that each sample index is specific to a given sample library, further enhancing the accuracy and reliability of sequencing results.

Universal Overgo-Hybridization Probes (Uprobe)

Targeted Physical Mapping

The expansive array of >70 public vertebrate genomic bacterial artificial chromosome (BAC) libraries can provide a means of comparative mapping, sequencing, and functional analysis of targeted chromosomal segments that is independent and complementary to whole-genome sequencing. However, at the present time, no complementary resource exists for the efficient targeted physical mapping of the majority of these BAC libraries.

Universal Probe Design

Universal overgo-hybridization probes, designed from regions of sequenced genomes that are highly conserved between species, have been demonstrated to be an effective resource for the isolation of orthologous regions from multiple BAC libraries in parallel. The application of the universal probe design principal across entire genomes, and the subsequent creation of a complementary probe resource, Uprobe, for screening vertebrate BAC libraries.

Uprobe Composition

Uprobe currently consists of whole-genome sets of universal overgo-hybridization probes designed for screening mammalian or avian/reptilian libraries. Retrospective analysis, experimental validation of the probe design process on a panel of representative BAC libraries, and estimates of probe coverage across the genome indicate that the majority of all eutherian and avian/reptilian genes or regions of interest can be isolated using Uprobe.

Inter species Sequence Comparisons

One simple and powerful approach for detecting putative functional elements in vertebrate genomes is through interspecies sequence comparisons. The power of interspecies sequence comparisons to detect putative functional elements is strongly correlated with the number of species and the divergence among the species included in the comparison.

Targeted Comparative Mapping and Sequencing

While whole-genome sequencing will yield a survey of many more vertebrates in the near future, targeted comparative mapping and sequencing is an efficient, rapid, and complementary approach for addressing directed biological questions in an even greater breadth of species. In addition, by providing high-quality sequence across specific regions of interest, targeted comparative mapping and sequencing can also supplement low coverage draft whole-genome sequence assemblies.

Universal Probes for Comparative Mapping

Previously, as a means to facilitate targeted comparative physical mapping in placental mammals, the concept of âuniversalâ sequence-tagged sites, which are PCR-based markers designed from sequences conserved between two or more species that can be used for mapping in multiple species, with overgo-hybridization probes, which are efficient at screening arrayed genomic libraries. The resulting universal overgo-hybridization probes designed from sequences highly conserved in human and mouse were very efficient at isolating orthologous genomic segments from chimpanzee, baboon, cat, dog, cow, and pig bacterial artificial chromosome (BAC) libraries. The use of these universal probes ultimately facilitated the direct comparison of megabases of orthologous sequence between these and other species.

Whole-Genome Universal Probe Sets

Here we report the implementation of the universal probe design concept across entire genomes for the creation of whole-genome universal probe sets that can be used to isolate orthologous chromosomal segments in specific evolutionary clusters of species. Based on a detailed retrospective analysis of prior universal probe hybridization results and new probe design algorithms, we have generated and experimentally validated hybridization-based whole-genome probe sets that can be used to effectively identify the vast majority of all eutherian (placental mammals) or avian/reptilian genes or regions of interest. Additional information is also accessible through the Uprobe Web site, including experimental protocols for using the universal probes, as well as all computational resources related to the creation of the whole-genome universal probe sets.

Mismatch Tolerance

Overgo-hybridization probes (n = 341) designed from human sequence and used to screen chimpanzee, baboon, cat, dog, cow, and pig BAC libraries were compared to newly available genomic sequence from each of these six target species. Local probeâtarget species sequence alignments were extracted from long-range humanâtarget species genomic sequence alignments, and the number of mismatches recorded for each probeâtarget sequence comparison. The probeâtarget species sequence mismatch information was then combined with the probe hybridization results. As expected, there was a clear correlation between the number of probeâtarget sequence mismatches and the presence of a positive hybridization signal. Probes with zero, three, or more than four mismatches with the target sequence were associated with positive hybridization signals 96%, 61%, and 26% of the time, respectively. We therefore conclude that the majority of individual 36-bp probes with three or fewer mismatches to a target sequence will result in a hybridization signal that is readily detectable using our BAC library hybridization protocol.

Genome Coverage

To estimate the effective genome coverage of this probe set, i.e., the ability to isolate the orthologous genomic segment from another mammal to any given region of the human genome. To do so, we determined the number of unique probes in 200-kb intervals (50-kb slide) across the genome, assuming an optimal spacing of one probe every 30 kb. Assuming a probe success rate of at least 50%, the number of optimally spaced unique probes (as opposed to all unique probes) within the interval should therefore provide a conservative estimate as to the ability to isolate the orthologous region from a given BAC library. Using these criteria, we found that 95%, 86%, and 50% of all 200-kb intervals had at least one, three, or six optimally spaced unique probes, respectively. Overall, the average number of optimally spaced unique probes in each 200-kb interval was estimated to be 5.07 Â± 2.12.

Experimental Validation

To validate the probe design methods and estimate a probe success rate in different mammalian lineages, a sample of representative unique mammalian universal probes (n = 95) and one nonunique probe were selected for experimental validation. Specifically, the probes selected for experimental validation had an average probe score (32.47 Â± 0.42) between that of all unique probes (32.38 Â± 0.43), and an optimal subset of probes spaced at 30-kb intervals (32.56 Â± 0.44). The test set of universal probes was hybridized to a panel of nine BAC libraries (marmoset, galago, rabbit, bat, shrew, armadillo, elephant, wallaby, and platypus) selected to represent the major mammalian lineages. Among the placental mammals, the universal probes were associated with success rates between 58% (armadillo) and 95% (marmoset), suggesting that the probe design criteria were appropriate for effectively screening BAC libraries from placental mammals. In contrast, a statistically significant lower success rate of 36% was observed for wallaby (marsupial) and platypus (monotreme) compared with the minimum success rate observed in placental mammals (Ï2 test, P < 0.05). Thus, alignments that include additional species will likely be necessary to realize a similar probe success rate in marsupials and monotremes.

Luna Universal Probe One-Step RT-qPCR Kit

RNA Detection and Quantitation

The Luna Universal Probe One-Step RT-qPCR Kit is optimized for real-time quantitation of target RNA sequences using hydrolysis probes. One-Step RT-qPCR provides a convenient and powerful method for RNA detection and quantitation. In a single tube, RNA is first converted to cDNA by a reverse transcriptase, and then a DNA-dependent DNA polymerase amplifies the cDNA, enabling quantitation via qPCR. Probe-based qPCR/RT-qPCR monitors an increase in fluorescence upon 5Â´ â 3Â´ exonuclease cleavage of a quenched, target-specific probe to measure DNA amplification at each cycle of a PCR. At a point where the fluorescence signal is confidently detected over the background fluorescence, a quantification cycle or Cq value can be determined.

WarmStart Enzymes

In the Luna Universal One-Step Probe RT-qPCR Kit, Hot Start Taq DNA Polymerase is combined with a novel WarmStart-activated reverse transcriptase, allowing dual control of enzyme activity via reversible, aptamer-based inhibition. This temperature-dependent activation helps to prevent undesirable non-specific priming and extension prior to thermocycling, providing added security for setting up reactions at room temperature. The engineered Luna WarmStart Reverse Transcriptase also possesses higher thermostability than many other RTs, allowing an optimal reaction temperature of 55Â°C. Note that to ensure full activation of the Luna WarmStart RT, incubation at temperatures lower than 50Â°C is not recommended.

Reaction Mix and Enzyme Mix

The Luna Universal Probe One-Step Reaction Mix is supplied at 2X concentration and contains Hot-Start Taq DNA Polymerase, dNTPs, and all required buffer components. It is formulated with a unique passive reference dye that is compatible across a variety of instrument platforms, including those that require a high or low ROX reference signal. The Reaction Mix also features dUTP for carryover prevention and a non-fluorescent visible dye for monitoring reaction setup. The Luna WarmStart RT Enzyme Mix is supplied at 20X concentration and contains Luna WarmStart Reverse Transcriptase as well as Murine RNase Inhibitor to aid in preventing RNA degradation (see also template preparation in product manual). It is compatible with various RNA sample types (total RNA, poly(A)-RNA, etc.) and sources.

Performance Evaluation

RT-qPCR targeting human GAPDH was performed using the Luna Universal Probe One-Step RT-qPCR Kit over an 8-log range of input template concentrations (1 Î¼g â 0.1 pg Jurkat total RNA) with 8 replicates at each concentration. Multiplex RT-qPCR targeting human GAPDH, ribosomal protein L32g and PI3-Kinase-Related Kinase SMG1 was performed using the Luna Universal Probe One-Step RT-qPCR Kit over a 7-log range of input template concentrations (1 Î¼g â 1 pg Jurkat total RNA) with 4 replicates at each concentration. To account for copy number differences, 0.4 ÂµM primer was used for lower-copy target (SMG1) and 0.2 ÂµM primer for higher-copy targets (L32g and GAPDH). Commercially-available RT-qPCR reagents were tested on 7 RT-qPCR targets varying in abundance, length, and %GC. Data was collected by 2 users and according to manufacturerâs recommendations. Results were evaluated for efficiency, low input detection and lack of non-template amplification (where ÎCq = average Cq of non-template control â average Cq of lowest input). In addition, consistency, reproducibility and overall curve quality were assessed (Quality Score). Bar graph indicates % of targets that met acceptable performance criteria (indicated by green box on dot plot and Quality Score > 3).

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