Tandem Repeat DNA Research Articles

RGFP966 Suppresses Tumor Growth and Migration Through Inhibition of EGFR Expression in Hepatocellular Carcinoma Cells in vitro.

PurposeHistone deacetylase 3 (HDAC3) has been suggested to play a role in hepatocellular carcinoma (HCC). In the present report, we aimed to identify the effects of RGFP966, a specific HDAC3 inhibitor, on the cell proliferation and migration of HCC cell lines.MethodsHuman HCC cell lines, which were identified using short tandem repeat (STR) DNA profiling analysis, were used in this report. Cell proliferation assay was used to identify the growth viability of cells. Wound healing and transwell assay were used to identify the migration ability of cells. Further, a human phospho-receptor tyrosine kinases array kit was used to screen out RGFP966 effects on key receptor tyrosine kinases. Then, the mRNA expression was quantified by real-time PCR, and protein expression was identified by Western blot immunoassay.ResultsWe found that RGFP966 inhibited both proliferation and migration of HCC cells. Further, RGFP966 represses the expression and phosphorylation levels of epidermal growth factor receptor (EGFR) in HCC cells. Moreover, HDAC3 is involved in the inhibition of EGFR by RGFP966. Overall, we elucidated an inhibitive function of RGFP966 in HCC progression.ConclusionRGFP966 inhibits EGFR signaling pathway and suppresses proliferation and migration of HCC cells.

Characteristics of a novel cell line ZJU-0430 established from human gallbladder carcinoma

BackgroundGallbladder cancer is the most common malignant neoplasm of the biliary tract, responsible for 80–95% of cases. Appropriate models are required for investigating the molecular pathogenesis of gallbladder cancer.MethodsIn this study, we aimed to establish a gallbladder cancer cell line from primary tumour. Single cell RNA sequencing, Light and electron microscopy, DNA content analysis, cytogenetic analysis, short tandem repeat (STR) DNA fingerprint analysis, immunophenotypic characterization, and xeno-transplantation were utilized to characterize the novel ZJU-0430 cell line in vitro and in vivo.ResultsThe cell line showed multiple cell shapes and characteristic epithelial morphologies under the microscope, but no too much heterogeneity by scRNA-Seq, with a population doubling time (PDT) of 19.81 h, which was shorter than that for GBC-SD cells. An immunophenotypic analysis revealed that ZJU-0430 cells were positive for CD24, CD44, CD29 and CD133 expression, and partially positive for CD184, and CD326 expression, and negative for CD34, CD90, CD117, and CD338 expression, similar to the primary cancer cells. A pathological analysis confirmed the origination of cell line from gallbladder tumour. ZJU-0430 cells had higher migration, invasion and proliferation properties than GBC-SD cells in vitro, and showed in vivo tumorigenicity in nude mouse xenograft settings.ConclusionsThe results confirm the potential utility of ZJU-0430 cell line as a representative model of gallbladder cancer and suggest that it could be used in the in vitro and in vivo studies of gallbladder cancer pathogenesis and to develop new therapeutics.

Variable Number Tandem Repeats in the Mitochondrial DNA of Lentinula edodes

Variable number tandem repeats (VNTRs) in mitochondrial DNA (mtDNA) of Lentinula edodes are of interest for their role in mtDNA variation and their application as genetic marker. Sequence analysis of three L. edodes mtDNAs revealed the presence of VNTRs of two categories. Type I VNTRs consist of two types of repeat units in a symmetric distribution, whereas Type II VNTRs contain tandemly arrayed repeats of 7- or 17-bp DNA sequences. The number of repeat units was variable depending on the mtDNA of different strains. Using the variations in VNTRs as a mitochondrial marker and the A mating type as a nuclear type marker, we demonstrated that one of the two nuclei in the donor dikaryon preferentially enters into the monokaryotic cytoplasm to establish a new dikaryon which still retains the mitochondria of the monokaryon in the individual mating. Interestingly, we found 6 VNTRs with newly added repeat units from the 22 mates, indicating that elongation of VNTRs occurs during replication of mtDNA. This, together with comparative analysis of the repeating pattern, enables us to propose a mechanistic model that explains the elongation of Type I VNTRs through reciprocal incorporation of basic repeat units, 5’-TCCCTTTAGGG-3’ and its complementary sequence (5’-CCCTAAAGGGA-3’).

Leucocyte Telomere Length and Glucose Tolerance Status in Mixed-Ancestry South Africans.

Telomeres are DNA-tandem repeats situated at the ends of chromosomes and are responsible for genome stabilization. They are eroded by increased cell division, age and oxidative stress with shortened leucocyte telomeres (LTL) being associated with inflammatory disorders, including Type II diabetes. We assessed LTL in 205 participants across glucose tolerance groups at baseline and after three years in the mixed ancestry population of South Africa which have been shown to have high rates of obesity and T2DM. Baseline and follow-up data included glucose tolerance status, anthropometric measurements, lipids, insulin, γ-glutamyl transferase (GGT), cotinine, and HbA1c. Telomere length was measured using the absolute telomere q-PCR method performed on a Bio-Rad MiniOpticon Detector. No significant difference was detected in LTL across glucose tolerance groups at both time points, including in subjects who showed a deterioration of their glucose tolerance status. There was, however, a significant negative correlation between LTL and age which was more pronounced in diabetes (r = −0.18, p = 0.04) and with GGT (r = −0.16, p = 0.027). This longitudinal study has demonstrated that LTL shortening is not evident within three years, nor is it associated with glycaemia. Further studies in a larger sample and over a longer time period is required to confirm these results.

Genetic diversity and drug resistance of Mycobacterium tuberculosis in Yunnan, China.

BackgroundChina is a country with high burden of tuberculosis (TB), especially drug‐resistant TB (DR‐TB), which is still a serious health problem in Yunnan Province. Mycobacterium tuberculosis (MTB) is the pathogenic microorganism of TB. The epidemiological characteristics of MTB strains in local areas need to be described.MethodsA total of 430 clinical MTB isolates were collected from Yunnan Province and genotyped through the method of 24‐locus mycobacterial interspersed repetitive unit‐variable number tandem DNA repeats (MIRU‐VNTR).ResultsThe genotypes of the 24 loci showed abundantly genetic diversity, and allelic diversity index (h) of these loci varied from 0.012 to 0.817. Among the 430 strains, 30 clusters and 370 unique genotypes were identified. Beijing family was the predominant lineage (70.47%) in Yunnan MTB strains, and the other lineages contained T family (5.81%), MANU2 (0.70%), LAM (3.26%), CAS (0.23%), New‐1 (8.37%), and some unknown clades (11.16%). A total of 74 TB strains were identified as drug resistance through drug susceptibility testing (DST), including 38 multidrug‐resistant TB (MDR‐TB) and 36 single‐drug‐resistant TB (SDR‐TB). The frequency of MDR‐TB strains was significantly higher in Beijing family (10.89%) than that in non‐Beijing family (3.94%, P = 0.032).ConclusionsAlthough MTB strains showed high genetic diversity in Yunnan, China, the Beijing family was still the dominant strain. A high frequency of MDR‐TB strains was recorded in the Beijing family.

Generation of immortalized human endometrial stromal cell lines with different endometriosis risk genotypes.

Endometriotic lesions are composed in part of endometrial-like stromal cells, however, there is a shortage of immortalized human endometrial stromal cultures available for research. As genetic factors play a role in endometriosis risk, it is important that genotype is also incorporated into analysis of pathological mechanisms. Human telomerase reverse transcriptase (hTERT) immortalization (using Lenti-hTERT-green fluorescent protein virus) took place following genotype selection; 13 patients homozygous for either the risk or non-risk 'other' allele for one or more important endometriosis risk single nucleotide polymorphism on chromosome 1p36.12 (rs3820282, rs56318008, rs55938609, rs12037376, rs7521902 or rs12061255). Short tandem repeat DNA profiling validated that donor tissue matched that of the immortalized cell lines and confirmed that cultures were genetically novel. Expression of morphological markers (vimentin and cytokeratin) and key genes of interest (telomerase, estrogen and progesterone receptors and LINC00339) were examined and functional assays for cell proliferation, steroid hormone and inflammatory responses were performed for 7/13 cultures. All endometrial stromal cell lines maintained their fibroblast-like morphology (vimentin-positive) and homozygous endometriosis-risk genotype following introduction of hTERT. Furthermore, the new stromal cultures demonstrated positive and diverse responses to hormones (proliferation and decidualisation changes) and inflammation (dose-dependent response), while maintaining hormone receptor expression. In conclusion, we successfully developed a range of human endometrial stromal cell lines that carry important endometriosis-risk alleles. The wider implications of this approach go beyond advancing endometriosis research; these cell lines will be valuable tools for multiple endometrial pathologies offering a level of genetic and phenotypic diversity not previously available.

At the Beginning of the End and in the Middle of the Beginning: Structure and Maintenance of Telomeric DNA Repeats and Interstitial Telomeric Sequences.

Tandem DNA repeats derived from the ancestral (TTAGGG)n run were first detected at chromosome ends of the majority of living organisms, hence the name telomeric DNA repeats. Subsequently, it has become clear that telomeric motifs are also present within chromosomes, and they were suitably called interstitial telomeric sequences (ITSs). It is well known that telomeric DNA repeats play a key role in chromosome stability, preventing end-to-end fusions and precluding the recurrent DNA loss during replication. Recent data suggest that ITSs are also important genomic elements as they confer its karyotype plasticity. In fact, ITSs appeared to be among the most unstable microsatellite sequences as they are highly length polymorphic and can trigger chromosomal fragility and gross chromosomal rearrangements. Importantly, mechanisms responsible for their instability appear to be similar to the mechanisms that maintain the length of genuine telomeres. This review compares the mechanisms of maintenance and dynamic properties of telomeric repeats and ITSs and discusses the implications of these dynamics on genome stability.

Evaluation genotypes of cancer cell lines HCC1954 and SiHa by short tandem repeat (STR) analysis and DNA sequencing.

Cancer cell lines are used worldwide in biomedical researches, and data interpretation solely depends on unambiguous attribution of those respective cell lines to its original sources. Approximately one-third of all cell lines have an origin other than that assumed, leading to invalid results. It is necessary to characterize the origin of cell lines. Short-tandem-repeat (STR) fingerprinting (DNA fingerprinting) is the method for characterization of genetic identity in cultured cell lines under certain experimental conditions. We showed the fingerprinting profiles in a summed and unidentified human cancer cell line comparison to HCC1954 cell line, revealing marked alterations in DNA fingerprinting profiles up to fourteen STR loci from 16 loci. Furthermore, Sanger DNA sequencing showed no c.3140A > G heterozygous mutation in the PIK3CA gene of this suspected HCC1954 cell line. In addition, we showed the fingerprinting profiles in an unidentified cancer cell line comparison to SiHa cervical cell line, revealing same DNA fingerprinting profiles. In conclusion, we have successfully authenticated and identified both suspected HCC1954 and SiHa cell lines by STR analysis and DNA sequencing. STR analysis combined DNA sequencing may be very useful to evaluate genotypes of cancer cell lines in our cancer studies, as well as in judicial authentication and forensic sciences.

3D Molecular Cytology of Hop (Humulus lupulus) Meiotic Chromosomes Reveals Non-disomic Pairing and Segregation, Aneuploidy, and Genomic Structural Variation.

Hop (Humulus lupulus L.) is an important crop worldwide, known as the main flavoring ingredient in beer. The diversifying brewing industry demands variation in flavors, superior process properties, and sustainable agronomics, which are the focus of advanced molecular breeding efforts in hops. Hop breeders have been limited in their ability to create strains with desirable traits, however, because of the unusual and unpredictable inheritance patterns and associated non-Mendelian genetic marker segregation. Cytogenetic analysis of meiotic chromosome behavior has also revealed conspicuous and prevalent occurrences of multiple, atypical, non-disomic chromosome complexes, including those involving autosomes in late prophase. To explore the role of meiosis in segregation distortion, we undertook 3D cytogenetic analysis of hop pollen mother cells stained with DAPI and FISH. We used telomere FISH to demonstrate that hop exhibits a normal telomere clustering bouquet. We also identified and characterized a new sub-terminal 180 bp satellite DNA tandem repeat family called HSR0, located proximal to telomeres. Highly variable 5S rDNA FISH patterns within and between plants, together with the detection of anaphase chromosome bridges, reflect extensive departures from normal disomic signal composition and distribution. Subsequent FACS analysis revealed variable DNA content in a cultivated pedigree. Together, these findings implicate multiple phenomena, including aneuploidy, segmental aneuploidy, or chromosome rearrangements, as contributing factors to segregation distortion in hop.

Characterization of the ICCE Repeat in Mammals Reveals an Evolutionary Relationship with the DXZ4 Macrosatellite through Conserved CTCF Binding Motifs.

Appreciation is growing for how chromosomes are organized in three-dimensional space at interphase. Microscopic and high throughput sequence-based studies have established that the mammalian inactive X chromosome (Xi) adopts an alternate conformation relative to the active X chromosome. The Xi is organized into several multi-megabase chromatin loops called superloops. At the base of these loops are superloop anchors, and in humans three of these anchors are composed of large tandem repeat DNA that include DXZ4, Functional Intergenic Repeating RNA Element, and Inactive-X CTCF-binding Contact Element (ICCE). Each repeat contains a high density of binding sites for the architectural organization protein CCCTC-binding factor (CTCF) which exclusively associates with the Xi allele in normal cells. Removal of DXZ4 from the Xi compromises proper folding of the chromosome. In this study, we report the characterization of the ICCE tandem repeat, for which very little is known. ICCE is embedded within an intron of the Nobody (NBDY) gene locus at Xp11.21. We find that primary DNA sequence conservation of ICCE is only retained in higher primates, but that ICCE orthologs exist beyond the primate lineage. Like DXZ4, what is conserved is organization of the underlying DNA into a large tandem repeat, physical location within the NBDY locus and conservation of short DNA sequences corresponding to specific CTCF and Yin Yang 1 binding motifs that correlate with female-specific DNA hypomethylation. Unlike DXZ4, ICCE is not common to all eutherian mammals. Analysis of certain ICCE CTCF motifs reveal striking similarity with the DXZ4 motif and support an evolutionary relationship between DXZ4 and ICCE.

Correction to: Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization, by Uzarski JS, Bijonowski BM, Wang B, Ward HH, Wandinger-Ness A, Miller WM, and Wertheim JA. Tissue Eng Part C Methods 2015;21(10):1032–1043. DOI: 10.1089/ten.tec.2014.0665

[This corrects the article DOI: 10.1089/ten.tec.2014.0665.].

PO-330 Investigating tissue- and age-specific microsatellite instability in mice with different levels of MLH1, a central mismatch repair protein

IntroductionLynch syndrome (LS) is an inherited condition in which individuals harbour germline mutations in one or more of the DNA mismatch repair (MMR) genes, typically MLH1 or MSH2, predisposing them to cancers, in particular non-polyposis colorectal cancer. Tumours in LS are characterised by high microsatellite instability (MSI), which reflects defective MMR. MSI in colorectal cancer results from epigenetic inactivation or loss of heterozygosity (LOH) of the wild-type MMR allele, leading to absence of MMR proteins. However, some evidence suggests haploinsufficiency of MMR proteins, i.e. heterozygosity may lead to loss of genome integrity. For instance, Mlh1 +/-mice display increased tumour incidence and individuals with LS show subtle MSI in their peripheral blood leukocytes.Our aim is to understand how MMR protein levels affect in vivo tandem DNA repeat instability. A key outstanding question is why certain tissues, in particular the intestine and colon in humans, are especially prone to MSI. We hypothesise that normal levels of MMR proteins are particularly critical for genome stability in these tissues.Material and methodsWe are using mouse genetics to find out how the levels of MMR proteins contribute to in vivo tandem DNA repeat instability. We are analysing tissue- and age- specific MSI in mice expressing different levels of MMR proteins. We are using single molecule PCR followed by capillary electrophoresis to investigate microsatellite instability. Data is being analysed using Fragman R package. Our group holds a National Animal Experiment Board license to perform animal experiments.Results and discussionsOur preliminary results in old Mlh1 +/-mice show a very subtle but detectable MSI increase in small intestine compared to Mlh1+/+mice, the observed difference being predominantly due to an increase in deletion-mutant alleles.ConclusionOur result show subtle increase in MSI in macroscopically normal intestine of Mlh1 +/-mice, which indicates inactivation of just one Mlh1 allele is sufficient to induce genome instability.

Biological Roles of Protein-Coding Tandem Repeats in the Yeast Candida Albicans.

Tandem repeat (TR) DNA mutates faster than other DNA by insertion and deletion of repeats. Large parts of eukaryotic proteomes are encoded by ORFs containing protein-coding TRs (TR-ORFs, pcTRs) with largely unknown biological consequences. We explored these in the yeast Candida albicans, an opportunistic human pathogen. We found that almost half of C. albicans’ proteins are encoded by TR-ORFs. pcTR frequency differed only moderately between different gene (GO) categories. Bioinformatic predictions of genome-wide mutation rates and clade-specific differences in pcTR allele frequencies indicated that pcTRs (i) significantly increase the genome-wide mutation rate; (ii) significantly impact on fitness and (iii) allow the evolution of selectively advantageous clade-specific protein variants. Synonymous mutations reduced the repetitiveness of many amino acid repeat-encoding pcTRs. A survey, in 58 strains, revealed that in some pcTR regions in which repetitiveness was not significantly diminished by synonymous mutations the habitat predicted which alleles were present, suggesting roles of pcTR mutation in short-term adaptation and pathogenesis. In C. albicans pcTR mutation apparently is an important mechanism for mutational advance and possibly also rapid adaptation, with synonymous mutations providing a mechanism for adjusting mutation rates of individual pcTRs. Analyses of Arabidopsis and human pcTRs showed that the latter also occurs in other eukaryotes.

Comparative genome-wide characterization leading to simple sequence repeat marker development for Nicotiana

BackgroundSimple sequence repeats (SSRs) are tandem repeats of DNA that have been used to develop robust genetic markers. These molecular markers are powerful tools for basic and applied studies such as molecular breeding. In the model plants in Nicotiana genus e.g. N. benthamiana, a comprehensive assessment of SSR content has become possible now because several Nicotiana genomes have been sequenced. We conducted a genome-wide SSR characterization and marker development across seven Nicotiana genomes.ResultsHere, we initially characterized 2,483,032 SSRs (repeat units of 1–10 bp) from seven genomic sequences of Nicotiana and developed SSR markers using the GMATA® software package. Of investigated repeat units, mono-, di- and tri-nucleotide SSRs account for 98% of all SSRs in Nicotiana. More complex SSR motifs, although rare, are highly variable between Nicotiana genomes. A total of 1,224,048 non-redundant Nicotiana (NIX) markers were developed, of which 99.98% are novel. An efficient and uniform genotyping protocol for NIX markers was developed and validated. We created a web-based database of NIX marker information including amplicon sizes of alleles in each genome for downloading and online analysis.ConclusionsThe present work constitutes the first deep characterization of SSRs in seven genomes of Nicotiana, and the development of NIX markers for these SSRs. Our online marker database and an efficient genotyping protocol facilitate the application of these markers. The NIX markers greatly expand Nicotiana marker resources, thus providing a useful tool for future research and breeding. We demonstrate a novel protocol for SSR marker development and utilization at the whole genome scale that can be applied to any lineage of organisms.The Tobacco Markers & Primers Database (TMPD) is available at http://biodb.sdau.edu.cn/tmpd/index.html

Locus specific engineering of tandem DNA repeats in the genome of Saccharomyces cerevisiae using CRISPR/Cas9 and overlapping oligonucleotides

DNA repeats constitute a large part of genomes of multicellular eucaryotes. For a longtime considered as junk DNA, their role in genome organization and tuning of gene expression is being increasingly documented. Synthetic biology has so far largely ignored DNA repeats as regulatory elements to manipulate functions in engineered genomes. The yeast Saccharomyces cerevisiae has been a workhorse of synthetic biology, owing to its genetic tractability. Here we demonstrate the ability to synthetize, in a simple manner, tandem DNA repeats of various size by Cas9-assisted oligonucleotide in vivo assembly in this organism. We show that long tandem DNA repeats of several kilobases can be assembled in one step for different monomer size and G/C content. The combinatorial nature of the approach allows exploring a wide variety of design for building synthetic tandem repeated DNA directly at a given locus in the Saccharomyces cerevisiae genome. This approach provides a simple way to incorporate tandem DNA repeat in synthetic genome designs to implement regulatory functions.

Co-optation of Tandem DNA Repeats for the Maintenance of Mesenchymal Identity

Tandem repeats (TRs) are generated by DNA replication errors and retain a high level of instability, which in principle would make them unsuitable for integration into gene regulatory networks. However, the appearance of DNA sequence motifs recognized by transcription factors may turn TRs into functional cis-regulatory elements, thus favoring their stabilization in genomes. Here, we show that, in human cells, the transcriptional repressor ZEB1, which promotes the maintenance of mesenchymal features largely by suppressing epithelial genes and microRNAs, occupies TRs harboring dozens of copies of its DNA-binding motif within genomic loci relevant for maintenance of epithelial identity. The deletion of one such TR caused quasi-mesenchymal cancer cells to reacquire epithelial features, partially recapitulating the effects of ZEB1 gene deletion. These data demonstrate that the high density of identical motifs in TRs can make them suitable platforms for recruitment of transcriptional repressors, thus promoting their exaptation into pre-existing cis-regulatory networks.

Potential effects of ionizing radiation on the evidentiary value of DNA, latent fingerprints, hair, and fibers: A comprehensive review and new results

An extensive literature review and new post-irradiation experimental results are presented of genotyping blood stains and hair, and physical examinations of latent fingerprints, hairs, and fibers. Results indicate that successful development of nuclear short tandem repeat (STR) and mitochondrial DNA sequence profiles from human blood and hair evidence is possible—up to a point—following exposure to gamma, neutron, beta, and alpha radiation at several levels that would most likely be present at this type of crime scene (i.e., a “dirty bomb,” etc.). Commencing at gamma radiation levels between 90 and 900kGy, DNA analysis using conventional DNA techniques was unsuccessful. In general, irradiation negatively affected the quality of latent fingerprints. All four radiation types degraded most fingerprint samples at all doses; nevertheless, many fingerprints remained of value for potential use in comparison. Although variable from one hair to another, microscopic changes observed for all types and levels of irradiation could potentially result in false exclusions. Negligible microscopic changes were observed in papers and fibers (used as substrates for fingerprints and bloodstains) up to 90kGy gamma, but fluorescence of fibers began to change above that dose. Paper and fibers, as well as plastic evidence enclosures, became extremely brittle leading to breakage after a gamma dose of 900kGy.

Organ reserve, excess metabolic capacity, and aging

"Organ reserve" refers to the ability of an organ to successfully return to its original physiological state following repeated episodes of stress. Clinical evidence shows that organ reserve correlates with the ability of older adults to cope with an added workload or stress, suggesting a role in the process of aging. Although organ reserve is well documented clinically, it is not clearly defined at the molecular level. Interestingly, several metabolic pathways exhibit excess metabolic capacities (e.g., bioenergetics pathway, antioxidants system, plasticity). These pathways comprise molecular components that have an excess of quantity and/or activity than that required for basic physiological demand in vivo (e.g., mitochondrial complex IV or glycolytic enzymes). We propose that the excess in mtDNA copy number and tandem DNA repeats of telomeres are additional examples of intrinsically embedded structural components that could comprise excess capacity. These excess capacities may grant intermediary metabolism the ability to instantly cope with, or manage, added workload or stress. Therefore, excess metabolic capacities could be viewed as an innate mechanism of adaptability that substantiates organ reserve and contributes to the cellular defense systems. If metabolic excess capacities or organ reserves are impaired or exhausted, the ability of the cell to cope with stress is reduced. Under these circumstances cell senescence, transformation, or death occurs. In this review, we discuss excess metabolic and structural capacities as integrated metabolic pathways in relation to organ reserve and cellular aging.

A Standard Nomenclature for Referencing and Authentication of Pluripotent Stem Cells.

Unambiguous cell line authentication is essential to avoid loss of association between data and cells. The risk for loss of references increases with the rapidity that new human pluripotent stem cell (hPSC) lines are generated, exchanged, and implemented. Ideally, a single name should be used as a generally applied reference for each cell line to access and unify cell-related information across publications, cell banks, cell registries, and databases and to ensure scientific reproducibility. We discuss the needs and requirements for such a unique identifier and implement a standard nomenclature for hPSCs, which can be automatically generated and registered by the human pluripotent stem cell registry (hPSCreg). To avoid ambiguities in PSC-line referencing, we strongly urge publishers to demand registration and use of the standard name when publishing research based on hPSC lines.

Sensitive detection of formamidopyrimidine-DNA glycosylase activity based on target-induced self-primed rolling circle amplification and magnetic nanoprobes.

We developed a novel approach to determine formamidopyrimidine DNA glycosylase (FPG) activity by taking advantage of target-induced self-primed rolling circle amplification (RCA) and magnetic nanoprobes. Herein, a unique nick (8-oxoguanine, 8-oxoG) was positioned in duplex DNA containing P-circle and P1, which together serve as a FPG substrate, RCA template, and RCA primer probe. The presence of FPG specifically binds 8-oxoG and cleaves the P-circle into two parts, producing 5'-phosphoryl termini. A phosphodiester bond between the 5'-phosphoryl and 3'-hydroxyl termini was formed with the addition of T4 DNA ligase, producing an unnicked circular strand. Using the unnicked strand as the RCA template, the P1 hybridized with the circle probe as a primer will trigger the RCA process. The RCA reaction produces amounts of long tandem-repeat DNA tiles with multiple recognizing regions for the FAM modified DNA probes (FP) and biotin-modified DNA probes (BP). With the streptavidin-biotin interaction, the BPs and FPs can be easily immobilized on the surface of streptavidin-modified magnetic microbeads (MBs). Due to the RCA enhanced and highly-concentrated fluorescence accumulation on the MBs, an ultralow detection limit of 1.033 U mL-1 for FPG was obtained. Combined with the high tolerance capability of human blood serum owing to magnetic isolation, the FPG assays in human blood serum were also obtained using fluorescence and confocal laser scanning microscopy. These results indicate that this robust self-primed RCA combined with magnetic nanoprobes is an excellent candidate for quantitatively monitoring the FPG activity responsible for DNA oxidative damage-related clinical diagnosis and therapy.