One-step reverse transcriptase-free miRNA detection system and its application for detection of gastrointestinal cancers.

MicroRNAs (miRNAs) play pivotal roles in gene regulation and their dysregulation is implicated in various diseases, including cancer. Current methods for miRNA analysis often involve complex procedures and high costs, limiting their clinical utility. Therefore, there is a critical need for the development of simpler and more cost-effective miRNA detection techniques to enable early disease diagnosis. In this study, we introduce a novel one-enzyme for miRNA one-step detection method using Taq DNA polymerase, termed OSMOS-qPCR. We optimized the PCR buffer, PCR program, Taq DNA Polymerase concentrations and reverse PCR primer concentrations, resulted in a wide linear range from 100 fM to 0.001 fM (R2 > 0.98 for each miRNA), the detection limit for OSMOS-qPCR was 0.0025 fM. Furthermore, OSMOS-qPCR demonstrates excellent specificity to differentiation of less than 0.1 % nonspecific signal. Finally, we demonstrated the robust amplification efficiency, enabling the detection of trace amounts of cell-free miRNA in serum samples, and the excellent discrimination ability between gastrointestinal cancers and control subjects (AUC value = 1.0) if combined two miRNAs. The development of OSMOS-qPCR offering a simpler, cost-effective, and efficient detection method, has the potential to be non-invasive strategy for early detection of gastrointestinal cancers.

Enhancing APE1 detection through apurinic/apyrimidinic site inhibition of DNA polymerase: an innovative, highly sensitive approach.

This study presents an innovative method for the highly sensitive detection of apurinic/apyrimidinic endonuclease 1 (APE1), a crucial biomarker and target for cancer diagnosis and treatment. The method is predicated on our discovery that the apurinic or apyrimidinic site (AP site) can inhibit the activity of Taq DNA polymerase. Subsequent experiments further led to the development of a new amplification method based on the digestion activity of Lambda exonuclease. This approach showed potential to detect trace amounts of APE1 in biological samples with high sensitivity.

Cloning Polymerase Chain Reaction (PCR) Products: Making T Vectors.

During polymerase chain reaction (PCR), DNA polymerases such as Taq add a single, unpaired residue-preferentially an adenosyl residue-to each 3' end of a double-stranded amplified product. The unpaired terminal (A) residues can pair with a linear T vector that carries an unpaired 3'-thymidyl residue at each end. T vectors can be prepared in the laboratory or they may be purchased ready-made from commercial suppliers. This protocol outlines two methods in common use to generate T vectors.

Cloning Polymerase Chain Reaction (PCR) Products: TA Cloning.

The nontemplate-dependent terminal transferase activity inherent in nonproofreading DNA polymerases such as Taq provides a highly efficient method to clone PCR products. The enzyme adds a single, unpaired residue-preferentially an adenosyl residue-to each 3' end of a double-stranded amplified product. The unpaired terminal (A) residues can pair with a linear T vector that carries an unpaired 3'-thymidyl residue at each end. The two chief advantages of TA cloning are speed and lack of reliance on restriction enzymes. The major disadvantage is an inability to clone directionally. For this reason, it is important to pick and analyze several transformed clones when a particular orientation of the amplified fragment is required.

TaqMan quantitative real-time PCR for detecting Avipoxvirus DNA in various sample types from hummingbirds.

BACKGROUND: Avian pox is a viral disease documented in a wide range of bird species. Disease-related detrimental effects can cause dyspnea and dysphagia, and birds with high metabolic requirements, such as hummingbirds, are thus especially vulnerable to the pathogen. Hummingbirds have a strong presence in California, especially in urban environments. However, little is understood regarding the impact of pox virus on hummingbird populations. Currently, diagnosing a pox infection relies on obtaining a tissue biopsy, which poses significant risks to birds and challenges in the field. Understanding the ecology of hummingbird pox viral infections could be advanced by a minimally invasive ante-mortem diagnostic method. Our aim was to address whether pox infections can be diagnosed using integumentary system samples besides tissue biopsies. To meet this goal, we tested multiple integumentary sample types using a quantitative real-time PCR assay. A secondary study goal was to determine which sample types (ranging from minimally to highly invasive sampling) were optimal for identifying infected birds. METHODOLOGY AND PRINCIPAL FINDINGS: Pox-like lesion tissue, pectoral muscle, feathers, toenail clippings, blood, and swabs (both pox-like lesion tissue and non pox-like lesion tissue) were taken from live birds and carcasses of two species of hummingbirds found in California. To maximize successful diagnosis, especially for samples with low viral load, a real-time quantitative PCR assay was developed for detecting the hummingbird-specific Avipoxvirus 4b core protein gene. Avipoxvirus DNA was successfully amplified from all sample types obtained from 27 individuals. These results were compared to those of conventional PCR and comparisons were also made among sample types, utilizing lesion tissue samples as the gold standard. CONCLUSIONS AND SIGNIFICANCE: Hummingbird avian pox can be diagnosed without relying on tissue biopsies. We identify that feather samples, of which contour feathers yielded the best results, can be used for diagnosing infected birds, thus reducing sampling risk. In sum, the real-time PCR assay detected viral DNA in various integumentary system sample types and will be useful in future studies of hummingbird disease ecology.

Dual coding potential of a 2',5'-branched ribonucleotide in DNA.

Branchpoints in RNA templates are highly mutagenic, but it is not known yet whether this also applies to branchpoints in DNA templates. Here, we report how nucleic acid polymerases replicate a 2',5'-branched DNA (bDNA) molecule. We constructed long-chained bDNA templates containing a branch guanosine and T7 promoters at both arms by splinted ligation. Quantitative real-time PCR analysis was used to investigate whether a branchpoint blocks DNA synthesis from the two arms in the same manner. We find that the blocking effect of a branchpoint is arm-specific. DNA synthesis from the 2'-arm is more than 20,000-fold decreased, whereas from the 3'-arm only 15-fold. Our sequence analysis of full-length nucleic acid generated by Taq DNA polymerase, Moloney murine leukemia virus reverse transcriptase, and T7 RNA polymerase from the 2'-arm of bDNA shows that the branched guanine has a dual coding potential and can base-pair with cytosine and guanine. We find that branchpoint templating is influenced by the type of the surrounding nucleic acid and is probably modulated by polymerase and RNase H active sites. We show that the branchpoint bypass by the polymerases from the 3'-arm of bDNA is predominantly error-free, indicating that bDNA is not as highly mutagenic as 2',5'-branched RNA.

5'-Terminal nucleotide variations in human cytoplasmic tRNAHisGUG and its 5'-halves.

Transfer RNAs (tRNAs) are fundamental adapter components of translational machinery. tRNAs can further serve as a source of tRNA-derived noncoding RNAs that play important roles in various biological processes beyond translation. Among all species of tRNAs, tRNAHisGUG has been known to uniquely contain an additional guanosine residue at the -1 position (G-1) of its 5'-end. To analyze this -1 nucleotide in detail, we developed a TaqMan qRT-PCR method that can distinctively quantify human mature cytoplasmic tRNAHisGUG containing G-1, U-1, A-1, or C-1 or lacking the -1 nucleotide (starting from G1). Application of this method to the mature tRNA fraction of BT-474 breast cancer cells revealed the presence of tRNAHisGUG containing U-1 as well as the one containing G-1 Moreover, tRNA lacking the -1 nucleotide was also detected, thus indicating the heterogeneous expression of 5'-tRNAHisGUG variants. A sequence library of sex hormone-induced 5'-tRNA halves (5'-SHOT-RNAs), identified via cP-RNA-seq of a BT-474 small RNA fraction, also demonstrated the expression of 5'-tRNAHisGUG halves containing G-1, U-1, or G1 as 5'-terminal nucleotides. Although the detected 5'-nucleotide species were identical, the relative abundances differed widely between mature tRNA and 5'-half from the same BT-474 cells. The majority of mature tRNAs contained the -1 nucleotide, whereas the majority of 5'-halves lacked this nucleotide, which was biochemically confirmed using a primer extension assay. These results reveal the novel identities of tRNAHisGUG molecules and provide insights into tRNAHisGUG maturation and the regulation of tRNA half production.

Binding-responsive catalysis of Taq DNA polymerase for the sensitive and selective detection of cell-surface proteins.

Here we develop a new method for the sensitive and selective detection of cell-surface proteins with an aptamer probe designed for binding-responsive catalysis of Taq DNA polymerase. Taking the biotin receptor as a model, the method allows the detection of target protein on surfaces of different types of cancer cells.

The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-alkylguanine DNA adducts.

Enzymatic approaches for locating alkylation adducts at single-base resolution in DNA could enable new technologies for understanding carcinogenesis and supporting personalized chemotherapy. Artificial nucleotides that specifically pair with alkylated bases offer a possible strategy for recognition and amplification of adducted DNA, and adduct-templated incorporation of an artificial nucleotide has been demonstrated for a model DNA adduct O(6)-benzylguanine by a DNA polymerase. In this study, DNA adducts of biological relevance, O(6)-methylguanine (O(6)-MeG) and O(6)-carboxymethylguanine (O(6)-CMG), were characterized to be effective templates for the incorporation of benzimidazole-derived 2'-deoxynucleoside-5'-O-triphosphates ( BENZI: TP and BIM: TP) by an engineered KlenTaq DNA polymerase. The enzyme catalyzed specific incorporation of the artificial nucleotide BENZI: opposite adducts, with up to 150-fold higher catalytic efficiency for O(6)-MeG over guanine in the template. Furthermore, addition of artificial nucleotide BENZI: was required for full-length DNA synthesis during bypass of O(6)-CMG. Selective incorporation of the artificial nucleotide opposite an O(6)-alkylguanine DNA adduct was verified using a novel 2',3'-dideoxy derivative of BENZI: TP. The strategy was used to recognize adducts in the presence of excess unmodified DNA. The specific processing of BENZI: TP opposite biologically relevant O(6)-alkylguanine adducts is characterized herein as a basis for potential future DNA adduct sequencing technologies.

The Use of Real-Time Reverse Transcription-PCR for Assessing Estrogen Receptor and Estrogen-Responsive Gene Expression.

Real-time reverse transcription-polymerase chain reaction (RT-PCR), also known as quantitative RT-PCR (qRT-PCR), is a powerful tool for assessing gene transcription levels. The technique is especially useful for measuring estrogen receptor transcript levels as well as gene expression changes in response to estrogen stimulation as it is quick, accurate, robust, and allows the measurement of gene expression in a variety of tissues and cells. This chapter describes the protocols used for the real-time RT-PCR assay using hydrolysis (TaqMan-type) probes.

Phytoestrogens Activate the Estrogen Receptor in HepG2 Cells.

Phytoestrogens are popular alternatives to estrogen therapy however their effects on hemostasis in postmenopausal women are unknown. This chapter describes a protocol to determine the effect of the phytoestrogens genistein, daidzein and equol, on the expression of key genes from the hemostatic system in human hepatocyte cell models and to determine the role of estrogen receptors in mediating any response seen using in vitro culture systems and Taqman(®) gene expression analysis.

A polymerase engineered for bisulfite sequencing.

Bisulfite sequencing is a key methodology in epigenetics. However, the standard workflow of bisulfite sequencing involves heat and strongly basic conditions to convert the intermediary product 5,6-dihydrouridine-6-sulfonate (dhU6S) (generated by reaction of bisulfite with deoxycytidine (dC)) to uracil (dU). These harsh conditions generally lead to sample loss and DNA damage while milder conditions may result in incomplete conversion of intermediates to uracil. Both can lead to poor recovery of bisulfite-treated DNA by the polymerase chain reaction (PCR) as either damaged DNA and/or intermediates of bisulfite treatment are poor substrate for standard DNA polymerases. Here we describe an engineered DNA polymerase (5D4) with an enhanced ability to replicate and PCR amplify bisulfite-treated DNA due to an ability to bypass both DNA lesions and bisulfite intermediates, allowing significantly milder conversion conditions and increased sensitivity in the PCR amplification of bisulfite-treated DNA. Incorporation of the 5D4 DNA polymerase into the bisulfite sequencing workflow thus promises significant sensitivity and efficiency gains.

The addition of a spin column step in the telomeric repeat application protocol removes telomerase inhibitors.

Telomerase activity in cancer cells is commonly analyzed by a polymerase chain reaction (PCR)-based assay termed the telomeric repeat amplification protocol (TRAP). However, nonspecific inhibition of Taq polymerase during the PCR step is frequently observed in inhibitor analysis or drug screening. Thus, the removal of excess inhibitors prior to PCR is an essential step for the proper evaluation of telomerase inhibitory effects. Here, a size exclusion spin column was applied to remove small molecular weight inhibitors from the telomerase extension products. The spin column-added protocol, termed sTRAP, provides a more reliable estimation of the inhibitory effects of telomerase activity.

A simple PCR-based fluorometric system for detection of mutant fusion DNAs using a quencher-free fluorescent DNA probe and graphene oxide.

We propose a facile fluorometric system for detection of gene mutations using graphene oxide (GO). A fluorescent probe DNA anneals to a specific mutant gene and is degraded by the 5'→ 3' exonuclease activity of Taq polymerase during PCR, and the released fluorophore retains fluorescence after addition of GO without quenching.

MicroRNA function in mast cell biology: protocols to characterize and modulate microRNA expression.

MicroRNAs (miRNAs) are small noncoding RNA molecules that can modulate mRNA levels through RNA-induced silencing complex (RISC)-mediated degradation. Recognition of target mRNAs occurs through imperfect base pairing between an miRNA and its target, meaning that each miRNA can target a number of different mRNAs to modulate gene expression. miRNAs have been proposed as novel therapeutic targets and many studies are aimed at characterizing miRNA expression patterns and functions within a range of cell types. To date, limited research has focused on the function of miRNAs specifically in mast cells; however, this is an emerging field. In this chapter, we will briefly overview miRNA synthesis and function and the current understanding of miRNAs in hematopoietic development and immune function, emphasizing studies related to mast cell biology. The chapter will conclude with fundamental techniques used in miRNA studies, including RNA isolation, real-time PCR and microarray approaches for quantification of miRNA expression levels, and antagomir design to interfere with miRNA function.

Detection of circulatory microRNAs in prostate cancer.

Prostate cancer (PCa) is one of the most common cancer worldwide and accounts for 14.4 % of all new cancer cases. The clinical outcome and management of PCa can be significantly improved by use of biomarker assays for early detection, prognosis and also for prediction and monitoring of treatment response. MiRNAs are short, endogenous, single-stranded RNA molecules that play important role in regulation of gene expression and can modulate a number of cellular processes. Discovery of miRNAs in circulation has not only facilitated understanding their role in various diseases but also paved new avenues for biomarker discovery due to their ease of access and stability. The fact that a minimally invasive test based on miRNAs profiles can distinguish the presence or absence of disease illustrates immense potential of these molecules as predictive biomarkers.In this chapter, we have summarized the presumed mechanisms of miRNA release into the circulation and systematically summarized the studies of circulatory miRNAs in PCa. Also, we have mainly focused on the methodology of identification of circulatory miRNAs from biofluids.

Measuring microRNA expression in mouse hematopoietic stem cells.

MicroRNAs (miRNAs) are important regulators of diverse biologic processes. In the hematopoietic system, miRNAs have been shown to regulate lineage fate decisions, mature immune effector cell function, apoptosis, and cell cycling, and a more limited number of miRNAs has been shown to regulate hematopoietic stem cell (HSC) self-renewal. Many of these miRNAs were initially identified as candidate regulators of HSC function by comparing miRNA expression in hematopoietic stem and progenitors cells (HSPCs) to their mature progeny. While the measurement of miRNA expression in rare cell populations such as HSCs poses practical challenges due to the low amount of RNA present, a number of techniques have been developed to measure miRNAs in small numbers of cells. Here, we describe our protocol for measuring miRNAs in purified mouse HSCs using a highly sensitive real-time quantitative PCR strategy that utilizes microfluidic array cards containing pre-spotted TaqMan probes that allows the detection of mature miRNAs in small reaction volumes. We also describe a simple data analysis method to evaluate miRNA expression profiling data using an open-source software package (HTqPCR) using mouse HSC miRNA profiling data generated in our lab.

Real-time quantitative (RQ-)PCR approach to quantify the contribution of proliferation to B lymphocyte homeostasis.

The cells of the adaptive immune system, B and T lymphocytes, each generate a unique antigen receptor through V(D)J recombination of their immunoglobulin (Ig) and T-cell receptor (TCR) loci, respectively. Such rearrangements join coding elements to form a coding joint and delete the intervening DNA as circular excision products containing the signal joint. These excision circles are relatively stable structures that cannot replicate and have no function in the cell. Since the coding joint in the genome is replicated with each cell division, the ratio between coding joints and signal joints in a population of B cells can be used as a measure for proliferation. This chapter describes a real-time quantitative polymerase chain reaction (RQ-PCR)-based approach to quantify proliferation through calculating the ratio between coding joints and signal joints of the frequently occurring kappa-deleting rearrangements in the IGK light chain loci in man and mouse. The approach is useful to study the contribution of proliferation to B-cell homeostasis in health and disease.

[Detection of EGFR gene mutations in 100 non-small cell lung cancer clinical samples by a real-time polymerase chain reaction method using amplification refractory mutation system specific primers and Taqman fluorescence probes].

BACKGROUND: Epidermal growth factor receptor (EGFR) gene mutation is the most important predictor of the efficiency of EGFR-tyrosine kinase inhibitors in the treatment of non-small cell lung cancer (NSCLC). The detection of EGFR gene mutations can guide individual therapies for NSCLC. Numerous methods are used to detect EGFR gene mutation and each method has different features. This study aims to establish a real-time polymerase chain reaction (PCR) method for the detection of EGFR gene mutations using amplification refractory mutation system (ARMS) specific primers and Taqman fluorescence probes. METHODS: ARMS specific primers for the two EGFR gene mutations (E746_A750 and L858R) and Taqman fluorescence probes for the detection of the target sequence were carefully designed by the Primer Premier 5.0 software. Then, using the recombinants containing E746_A750 and L858R mutations as the study objects, we further analyzed the sensitivity and lower limit of this method, and then determined the cutoff ΔCt value to evaluate specific or non-specific amplification. A total of 100 clinical samples were collected and used to detect the EGFR gene mutations using this method. RESULTS: The lower limit of this method for the detection of EGFR gene mutation was 10 copies if no interference of wild-type EGFR gene or background DNA existed. Regarding the method sensitivity, the detection resolution was as high as 1% and 0.1%-0.5% in the background of 500 and 5,000 copies/µL wild-type EGFR gene, respectively. Regarding the method specificity, non-specific amplifications were found when it was used to detect 21 L858R mutations in leukocyte DNA samples from healthy volunteers. However, the minimal ΔCt value was 14.48. Non-specific amplifications were not found when detecting 19 Del mutations. Among the 100 clinical samples, 39 mutations were detected (19 Del and 21 L858R were 21 and 18, respectively) using this method. The total mutation rate was 39.0%. CONCLUSIONS: The proposed ARMS-TaqMan real-time PCR method for the detection of 19 Del and 21 L858R mutations in EGFR gene was rapid, simple, sensitive, specific, and applicable in the clinical setting.

Homogeneous assay for real-time and simultaneous detection of thymidine kinase 1 and deoxycytidine kinase activities.

Measurement of thymidine kinase-1 (TK1) and deoxycytidine kinase (dCK) activity may be useful in cancer disease management. Therefore, a one-step homogeneous assay for real-time determination of TK1 and dCK was developed by combining enzyme complementation with fluorescent signal generation using primer extension and a quenched probe oligodeoxyribonucleotide system at 37 °C. Complementation, for producing dCTP and TTP from nucleoside substrates, was carried out by dTMP kinase and/or UMP/CMP kinase and nucleoside diphosphate kinase. dNTP was continuously incorporated into a fixed oligodeoxyribonucleotide primer, template, and probe system, and the fluorescent signal was generated by using the combined actions of primer extension and 5' exonuclease activity of Thermophilus aquaticus (Taq) DNA polymerase for specific relief of fluorescent quenching. Fluorescence was captured at 1-min intervals using a real-time polymerase chain reaction (PCR) instrument. A horizontal threshold line, crossing all sample relative fluorescent units (RFU) values at the level of the RFU of the blank sample at the end of the assay (i.e., 90 min), was drawn, obtaining RFU measurement data in minutes for each sample. Duplex proof of principle was demonstrated by the independent determination of different amounts of dCK and TK1 in combination. R(2) values of 0.90 were demonstrated with Prolifigen TK-REA U/L reference values obtained from pathological canine and human serum samples.