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Kai Li

Publications and source records attributed to Kai Li.

At least 19 recordsLinked to original sources
PubMed2026

E2F7 promotes lung adenocarcinoma progression by affecting phosphorylation and stabilization of β-catenin.

BACKGROUND: E2F transcription factor 7 (E2F7) has been implicated in the tumorigenesis and progression of multiple cancer types; however, the molecular mechanisms through which E2F7 regulates malignant phenotypes in cancer cells remain largely undefined. In this study, we investigated the biological functions and underlying mechanisms of E2F7 in lung adenocarcinoma (LUAD). METHODS: E2F7 expression in LUAD was analyzed using The Cancer Genome Atlas (TCGA) datasets and further validated in clinical specimens via quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry. The effects of E2F7 on cancer cell self‑renewal and epithelial-mesenchymal transition (EMT) were assessed using sphere formation and Transwell assays, respectively. In vivo tumorigenicity and metastasis were evaluated using xenograft models combined with extreme limiting dilution analysis to assess tumor-initiating capacity. Wnt/β‑catenin pathway activity was measured using T-cell factor optimal promoter luciferase reporter plasmid/far-from optimal promoter luciferase reporter plasmid (TOP/FOP) flash reporter assays. β‑Catenin expression, stability, and ubiquitination were examined via western blotting, cycloheximide chase assays, and ubiquitination assays. Protein-protein interactions among E2F7, β‑catenin, and glycogen synthase kinase 3 beta (GSK3β) were verified through co‑immunoprecipitation (Co‑IP), glutathione S‑transferase (GST) pull‑down, and immunofluorescence assays. Truncated mutants were generated to map the functional binding domains of E2F7. In vitro immunoprecipitation and kinase assays were further performed to confirm that E2F7 regulates GSK3β autophosphorylation and β‑catenin phosphorylation. RESULTS: Bioinformatic analyses revealed that E2F7 was significantly upregulated in LUAD tissues, and elevated E2F7 expression correlated with poor patient prognosis. Functional assays demonstrated that E2F7 promoted LUAD cell self‑renewal and EMT. Mechanistically, cytoplasmic E2F7 directly associated with β‑catenin through its DNA‑binding domain (DBD) and PHA03247 domain. E2F7 modulated β‑catenin phosphorylation at Ser675 and Ser33/37/T41, thereby inhibiting ubiquitin‑mediated degradation and enhancing β‑catenin protein stability. Furthermore, E2F7 interacted with GSK3β and suppressed its autophosphorylation at Tyr216, concomitant with reduced β-catenin phosphorylation at Ser33/37/T41 and its accumulation. CONCLUSION: Collectively, these findings indicate that E2F7 drives LUAD malignant progression through regulation of the GSK3β/β‑catenin signaling axis and stabilization of β‑catenin. This study unveils a novel oncogenic mechanism of E2F7 in LUAD and identifies E2F7 as a promising therapeutic target for clinical intervention in LUAD.

E2F7↗
PubMed2026

Improvement the accuracy and reproducibility of telomere length measurement utilizing qPCR.

Telomere length serves as a well-established molecular biomarker for evaluating aging and age-associated diseases. Among various methods, quantitative PCR for telomere length detection is convenient, rapid, cost-effective, and capable of high-throughput analysis in large epidemiological cohorts. However, numerous studies have indicated that issues related to differences in DNA quality caused by DNA extraction process significantly affect the accuracy and reproducibility of qPCR-based telomere length quantification. Initially, we established a model of DNA integrity variation, by utilizing nucleic acid endonucleases of serial activity units to cleave genomic DNA, generating DNA with varying degrees of degradation. The integrity of DNA templates decreases, the reduction of long fragments and the increase of short fragments in the mixed telomere products are the causes of the disruption in Ct values. Moreover, compared with longer reference gene amplicons, the short-segment internal gene reference can reduce the impact of genomic integrity on its amplification. Subsequently, we utilized additional gel excision purification to reduce degradation products. It was found that gel excision processing provides the best stability for telomere length detection with the lowest coefficient of variation. Additionally, the introduce of another calibrator sample, which is used for to adjust the T/S value of the test sample, narrows the deviation between qPCR-derived telomere length and gold-standard Terminal Restriction Fragment (TRF) measurements. Collectively, these results reveal that gel excision purification supports stable telomere detection. Calculating the correction coefficient incorporating the short internal reference and calibrator minimizes measurement deviations relative to sample TRF values.

Telomere↗
PubMed2026

Metabolic engineering of Candida yeasts for biotechnological applications.

Candida yeasts represent a versatile yet underexploited platform for industrial biotechnology. These yeasts utilize a remarkably broad range of carbon sources, particularly for hydrophobic carbon sources, coupled with robust growth and diverse biosynthetic capacities, making them promising hosts for sustainable production of chemicals, fuels, and proteins. Despite these advantages, industrial deployment of Candida species has been hindered by concerns regarding opportunistic pathogenicity and the historical lack of efficient genetic manipulation tools, leading to a substantial gap between metabolic potential and practical utilization. Recent advances in functional genomics, genome editing, and systems metabolic engineering are rapidly overcoming these barriers, enabling more precise and efficient strain development. In this review, we systematically summarize recent progress in the metabolic engineering of Candida species as microbial cell factories, with particular emphasis on expanding genetic toolkits, utilizting renewable and non-conventional carbon sources, and biosynthesizing high-value compounds. In addition, we propose a biosafety-oriented classification framework to support their safe industrial deployment. Finally, we discuss current challenges and emerging opportunities, emphasizing that the synergy of synthetic biology and artificial intelligence-driven design holds the key to unlocking the biotechnological potential of Candida yeasts.

Candida↗
PubMed2025

Plasma Proteomic Profiles Predict Individual Future Osteoarthritis Risk.

OBJECTIVE: Osteoarthritis (OA) is a widespread degenerative joint disease that causes a considerable socioeconomic burden. Despite progress in genetic and environmental insights, early diagnosis is still limited by the lack of evident symptoms during the initial phases and accurate biomarkers. This study aims to identify plasma proteins associated with future risk of OA and develop a predictive model. METHODS: We conducted a large-scale proteomic analysis of 45,307 participants from the UK Biobank, excluding those with baseline OA. Plasma samples were assayed using the Olink Explore Proximity Extension Assay targeting 1,463 unique proteins. Clinical variables and OA outcomes were extracted and linked to electronic health records. A predictive model was constructed using the LightGBM machine learning method, and SHapley Additive exPlanations (SHAP) were applied to evaluate the importance of variables. RESULTS: We identified a panel of proteins significantly associated with the risk of developing OA. Notably, after adjusting for multiple confounders, collagen type IX alpha 1 chain (COL9A1) and cartilage acidic protein 1 (CRTAC1) were the most significant predictors of incident OA, with hazard ratios of 1.54 (95% confidence interval [CI] 1.48-1.61) and 1.65 (95% CI 1.54-1.78), respectively. SHAP analysis allowed a profound interpretation of the contribution of each protein and clinical variable to the model, revealing the multifactorial nature of OA risk prediction. The temporal trajectories of plasma proteins indicated that the levels of COL9A1 and CRTAC1 began to deviate from normal for more than a decade before OA onset, suggesting their potential use in early detection strategies. The predictive model, developed using the LightGBM algorithm, integrated proteins with clinical covariates and demonstrated an area under the curve (AUC) of 0.729 for 5-year OA prediction, 0.721 for 10-year prediction, and 0.723 for all incident OA. The predictive accuracy of the model was further enhanced for hip and knee OA, achieving AUCs of 0.820 and 0.803 for 5-year predictions. CONCLUSION: Our study identified the role of plasma proteomics in predicting future OA risk, which could contribute to preemptive measures. The innovative model, which integrates proteomic biomarkers with clinical data, offers a potential tool for risk assessment, potentially optimizing OA management strategies and enhancing prevention efforts.

Humans↗
PubMed2025

Marek's disease virus-1 unique gene LORF1 is involved in viral replication and MDV-1/Md5-induced atrophy of the bursa of Fabricius.

Marek's disease virus (MDV), an alphaherpesvirus, causes severe immunosuppression and T cell lymphomas in chickens, known as Marek's disease (MD), an economically important poultry disease primarily controlled by vaccination. Importantly, it also serves as a comparative model for studying herpesvirus-induced tumor formation in humans. MDV encodes more than 100 genes, most of which have unknown functions. MDV LORF1 is unique to serotype I MDV (MDV-1), lacking homologs in other herpesviruses, and has not been explored yet. To this end, an infectious bacterial artificial chromosome (BAC) harboring the complete genome of the MDV-1 very virulent strain Md5 was generated, and the rescued rMd5 maintained biological properties similar to the parental virus both in vitro and in vivo. Subsequently, rMd5ΔLORF1, a recombinant Md5 virus deficient in pLORF1 expression, was generated by a frameshift mutation in the LORF1 gene. Chickens infected with rMd5ΔLORF1 exhibited a lower mortality rate and delayed bursal atrophy than those infected with the parental rMd5 and the revertant virus (rMd5-reLORF1). Consistently, viral loads of rMd5ΔLORF1 were obviously lower than those of rMd5 or rMd5-reLORF1 in the bursa, but not in the spleen. Importantly, we found that pLORF1 deficiency impairs viral replication in bursal B cells. Furthermore, we showed that pLORF1 associated with the cellular membrane, interacted with MDV structural proteins, and exhibited punctate colocalization with tegument or capsid proteins in the cytoplasm. Taken together, this study demonstrates for the first time that the MDV-1 unique gene LORF1 is involved in MDV-induced bursal atrophy but not in tumor formation.

Animals↗
PubMed2007

Somatic microindels: analysis in mouse soma and comparison with the human germline.

Microindels, defined as mutations that result in a colocalized microinsertion and microdeletion with a net gain or loss of between 1 and 50 nucleotides, may be an important contributor to cancer. We report the first comprehensive analysis of somatic microindels. Our large database of mutations in the lacI transgene of Big Blue((R)) mice contains 0.5% microindels, 2.8% pure microinsertions, and 11.5% pure microdeletions. There appears to be no age, gender, or tissue-type specificity in the frequency of microindels. Of the independent somatic mutations that result in a net in-frame insertion or deletion, microindels are responsible for 13% of protein expansions and 6% of protein contractions. These in-frame microindels may play a crucial role in oncogenesis and evolution via "protein tinkering" (i.e., modest expansion or contraction of proteins). Four characteristics suggest that microindels are caused by unique mechanisms, not just simple combinations of the same mechanisms that cause pure microinsertions and pure microdeletions. First, microinsertions and microdeletions commonly occur at hotspots, but none of the 30 microindels are recurrent. Second, the sizes of the deletions and insertions in microindels are larger and more varied than in pure microdeletions and pure microinsertions. Third, microinsertions overwhelmingly repeat the adjacent base (97%) while the insertions in microindels do so only infrequently (17%). Fourth, analysis of the sequence contexts of microindels is consistent with unique mechanisms including recruitment of translesion DNA synthesis polymerases. The mouse somatic microindels have characteristics similar to those of human germline microindels, consistent with similar causative mechanisms in mouse and human, and in soma and germline.

Age Factors↗
PubMed2006

[Accuracy of age estimation in 14-year-old females by two skeletal age standards].

OBJECTIVE: To study the accurate of Li Guozhen's Percent Numeration and the Development Standard of Chinese Wrist Bone CHN Numeration of age estimation in 14-year-old females. METHODS: Fifty-one 14-year-old females from three middle schools in Loudi City in Hunan were selected randomly, whose left wrists were photographed with X-rays under the same condition. Skeletal age was estimated following the two numerations. The two skeletal ages were compared with each other and with chronological age. RESULTS: The skeletal age from Percent Numeration and CHN Numeration have no significance difference, P > 0.05. And the skeletal ages of the two Numerations have no significance difference either, P > 0.05. CONCLUSION: The Percent Numeration and the CHN Numeration are still suited for age estimation in 14-year-old females.

Adolescent↗
PubMed2006

Acute respiratory distress syndrome induced by avian influenza A (H5N1) virus in mice.

RATIONALE AND OBJECTIVE: The acute respiratory distress syndrome (ARDS) caused by avian influenza H5N1 viral infection has been reported in many humans since this virus was found to infect humans in Hong Kong in 1997, but no studies regarding an animal model of ARDS with H5N1 viral infection have been found in the literature. Here we present a mouse model of ARDS induced by H5N1 virus. METHODS: Six- to 8-wk-old BALB/c mice were inoculated intranasally (50 micro l) with 1 x 10(2) 50% mouse infectious doses of A/Chicken/Hebei/108/2002 (H5N1) virus. Lung injury was assessed by observation of lung water content and histopathology. Arterial blood gas, white blood cell count in bronchial alveolar lavage fluid, and tumor necrosis factor-alpha and interleukin-6 in bronchoalveolar lavage fluid and serum were measured at the indicated time points. RESULTS: Our data showed that H5N1 viral infection in mice resulted in typical ARDS, which was characterized by the following features: (1) about 80% of mice (13 of 16) dead on Days 6 to 8 postinoculation; (2) highly edematous lungs and dramatically increased lung wet:dry weight ratios and lung wet weight:body weight ratios; (3) inflammatory cellular infiltration, alveolar and interstitial edema, and hemorrhage in lungs; (4) progressive and severe hypoxemia; and (5) significant increase in neutrophils, tumor necrosis factor-alpha, and interleukin-6 in BALF. CONCLUSION: These results suggested that we successfully established a mouse model of ARDS with H5N1 viral infection, which may benefit further investigation into the pathogenesis of human ARDS induced by H5N1 virus.

Animals↗
PubMed2006

Enantioselective synthesis of vinylcyclopropanes and vinylepoxides mediated by camphor-derived sulfur ylides: rationale of enantioselectivity, scope, and limitation.

By a sidearm approach, camphor-derived sulfur ylides 1 were designed and synthesized for the cyclopropanation of electron-deficient alkenes and epoxidation of aldehydes. Under the optimal conditions, the exo-type sulfonium salts 4a and 4b reacted with beta-aryl-alpha,beta-unsaturated esters, amides, ketones, and nitriles to give 1,3-disubstituted-2-vinylcyclopropanes with high diastereoselectivities and enantioselectivities. When the endo-type sulfonium salts 5a and 5b were used, the diastereoselectivities were not changed, whereas the absolute configurations of the products became the opposite to those of the reactions of 4a and 4b. An ylide cyclopropanation of chalcone derivatives with phenylvinyl bromide in the presence of catalytic amount of chiral sulfonium salts 4b and 5b has been developed. The sidearmed hydroxyl group was found to play a key role in the control of enantioselectivity and diastereoselectivity. The origins of the high diastereoselectivity and enantioselectivity were also studied by density functional theory calculations, which reveal the importance of the hydrogen-bonding between the sidearmed hydroxyl group and the substrate in determining the diastereoselectivity and enantioselectivity. The ylides 1 were also successfully applied for the epoxidation of aromatic aldehydes.

Journal Article↗
PubMed2006

Correlation between protein expression of PTEN in human pancreatic cancer and the proliferation, infiltration, metastasis and prognosis.

In order to investigate the correlation between protein expression of PTEN and the proliferation, infiltration, metastasis and prognosis in pancreatic cancer, immunohistochemical SP method was used to examine the protein expression of PTEN, PCNA, MVD, MMP-2, MMP-9 and TUNEL method to detect the levels of apoptosis of pancreatic cells in 41 pancreatic head cancers from regional pancreatectomy (RP) and 10 normal pancreatic tissues. The results showed that among 41 cases of pancreatic cancers, the positive staining of PTNE (39.02%) was significantly weaker than that in normal pancreatic tissues (P < 0.05). The levels of PCNA labeling index (LI), apoptotic index (AI), microvessel density (MVD), MMP-2 LI and MMP-9 LI were decreased gradually with the increase of the expression intensity of PTEN, and there was a significant difference in the above parameters among the patients having different expression levels of PTEN (P < 0.01 or P < 0.05). There was a negative correlation between the expression of PTEN and PCNA LI, MVD, MMP-2 LI, MMP-9 LI, and a positive correlation between AI and the expression of PTEN. The expression intensity of PTEN was correlated with the postoperative survival of the patients with pancreatic cancer (chi2 = 22.3400, P < 0.0001, RR = 2.030). It was suggested that the expression levels of PTEN protein were closely related with proliferation, infiltration and metastasis in human pancreatic cancer, and the expression of PTEN protein was one of the prognostic factors for pancreatic cancer following RP.

Adult↗
PubMed2006

Effect of HIF-1alpha on VEGF-C induced lymphangiogenesis and lymph nodes metastases of pancreatic cancer.

The effect of hypoxia inducible factor-1alpha (HIF-1alpha) on vascular endothelial growth factor C (VEGF-C) and the correlation between HIF-1alpha and lymphangiogenesis and lymph nodes metastases (LNM) in pancreatic cancer were investigated. Immunohistochemical SP method was used to detect the protein expression of HIF-1alpha and VEGF-C, and Lymphatic vessel density (LVD) was determined by stain of VEGFR-3, collagen type IV in 75 pancreatic head cancers from regional pancreatectomy (RP) during Dec. 2001 to Dec. 2003. The relationship between HIF-1alpha and VEGF-C, lymphangiogenesis, LNM was analyzed statistically. The results showed that the positive expressionrate of HIF-1alpha and VEGF-C in pancreatic cancer tissues was 48.00 % (36/75) and 65.33 % (49/75) respectively. In positive group of HIF-1alpha, the positive rate of VEGF-C and LVD, and LVD rate was 80.56 % (29/36), 13.22+/-3.76 and 88.89 % (32/36) respectively, and in negative group of HIF-1alpha, positive rate of VEGF-C and LVD was 51.28 % (20/39), 5.98+/-2.17 and 66.67 % (26/39) respectively (P<0.01 or P<0.05). It was suggested that HIF-1alpha could promote the expression of VEGF-C, lymphangiogenesis and LNM in pancreatic cancer.

Adult↗
PubMed2006

[Predation and dispersal of Lithocarpus glaber seeds by rodents in Tiantong National Forest Park, Zhejiang Province].

This paper studied the predation and dispersal of Lithocarps glaber seeds by rodents in the Tiantong National Forest Park (29 degrees 48'N, 121 degrees 47'E) of Ningbo City, Zhejiang Province. Five seeds release transects (three in evergreen broad-leaved forest, one in Pinus messoniana forest, and one in shrub), and two seeds release states (covered/uncovered and tagged/untagged) were set up. Iron snare traps were baited to determine the effects of rodents on seeds disappearance. The results showed that two species of rodents, Niviventer confucianus and N. fulvescensi, were the potential predators and dispersers of the seeds. The lost seeds labeled with small piece of tintags could be classified into three categories, i. e., 1) seeds consumed by rodents, with tags stuck seed fragments; 2) seeds cut off from tags, with tags kept only; and 3) both seeds and tags disappeared. The loss rate of seeds was high at the beginning of the observation, and then decreased gradually. The daily seeds disappearance rate (DS-DR) was variable in different habitats, with the highest (1.03%) in evergreen broad-leaved forest and the lowest (0.32%) in shrub. The proportion of these three categories was different, and related with habitat types. The difference of DSDR between covered and uncovered states was statistically significant (P < 0.05), with a higher DSDR under uncovered state. No significant difference was observed between the DSDR of tagged and untagged. The distance of seeds dispersal by rodents was within 6.7 m.

Animals↗
PubMed2005

Augmented humoral and cellular immune responses to hepatitis B DNA vaccine adsorbed onto cationic microparticles.

Plasmid expressing HBV small envelope antigen was formulated with poly(lactide-co-glycolide-acid) (PLGA) and cetyltrimethylammonium bromide (CTAB) to generate highly uniform microparticles. Controlled release of DNA from these microparticles was demonstrated in vitro and in vivo using flow cytometry and confocal laser scanning microscopy with the focus on localization and quantitatively evaluation of antigen-presenting cells (APCs) involved in the expression of target antigen. Compared to mice vaccinated with naked DNA, mice immunized with PLGA-CTAB-DNA microparticles displayed a much higher percentage of CD11c+, HBsAg-expressing APCs in the draining lymph nodes at 24 h and day 14 postinoculation. In addition, a prolonged transcription of plasmid DNA was detected by RT-PCR in mice immunized with the microparticles. A significantly enhanced immunogenicity of PLGA-CTAB-DNA over naked DNA was observed in immunized mice, including higher levels of antibody production, interferon gamma (IFN-gamma) secretion and cytotoxic T lymphocyte activity. Mice immunized with PLGA-CTAB-DNA microparticles also showed greater efficacy of immunoprotection against challenge of transplanted HBsAg-expressing tumor cells. Our data suggest that controlled release of the PLGA-CTAB-DNA microparticles might involve in the mechanisms of its augmented immunogenicity and enhanced immunoprotection.

Adsorption↗
PubMed2005

[Relationship between metastatic rate and amount of lymph nodes and prognosis of gastric cancer].

OBJECTIVE: to investigate the relationship between metastatic rate and amount of lymph nodes and prognosis of gastric cancer. METHODS: The clinical data of 672 patients of gastric cancer with 22.7 regional metastatic lymph nodes on average were analyzed retrospectively. The patients were divided into 3 groups according to the TNM staging system (1997, 5th edition): pN0 (without metastatic lymph node), pN1 group (with 1-6 metastatic lymph nodes), pN2 group (with 7-15 metastatic lymph nodes), and pN3 group (with more than 215 metastatic lymph nodes), or into 4 groups according to the lymph node metastasis rate: rN0 group (without metastasis of lymph node), rN1 group (with a lymph node metastasis rate 0 < 10%), rN2 group (with a lymph node metastasis rate of 10%-30%), and rN3 group with a lymph node metastasis rate > 30%). The 5-year survival rates of different groups in these 2 classification systems and the relationship between metastatic rate and amount of lymph nodes and prognosis of gastric cancer were analyzed. RESULTS: According to the lymph node metastasis grading system there were 93 patients in the pN0 group, 201 in the pN1 group, 108 in the pN2 group, and 32 in the pN3 group with the 5-year survival rates of 90.3%, 65.2%, 37.0%, and 18.9% respectively (chi(2) = 12.56, P < 0.01). According to the lymph node metastasis system 93 patients were in the rN0 group, 183 in the rN1 group, 92 in the rN2 group, and 66 in the rN3 group, with the 5-year survival rates of 90.3%, 69.9%, 45.7%, and 10.6% respectively (chi(2) = 14.38, P < 0.01). If the cases in the same pN group were re-divided according to the lymph node metastasis rate, there were significant differences in the 5-year survival rate among the new rN groups (chi(2) = 5.26, P = 0.21), and there was a great difference in the distribution of prognosis among different groups. However, if the cases in the same group rN group were re-divided according to the amounts of metastatic lymph nodes, there was no significant difference in 5-year survival rate among different new pN groups (chi(2) = 0.14, P = 0.932). Multivariate COX model analysis showed that the influencing factors of the prognosis were rN grading, pN grading, infiltration depth of cancer, gross type, and size of the cancer, arranged according to the degree of closeness (P < 0.05); lymph nose metastasis rate was closely related with the size, infiltration depth, and gross type of the cancer (P < 0.05). In the same metastasis rate, the 5-year survivals rate decreased along with the increase of the amount of metastatic lymph nodes, and vice versa (chi(2) = 8.22, P = 0.004; chi(2) = 4.52, P = 0.033). CONCLUSION: The staging system based on metastatic lymph node rate is more predictable the number of metastatic lymph nodes for the prognosis of gastric cancer.

Adult↗
PubMed2005

Visualization methods for statistical analysis of microarray clusters.

BACKGROUND: The most common method of identifying groups of functionally related genes in microarray data is to apply a clustering algorithm. However, it is impossible to determine which clustering algorithm is most appropriate to apply, and it is difficult to verify the results of any algorithm due to the lack of a gold-standard. Appropriate data visualization tools can aid this analysis process, but existing visualization methods do not specifically address this issue. RESULTS: We present several visualization techniques that incorporate meaningful statistics that are noise-robust for the purpose of analyzing the results of clustering algorithms on microarray data. This includes a rank-based visualization method that is more robust to noise, a difference display method to aid assessments of cluster quality and detection of outliers, and a projection of high dimensional data into a three dimensional space in order to examine relationships between clusters. Our methods are interactive and are dynamically linked together for comprehensive analysis. Further, our approach applies to both protein and gene expression microarrays, and our architecture is scalable for use on both desktop/laptop screens and large-scale display devices. This methodology is implemented in GeneVAnD (Genomic Visual ANalysis of Datasets) and is available at http://function.princeton.edu/GeneVAnD. CONCLUSION: Incorporating relevant statistical information into data visualizations is key for analysis of large biological datasets, particularly because of high levels of noise and the lack of a gold-standard for comparisons. We developed several new visualization techniques and demonstrated their effectiveness for evaluating cluster quality and relationships between clusters.

Algorithms↗
PubMed2005

High fidelity PCR with an off/on switch mediated by proofreading polymerases combining with phosphorothioate-modified primer.

In the initial report, introducing a single phosphorothioate modification at the very 3' terminus of the oligodeoxynucleotide primer has been shown to effectively protect the oligodeoxynucleotide degradation due to the 3' exonuclease activity. In this study, we reported a novel finding that phosphorothioate modification at the 3' end of primers could not only effectively prevent the primer from degradation, but could also mediate an off-switch extension by Pfu polymerase when primers also carry single or multiple mismatched bases located in the first eight bases of the 3' terminus. This suggests that the combination of 3' phosphorothioate-modified primers with exo+ polymerases such as Pfu constituted an on/off switch, which allows perfectly matched primers to be extended but not mismatched primers. Furthermore, we found that polymerases with different fidelities showed different efficiencies in turning off mismatched-primer mediated extension. So we described here a SYBR green-based real-time quantitative PCR assay for the detection of abundance level of gene expression that did not require fluorescently labeled gene-specific probes or complicated primer combinations. The emergence of real-time quantitative RT-PCR technology is thus suited for a diverse application with a need for high-throughput methods to detect and quantify different gene expressions by way of simplicity, versatility, and accuracy, and thus could complement global microarray-based expression profiling strategies.

DNA Primers↗