Ligase Chain Reaction Research Articles

Entropy-Driven Catalytic G-Quadruple Cycle Amplification Integrated with Ligases for Label-Free Detection of Single Nucleotide Polymorphisms.

G-Quadruplex/thioflavin (G4/THT) has become a very promising label-free fluorescent luminescent element for nucleic acid detection due to its good programmability and compatibility. However, the weak fluorescence efficiency of single-molecule G4/THT limits its potential applications. Here, we developed an entropy-driven catalytic (EDC) G4 (EDC-G4) cycle amplification technology as a universal label-free signal amplification and output system by properly programming classical EDC and G4 backbone sequences, preintegrated ligase chain reaction (LCR) for label-free sensitive detection of single nucleotide polymorphisms (SNPs). First, the positive strand LCR enabled specific transduction and preliminary signal amplification from single-base mutation information to single-strand information. Subsequently, the EDC-G4 cycle amplification reaction was activated, accompanied by the production of a large number of G4/THT luminophores to output fluorescent signals. The EDC-G4 system was proposed to address the weak fluorescence of G4/THT and obtain a label-free fluorescence signal amplification. The dual-signal amplification effect enabled the LCR-EDC-G4 detection system to accurately detect mutant target (MT) at concentrations as low as 22.39 fM and specifically identify 0.01% MT in a mixed detection pool. Moreover, the LCR-EDC-G4 system was further demonstrated for its potential application in real biological samples. Therefore, this study not only contributes ideas for the development of label-free fluorescent biosensing strategies but also provides a high-performance and practical SNP detection tool in parallel.

Inverted Sandwich-Type e-LCR Aided by Lambda Exonuclease-RecJf Combination Enables Ultrasensitive Detection of Low-Frequency EGFR-L858R Mutation in NSCLC.

Highly sensitive detection of low-frequency EGFR-L858R mutation is particularly important in guiding targeted therapy of nonsmall-cell lung carcinoma (NSCLC). To this end, a ligase chain reaction (LCR)-based electrochemical biosensor (e-LCR) with an inverted sandwich-type architecture was provided by combining a cooperation of lambda exonuclease-RecJf exonuclease (λ-RecJf exo). In this work, by designing a knife-like DNA substrate (an overhang ssDNA part referred to the "knife arm") and introducing the λ-RecJf exo, the unreacted DNA probes in the LCR were specially degraded while only the ligated products were preserved, after which the ligated knife-like DNA products were hybridized with capture probes on the gold electrode surface through the "knife arms", forming the inverted sandwich-type DNA structure and bringing the methylene blue-label close to the electrode surface to engender the electrical signal. Finally, the sensitivity of the e-LCR could be improved by 3 orders of magnitude with the help of the λ-RecJf exo, and due to the mutation recognizing in the ligation site of the employed ligase, this method could detect EGFR-L858R mutation down to 0.01%, along with a linear range of 1 fM-10 pM and a limit detection of 0.8 fM. Further, the developed method could distinguish between L858R positive and negative mutations in cultured cell samples, tumor tissue samples, and plasma samples, whose accuracy was verified by the droplet digital PCR, holding a huge potential in liquid biopsy for precisely guiding individualized-treatment of NSCLC patients with advantages of high sensitivity, low cost, and adaptability to point-of-care testing.

Self-Replicating DNA-Based Nanoassemblies.

The properties of DNA that make it an effective genetic material also allow it to be ideal for programmed self-assembly. Such DNA-programmed assembly has been utilized to construct responsive DNA origami and wireframe nanoassemblies, yet replicating these hybrid nanomaterials remains challenging. Here we report a strategy for replicating DNA wireframe nanoassemblies using the isothermal ligase chain reaction lesion-induced DNA amplification (LIDA). We designed a triangle wireframe structure that can be formed in one step by ring-closing of its linear analog. Introducing a small amount of the wireframe triangle to an excess of the linear analog and complementary fragments, one of which contains a destabilizing abasic lesion, leads to rapid, sigmoidal self-replication of the wireframe triangle via cross-catalysis. Using the same cross-catalytic strategy we also demonstrate rapid self-replication of a hybrid wireframe triangle containing synthetic vertices as well as the self-replication of circular DNA. This work reveals the suitability of isothermal ligase chain reactions such as LIDA to self-replicate complex DNA architectures, opening the door to incorporating self-replication, a hallmark of life, into biomimetic DNA nanotechnology.

POSoligo software for in vitro gene synthesis

Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of nucleotides. The main approach to synthesizing long-chain DNA molecules involves linking short-chain oligonucleotides through ligase chain reaction (LCR) and polymerase chain reaction (PCR). Short-chain DNA molecules have low mutation rates, while LCR requires complementary interfaces at both ends of the two nucleic acid molecules or may alter the conformation of the nucleotide chain, leading to termination of amplification. Therefore, molecular melting temperature, length, and specificity must be considered during experimental design. POSoligo is a specialized offline tool for nucleotide fragment synthesis. It optimizes the oligonucleotide length and specificity based on input single-stranded DNA, producing multiple contiguous long strands (COS) and short patch strands (POS) with complementary ends. This process ensures free 5′- and 3′-ends during oligonucleotide synthesis, preventing secondary structure formation and ensuring specific binding between COS and POS without relying on stabilizing the complementary strands based on Tm values. POSoligo was used to synthesize the linear RBD sequence of SARS-CoV-2 using only one DNA strand, several POSs for LCR ligation, and two pairs of primers for PCR amplification in a time- and cost-effective manner.

Acoustic detection of a mutation-specific Ligase Chain Reaction based on liposome amplification.

Single nucleotide variants (SNVs) play a crucial role in understanding genetic diseases, cancer development, and personalized medicine. However, existing ligase-based amplification and detection techniques, such as Rolling Circle Amplification and Ligase Detection Reaction, suffer from low efficiency and difficulties in product detection. To address these limitations, we propose a novel approach that combines Ligase Chain Reaction (LCR) with acoustic detection using highly dissipative liposomes. In our study, we are using LCR combined with biotin- and cholesterol-tagged primers to produce amplicons also modified at each end with a biotin and cholesterol molecule. We then apply the LCR mix without any purification directly on a neutravidin modified QCM device Au-surface, where the produced amplicons can bind specifically through the biotin end. To improve sensitivity, we finally introduce liposomes as signal enhancers. For demonstration, we used the detection of the BRAF V600E point mutation versus the wild-type allele, achieving an impressive detection limit of 220 aM of the mutant target in the presence of the same amount of the wild type. Finally, we combined the assay with a microfluidic fluidized bed DNA extraction technology, offering the potential for semi-automated detection of SNVs in patients' crude samples. Overall, our LCR/acoustic method outperforms other LCR-based approaches and surface ligation biosensing techniques in terms of detection efficiency and time. It effectively overcomes challenges related to DNA detection, making it applicable in diverse fields, including genetic disease and pathogen detection.

Ligation-Based High-Performance Mimetic Enzyme Sensing Platform for Nucleic Acid Detection.

G-quadruplex (G4)/hemin DNAzyme is a promising candidate to substitute horseradish peroxidase in biosensing systems, especially for the detection of nucleic acids. However, the relatively suboptimal catalytic capacity limits its potential applications. This makes it imperative to develop an ideal signal for the construction of highly sensitive biosensing platforms. Herein, we integrated a novel chimeric peptide-DNAzyme (CPDzyme) with the ligase chain reaction (LCR) for the cost-efficient and highly sensitive detection of nucleic acids. By employing microRNA (miRNA) and single-nucleotide polymorphism detection as the model, we designed a G4-forming sequence on the LCR probe with a terminally labeled amino group. Subsequently, asymmetric hemin with carboxylic arms allowed assembly with the LCR products and peptide to form CPDzyme, followed by the magnetic separation of the extraneous components and chemiluminescence detection. Compared with the conventional G4/hemin signaling-based method, the LCR-CPDzyme system demonstrated 3 orders of magnitude improved sensitivity, with accurate quantification of as low as 25 aM miRNA and differentiation of 0.1% of mutant DNA from the pool containing a large amount of wild-type DNA. The proposed LCR-CPDzyme strategy is a potentially powerful method for in vitro diagnostics and serves as a reference for the development of other ligation- or hybridization-based nucleic acid amplification assays.

Demethylation-Driven ligase chain reaction for simultaneously sensitive detection of m6A demethylase FTO and m1A demethylase ALKBH3 in breast cancer at Single-Molecule level

RNA demethylases play a crucial role in gene expression regulation by controlling RNA methylation levels, and their abnormal expressions are associated with many human diseases. Herein, we develop a single-molecule detection method for simultaneous quantification of N6-methyladenosine (m6A) demethylase FTO and N1-methyladenosine (m1A) demethylase ALKBH3 based on RNA demethylation-directed multi-cyclic ligase chain reaction (LCR) cascades. We design a ssDNA containing m6A modification as the substrate of FTO and a ssDNA containing m1A modification as the substrate of ALKBH3. FTO and ALKBH3 catalyze the demethylation of m6A and m1A to generate normal adenine (A), respectively. Afterwards, Taq ligase initiates the LCR reaction with the demethylated ssDNAs as the templates to produce abundant biotinylated Cy3/Cy5-labeled signal probes. Subsequently, the resultant biotinylated probes are captured by streptavidin-coated magnetic beads (MBs) to form the MB-ssDNA-Cy3/Cy5 complexes. After magnetic separation, the ssDNAs in the MB-ssDNA-Cy3/Cy5 complexes are digested by DNase I, liberating abundant Cy3 and Cy5 fluorophores that can be counted by single-molecule detection. Remarkably, only a single DNA ligase is employed to achieve target signal conversion and multi-cyclic cascade amplification. This method displays extremely high sensitivity with a limit of detection of 6.33 × 10−18 mol/L for FTO and 1.44 × 10−17 mol/L for ALKBH3. Moreover, it can measure the enzyme kinetic parameters, screen inhibitors, and quantify cellular FTO and ALKBH3 activities with single-cell sensitivity. Furthermore, it can differentiate the FTO and ALKBH3 levels in the tissues of healthy human and breast cancer patients, offering a robust platform for biomedical research and clinical diagnosis.

Precise Differentiation of Wobble-Type Allele via Ratiometric Design of a Ligase Chain Reaction-Based Electrochemical Biosensor for CYP2C19*2 Genotyping of Clinical Samples.

Due to the comparable stability between the perfect-base pair and the wobble-base pair, a precise differentiation of the wobble-type allele has remained a challenge, often leading to false results. Herein, we proposed a ligase chain reaction (LCR)-based ratiometric electrochemical DNA sensor, namely, R-eLCR, for a precise typing of the wobble-type allele, in which the traditionally recognized "negative" signal of wobble-base pair-mediated amplification was fully utilized as a "positive" one and a ratiometric readout mode was employed to ameliorated the underlying potential external influence and improved its detection accuracy in the typing of the wobble-type allele. The results showed that the ratio between current of methylene blue (IMB) and current of ferrocene (IFc) was partitioned in three regions and three types of wobble-type allele were thus precisely differentiated (AA homozygote: IMB/IFc > 2; GG homozygote: IMB/IFc < 1; GA heterozygote: 1 < IMB/IFc < 2); the proposed R-eLCR successfully discriminated the three types of CYP2C19*2 allele in nine cases of human whole blood samples, which was consistent with those of the sequencing method. These results evidence that the proposed R-eLCR can serve as an accurate and robust alternative for the identification of wobble-type allele, which lays a solid foundation and holds great potential for precision medicine.

Ultrasensitive electrochemical biosensor based on triple signal amplification strategy for detection of serum cf-miR-181b: A possible biomarker related to osteosarcoma’s early diagnosis and chemotherapy monitoring

Osteosarcoma is one of the most common primary malignant bone tumors, however, the current diagnostic methods based on tissue biopsy suffers from invasive operation, high heterogeneity and non-early diagnosis. Herein, we proposed a new liquid biopsy method using triple signal amplification-based electrochemical biosensor (E-sensor) in detection of serum cf-miR-181b closely related to osteosarcoma. The E-sensor adopted an enzyme-linked DNA magnetic microbead (MMB), in which the miR-181b-triggered ligase chain reaction products were immobilized onto the surface of MMB followed by the attraction of horseradish peroxidase, to produce a catalytic current on the electrode surface. The E-sensor could detect miR-181b as low as 6.70 aM with a single-base resolution. Finally, the E-sensor was applied to detect serum cf-miR-181b in different individuals, which exhibited excellent performance in discriminating serum miR-181b between control group (healthy people/non-osteosarcoma patients) and osteosarcoma patients. Also, this assay could be used to distinguish the osteosarcoma patients before and after effective chemotherapy. In parallel, all results were in good agreement with that obtained by qRT-PCR. Therefore, the proposed E-sensor, due to its low cost, easy miniaturization and generalization, holds a huge application potential in the early auxiliary diagnosis and therapeutic monitoring of osteosarcoma and is of great value in clinical translation.

Simultaneous quantification of multiple single nucleotide variants in PIK3CA ctDNA using mass-tagged LCR probe sets.

Circulating tumor DNA (ctDNA) in blood carries genetic variations associated with tumors. There is evidence indicating that the abundance of single nucleotide variant (SNV) in ctDNA is correlated well with cancer progression and metastasis. Thus, accurate and quantitative detection of SNVs in ctDNA may benefit clinical practice. However, most current methods are unsuitable for the quantification of SNV in ctDNA that usually differentiates from wild-type DNA (wtDNA) only by a single base. In this setting, ligase chain reaction (LCR) coupled with mass spectrometry (MS) was developed to simultaneously quantify multiple SNVs using PIK3CA ctDNA as a model. Mass-tagged LCR probe set for each SNV including mass-tagged probe and three DNA probes was firstly designed and prepared. Then, LCR was initiated to discriminate SNVs specifically and amplify the signal of SNVs in ctDNA selectively. Afterward, a biotin-streptavidin reaction system was used to separate the amplified products, and photolysis was initiated to release mass tags. Finally, mass tags were monitored and quantified by MS. After optimizing conditions and verifying performance, this quantitative system was applied for blood samples from breast cancer patients, and risk stratification for breast cancer metastasis was also performed. This study is among the first to quantify multiple SNVs in ctDNA in a signal amplification and conversion manner, and also highlights the potential of SNV in ctDNA as a liquid biopsy marker to monitor cancer progression and metastasis.

Bacterial Vaginosis and Spontaneous Clearance of Chlamydia trachomatis in the Longitudinal Study of Vaginal Flora.

Up to 26% of urogenital Chlamydia trachomatis infections spontaneously resolve between detection and treatment. Mechanisms governing natural resolution are unknown. We examined whether bacterial vaginosis (BV) was associated with greater chlamydia persistence versus spontaneous clearance in a large, longitudinal study. Between 1999 and 2003, the Longitudinal Study of Vaginal Flora followed reproductive-age women quarterly for 1 year. Baseline chlamydia screening and treatment were initiated after ligase chain reaction testing became available midstudy, and unscreened endocervical samples were tested after study completion. Chlamydia clearance and persistence were defined between consecutive visits without chlamydia-active antibiotics (n = 320 persistence/n = 310 clearance). Associations between Nugent score (0-3, no BV; 4-10, intermediate/BV), Amsel-BV, and chlamydia persistence versus clearance were modeled with alternating and conditional logistic regression. Of chlamydia cases, 48% spontaneously cleared by the next visit (310/630). Nugent-intermediate/BV was associated with higher odds of chlamydia persistence (adjusted odds ratio [aOR] = 1.89; 95% confidence interval [CI], 1.30-2.74), and the findings were similar for Amsel-BV (aOR 1.39; 95% CI, .99-1.96). The association between Nugent-intermediate/BV and chlamydia persistence was stronger in a within-participant analysis of 67 participants with both clearance/persistence intervals (aOR = 4.77; 95% CI, 1.39-16.35). BV symptoms did not affect any results. BV is associated with greater chlamydia persistence. Optimizing the vaginal microbiome may promote chlamydia clearance.

Accuracy of pooling specimens from different individuals in the detection of genital Chlamydia trachomatis infection: a Meta analysis

Objective: To determine the accuracy of pooled specimens from multiple individuals for detection of infection with Chlamydia trachomatis. Methods: By April 2022, PubMed and Embase searched relevant studies published in peer-reviewed journals. The QUADAS-2 scale of a quality assessment tool was used to assess the quality of the studies. A curve of summary receiver operating characteristic was applied as a comprehensive assessment of diagnosed accuracy. A bivariate mixed-effects model was used for overall value merging in sensitivity and specificity. In addition, the subgroup analyses regarding sample type, testing method, and the number of samples per pool were performed. Results: A total of 14 846 subjects were included in the analysis. Three studies were from the United States, three from Canada, three from Denmark, two from Lithuania, two from India, two from the Netherlands, and one from Australia, Russia, and Singapore. Compared with the individual specimens, the pooled specimens of multiple individuals had an overall sensitivity of 0.98 (95%CI: 0.97-0.99) and specificity of 1.00 (95%CI: 1.00-1.00) for the detection of Chlamydia trachomatis. Results from the subgroup showed that the overall sensitivity of ligase chain reaction was significantly higher than that of PCR in the diagnosis of pooled samples. Conclusion: It is concluded from the published studies that the pooled specimens were substantially consistent with the single specimens in detecting infection with Chlamydia trachomatis.

Advances in ligase-based nucleic acid amplification technology for detecting gene mutations: a review.

Gene mutation has been a concern for researchers because it results in genetic variations with base changes in molecular structure. Researchers continue to explore methods to detect gene mutations, which may help in disease diagnosis, medication guidance, and so on. Currently, the detection methods, such as whole-genome sequencing and polymerase chain reaction, have some limitations in terms of cost and sensitivity. Ligase (an enzyme) can recognize base mismatch as a commonly used tool in genetic engineering. Therefore, the ligase-related nucleic acid amplification technology for detecting gene mutations has become a research hotspot. In this study, the main techniques explored for detecting gene mutations included the ligase detection reaction, ligase chain reaction, rolling circle amplification reaction, enzyme-assisted polymerase chain reaction, and loop-mediated isothermal amplification reaction. This review aimed to analyze the aforementioned techniques and mainly present their advantages and disadvantages, sensitivity, specificity, cost, detection time, applications, and so on. The findings may help develop sufficient grounds for further studies on detecting gene mutations.

A depth-first search algorithm for oligonucleotide design in gene assembly.

When synthesizing a gene with a long DNA sequence, it is usually necessary to divide it into several fragments. Based on these fragments, a set of oligonucleotides for gene assembly is produced. Each oligonucleotide is synthesized separately by the chemical reaction, and then the obtained oligonucleotides are assembled into the full gene sequence, in a specific environment, by polymerase chain reaction (PCR) or ligase chain reaction (LCR). In this paper, an effective and efficient algorithm to divide long genes into oligonucleotide sets is presented. First, according to the length of the overlapping oligonucleotide region, the long DNA sequence to be synthesized is divided into fragments of approximately equal length. Second, the length of these fragments is iterated to dynamically optimize the length of the overlapping regions to reduce melting temperature fluctuations. Then, the improved depth-first search algorithm is used according to the design principle of pruning optimization to obtain a uniform set of oligonucleotides with very close melting temperatures. This will decrease the errors in gene assembly with PCR or LCR. Lastly, the oligonucleotides that have homologous melting temperatures needed for PCR-based synthesis and two-step assembly of the target gene are deduced and outputted.

A zero-background electrochemical DNA sensor coupling ligase chain reaction with lambda exonuclease digestion for CYP2C19*2 allele genotyping in clinical samples

A zero-background electrochemical DNA sensor is badly desired for highly accurate real-sample assay. Herein, we built a lambda exonuclease (λ-Exo)-assisted ligase chain reaction (LCR)-based electrochemical biosensor, named λ-eLCR, for highly sensitive CYP2C19*2 allele genotyping with zero-background by using the methylene blue (MB)-attached DNA probe and an inverted hybridization format. Specifically, the ligated MB-labeled single-stranded DNAs (ssDNA) were generated exponentially by LCR amplification followed by λ-Exo digestion in the presence of target DNA, then they were invertedly hybridized with the upper sequence of capture probes on the gold electrode, bringing the redox reporter-MB close enough to the surface of the electrode. In this regard, the MB-derived signal detected by square wave voltammetry technique was quantitatively related with the target DNA concentration and guaranteed the zero background. Under the optimum condition, the proposed λ-eLCR exhibited excellent performance in terms of good linear range (100 fM–10 pM) and a low limit of detection (100 fM) for CYP2C19*2 gene. Finally, this λ-eLCR successfully distinguished the CYP2C19*2 allele genotypes in human whole blood samples, which has a great prospect in individualized medicine.

The AMR-ARRAY: A modular bead array detecting β-lactam, (fluoro) quinolone, colistin, aminoglycoside and macrolide resistance determinants in Gram-negative bacteria

The aim of this study was to develop a highly multiplexed bead array to detect genes and/or mutations frequently associated with resistance to antimicrobials of the β-lactam, (fluoro)quinolone, colistin, macrolide and aminoglycoside families in Enterobacteriaceae such as Escherichia coli, Shigella spp. and Salmonella spp. Ligase Chain Reaction and the Luminex® technology were combined in a 53-plex assay designed to target selected genetic markers with 3 internal controls. The AMR-ARRAY consistently detected resistance determinants as compared to phenotypically expressed resistance for 94.7% (856/904) of the assessed resistances. When compared to resistance profiles inferred from whole genome sequencing results, the AMR-ARRAY showed a selectivity and specificity of 99.3% and 100%, respectively. The strong features of the AMR-ARRAY are (i) its competitive cost, currently 18€/sample (ii) its wide analytical scope, currently 50 markers covering 5 antimicrobial families, (iii) its robust and user-friendly design consisting in a single-tube assay conducted in 4 successive steps (iv) its relatively short turnaround time, less than 8 h (v) its ability to detect allelic variability at critical SNPs (vi) its open access and easily upgradable design, with probes sequences, procedure and software source code freely available. The use of the AMR-ARRAY as a screening method in official antimicrobial resistance monitoring could improve the granularity of the collected data and pinpoint remarkable isolates harbouring unusual resistance determinants thereby enabling fit-for-purpose selection of isolates for Whole Genome analysis.

An Integrated Algorithm for Designing Oligodeoxynucleotides for Gene Synthesis.

The design and construction of large synthetic genes can be a slow, difficult, and confusing process, especially in the key step of oligodeoxynucleotide design. Herein we present an integrated algorithm to design oligonucleotide sets for gene synthesis by both ligase chain reaction and polymerase chain reaction. It offers much flexibility with no constraints on the gene to be synthesized. Firstly, it divides the long-input DNA sequence by a greedy algorithm based on the length of the oligodeoxynucleotide overlap region. Secondly, it tunes the length of the overlap region iteratively in an attempt to minimize the melting temperature variance of overlap. Thirdly, dynamic programming algorithm is used to achieve the uniform melting temperature of the oligodeoxynucleotide overlaps. Finally, the oligodeoxynucleotides with homologous melting temperature necessary for ligase chain reaction-based or two-step assembly PCR-based synthesis of the desired gene are outputted.

Integration of the Ligase Chain Reaction with the CRISPR-Cas12a System for Homogeneous, Ultrasensitive, and Visual Detection of microRNA.

The ligase chain reaction (LCR), as a classic nucleic acid amplification technique, is popular in the detection of DNA and RNA due to its simplicity, powerfulness, and high specificity. However, homogeneous and ultrasensitive LCR detection is still quite challenging. Herein, we integrate the LCR with a CRISPR-Cas12a system to greatly promote the application of the LCR in a homogeneous fashion. By employing microRNA as the model target, we design LCR probes with specific protospacer adjacent motif sequences and the guide RNA. Then, the LCR is initiated by target microRNA, and the LCR products specifically bind to the guide RNA to activate the Cas12a system, triggering secondary signal amplification to achieve ultrasensitive detection of microRNA without separation steps. Moreover, by virtue of a cationic conjugated polymer, microRNA can not only be visually detected by naked eyes but also be accurately quantified based on RGB ratio analysis of images with no need of sophisticated instruments. The method can quantify microRNA up to 4 orders of magnitude, and the determination limit is 0.4 aM, which is better than those of other reported studies using CRISPR-Cas12a and can be compared with that of the reverse-transcription polymerase chain reaction. This study demonstrates that the CRISPR-Cas12a system can greatly expand the application of the LCR for the homogeneous, ultrasensitive, and visual detection of microRNA, showing great potential in efficient nucleic acid detection and in vitro diagnosis.

AAUS guidelines 2021 revision sexually transmitted infection (STIs) diagnostic strategy for STI

AAUS guidelines 2021 revision sexually transmitted infection (STIs) diagnostic strategy for STI

A Guide to Using FASTPCR Software for PCR, In Silico PCR, and Oligonucleotide Analysis.

The FastPCR software is an integrated tool environment for PCR primer and probe design and for prediction of oligonucleotide properties. The software provides comprehensive tools for designing primers for most PCR and perspective applications, including standard, multiplex, long-distance, inverse, real-time with TaqMan probe, Xtreme Chain Reaction (XCR), group-specific, overlap extension PCR for multifragment assembling cloning, and isothermal amplification (Loop-mediated Isothermal Amplification). A program is available to design specific oligonucleotide sets for long sequence assembly by ligase chain reaction and to design multiplexed of overlapping and nonoverlapping DNA amplicons that tile across a region(s) of interest for targeted next-generation sequencing, competitive allele-specific PCR (KASP)-based genotyping assay for single-nucleotide polymorphisms and insertions and deletions at specific loci, among other features. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. FastPCR includes various bioinformatics tools for analysis and searching of sequences, restriction I-II-III-type enzyme endonuclease analysis, and pattern searching. The program also supports the assembly of a set of contiguous sequences, consensus sequence generation, and sequence similarity and conservancy analysis. FastPCR performs efficient and complete detection of various repeat types with visual display. FastPCR allows for sequence file batch processing that is essential for automation. The software is available for download at https://primerdigital.com/fastpcr.html and online version at https://primerdigital.com/tools/pcr.html .