DNA Template Sequence Research Articles

The natural non-protein amino acid N-β-methylamino-L-alanine (BMAA) is incorporated into protein during synthesis.

N-β-methylamino-L-alanine (BMAA) is an amino acid produced by cyanobacteria and accumulated through trophic levels in the environment and natural food webs. Human exposure to BMAA has been linked to progressive neurodegenerative diseases, potentially due to incorporation of BMAA into protein. The insertion of BMAA and other non-protein amino acids into proteins may trigger protein misfunction, misfolding and/or aggregation. However, the specific mechanism by which BMAA is associated with proteins remained unidentified. Such studies are challenging because of the complexity of biological systems and samples. A cell-free in vitro protein synthesis system offers an excellent approach for investigation of changing amino acid composition in protein. In this study, we report that BMAA incorporates into protein as an error in synthesis when a template DNA sequence is used. Bicinchoninic acid assay of total protein synthesis determined that BMAA effectively substituted for alanine and serine in protein product. LC-MS/MS confirmed that BMAA was selectively inserted into proteins in place of other amino acids, but isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) did not share this characteristic. Incorporation of BMAA into proteins was significantly higher when genomic DNA from post-mortem brain was the template. About half of BMAA in the synthetic proteins was released with denaturation with sodium dodecylsulfonate and dithiothreitol, but the remaining BMAA could only be released by acid hydrolysis. Together these data demonstrate that BMAA is incorporated into the amino acid backbone of proteins during synthesis and also associated with proteins through non-covalent bonding.

Next Generation Sequencing-Based Parallel Analysis of Melting Kinetics of 4096 Variants of a Bacterial Promoter

Promoter melting by bacterial RNA polymerase is a key step in transcription initiation. We used a next generation sequencing (NGS) based approach to analyze in parallel promoter melting of all 4096 sequence variants of the 6 bp -10 promoter element. We used NGS read count for each sequence of a promoter library containing a randomized -10 sequence as an observable to determine relative enrichment of -10 element sequence variants at different time points of the promoter melting reaction. The analysis reinforced the dominating role of consensus bases at positions -11 and -7, demonstrated an enhanced preference for A at -11 among sequences exhibiting the fastest melting kinetics, and showed higher overall importance of the T at -7 compared to the A at -11 for efficient promoter melting. Sequences lacking the consensus bases at -7 or -11 could still melt fast if they contained compensatory base patterns at other positions. We observed a significant correlation between the duplex melting energy of -10 element and the kinetics of promoter melting that became more pronounced when the dominating base-specific interactions with RNAP were diminished. These observations indicate that promoter melting kinetics is determined by a combination of base-specific effects/interactions and sequence-dependent stability of DNA duplex with the former playing a dominating role. Our data show that NGS can provide a reliable, quantitative readout for a highly parallel analysis of DNA template sequence dependence of activities of proteins that bind or operate on a DNA template.

A Microfluidic Microbeads Array Chip Integrated with Micro-fluid Driven Micro-pump for Discrimination of Gene Mutation

Abstract A novel approach for single-nucleotide detection based on the micropump-integrated microfluidic microbeads array chip and an apyrase-mediated primer extension process was developed. In this method, an integrated chip was constructed by a microfluidic chip, a primers-modified microbeads array and a micro-pump driven by evaporation and capillary effect. The target DNA flowed across the fabricated microfluidic beads array and hybridized with immobilized primer sequences. When the 3′ terminus of primer matched with the target DNA in the single-base mutation site of interest, under synergistic effect of apyrase and exonuclease-deficient Klenow DNA polymerase, the matched primer extended along the template DNA sequence and incorporated the biotin-dCTP into the extended primers and immobilized it onto the surface of microbeads. Then, the streptavidin-labeled quantum dots bond with deposited biotin moities of biotin-dCTP and generated a fluorescence signal. On the contrary, there is no signal when signal-base mismatched duplexes were present in the 3′ terminus of the primer. The limit of detection is 0.2 pM target DNA (S/N > 3) for micro-pump driven chip and 0.5 pM target DNA for liquid pressure driven chip respectively. The chip-based signal enhancement for single-nucleotide discrimination using micro-pump integrated microfluidic chip resulted in 500 times higher sensitivity than that of an off-chip test. Since the off-chip assay only detected 0.1 nM target DNA. The fluorescence signals are linear in the target DNA concentration ranging 0.5 pM to 30 pM. This method was also used to detect two multi-drug resistance gene 1 (MDR1)-associated SNP sites (C3435T and G2677T) from a human genomic sample. The fluorescence signals indicated the subject used here possessed both MDR1 3435CT and MDR1 2677TT genotypes, which were consistent with the results by DNA sequencing. This approach displays good specificity, sensitivity and stability for discrimination of gene mutation.

First Report of Pratylenchus neglectus on Winter Wheat in China.

Root-lesion nematodes are major pathogens of wheat and have been reported in the United States, Mexico, India, Australia, Egypt, Canary Islands, South Africa, Iran, Japan, the Netherlands, Belgium, Italy, Germany, and Yugoslavia (1). They can also cause injury in a large number of crops, including grasses, cereal grains, and vegetables. In 2009 and 2010, a survey was conducted for nematodes in winter wheat fields near Taian city, Shandong, northern China. Root tissues were stained via the acid fuchsin tissue stain technique, and nematode numbers were recorded under a stereo microscope. Sixty-eight root samples were collected during the winter wheat growing season, and root lesion nematode was found in all samples. The highest average lesion nematode populations in fresh roots were 154.3 nematodes/g in 2009 and 236.7 nematodes/g in 2010. Nematodes were collected from infested wheat roots by a modified Baermann funnel method. Dimensions of the nematodes were: length, 0.42 to 0.54 mm; a, 18.8 to 24.2; b, 4.4 to 5.7; c, 19.8 to 25.4; V, 80.4 to 84.8; and spear, 17.1 to 18.9 μm. DNA was extracted from individual nematodes using liquid nitrogen. Amplification of rDNA-internal transcribed spacer region using the forward primers 5'-CGTAACAAGGTAGCTGTAG-3' and the reverse primer 5'-TTTCACTCGCCGTTACTAAGG-3' yielded a PCR fragment of approximately 900 bp. PCR products were purified using Universal Plant DNA Purification Kit (Tiangen, China) and ligated to the pMD18-T vector system (TaKaRa Bio, Japan) and transformed to E. coli strain DH5α. Plasmid DNA carrying the insert was extracted and used as the template for DNA sequencing. DNA sequencing was carried out in an ABI 3730, compared and aligned using MEGA 5.0. Sequences showed 96% sequence identity with those of Pratylenchus neglectus (GenBank Accession No. FR692291.1). The sequence was submitted to the GenBank database (JX228136). To our knowledge, this is the first report of P. neglectus infesting winter wheat in China. P. neglectus has been reported as causing economically significant damage to wheat production of up to 70% yield loss in the Pacific Northwest. Damage from lesion nematode may therefore be potentially significant to wheat production in Shandong Province, and further information should be obtained on its prevalence.

An arrayed human genomic library constructed in the PAC shuttle vector pJCPAC-Mam2 for genome-wide association studies

Genomic libraries constructed in the original Bacterial Artificial Chromosome (BAC) and P1 Artificial Chromosome (PAC) cloning systems were very useful for completion of the Human Genome Project. Libraries constructed in these vector systems were used to generate physical maps of all twenty three-chromosome pairs and served as the templates for DNA sequencing. The PAC vector pJCPAC-Mam2 is a 23 kilobase (kb) shuttle vector that is highly versatile and useful for functional studies in human tissue culture cell lines. pJCPAC-Mam2 contains the P1 single-copy replicon for low copy expression and a multi-copy lytic replicon under the control of the lac repressor for high copy expression in Escherichia coli, wild type and mutant loxP sites for generation of bidirectional nested deletions in any clone of interest and the Epstein Barr Virus (EBV) latent replication origin oriP, the Epstein Barr Nuclear Antigen 1 (EBNA1) gene, and a puromycin-resistance gene for propagation in mammalian cells. We constructed an arrayed 115,000-member human genomic library in pJCPAC-Mam2 that is housed in twelve hundred 96 well dishes. Subsequent pooling using a column and rows strategy reduced the library to 19 final pools for Polymerase Chain Reaction (PCR) screening. We estimate that the average insert size is approximately 57 kb based on NotI digestion and Field Inversion Gel Electrophoresis (FIGE) analysis of 473 random PAC clones. The library appears to represent a two- to three-fold coverage of the human genome based on screening of the library with PCR primers representing nine genes. This library should serve as a valuable resource to validate potential disease genes identified by genome-wide association studies (GWAS) in human tissue culture cell lines and in animal models. Also, individual library members could potentially be used for gene therapy trials for a variety of recessive disorders.

Mir-373 Affects Human Lung Cancer Cells' Growth and its E-Cadherin Expression

The aims of this study was to elucidate whether the expression of E-cadherin can be affected by the recombinant has-mir-373 eukaryotic expression plasmid vector through tests in vitro, and to analyze the relationship between the expression of E-cadherin and tumor growth. According to the has-mir-373 sequence in miRBase database, two template DNA sequences were designed. The has-mir-373 sequence and a control sequence were synthesized and cloned into pGenesil-1 eukaryotic expression plasmid vector. The recombinant plasmids were transfected into human lung cancer A549 cells by liposome-mediated method. The mir-373 expression in A549 cells was detected by using real-time quantitative polymerase chain reaction (real-time PCR). MTT (methyl thiazolyl tetrazolium) was used to analyze the growth of cancer cell cycle. RT-PCR and Western blotting were used to evaluate the levels of E-cadherin mRNA and protein expression, respectively. The expression of E-cadherin in cells was determined by immunocytochemistry. The mobility capability of transfected cells were evaluated by using wound healing assay in vitro. The fluorescent light was observed under fluorescent microscope. RT-PCR indicated that the mRNA of E-cadherin increased, and the Western blotting results also displayed that mir-373 promoted the expression of the E-cadherin protein. Compared with the control groups, MTT method and wound healing assay demonstrated that both the growth rate and migration of A549 cells transfected with the recombinant has-mir-373 eukaryotic expression plasmid was also decreased significantly (p < 0.001). The differences between the other two control groups were not significant (p > 0.05). The immunocytochemistry demonstrated a significant increase of E-cadherin protein levels in the cells transfected with mir-373, but not in the cells of the control group. Mir-373 could increase the expression levels of the E-cadherin and decrease the migration ability of human lung cancer A549 cells in vitro.

Targeting DNA With Fingers and TALENs.

Researchers have long recognized that the random introduction of new DNA into the genome could result in unpredictable genetic effects due to bilateral influences between transgenic and endogenous sequences. Hence, precise editing or replacement of mutant genes has been a major goal of gene therapy since its inception. That goal looked possible with the publication of studies demonstrating that targeted cleavage of chromosomal sequences and enhanced homologous recombination (HR) could be achieved using chimeric molecules composed of a nuclease domain and separate, designer DNA-recognition domains.1,2 Zinc-finger nucleases (ZFNs) are artificial endonucleases that consist of a FokI cleavage domain tethered to engineered Cys2His2 zinc-finger, DNA-binding polypeptides.3,4,5 ZFNs have gained considerable momentum and are widely considered the most mature nuclease technology in the gene therapy field.6 However, after 15 years in the spotlight, a new chimeric nuclease has emerged, Transcription Activator-Like (TAL) Effector Nucleases (TALENs).7,8,9,10 Here, we describe functional and design characteristics of ZFNs and TALENs and discuss their expanding role as tools for research and gene therapy.

Construction and functional identification of siRNA adenoviral vector targeting HIF-1α in mouse

Objective To construct an adenoviral vector targeting hypoxic inducible factor-1α (HIF-1α) gene in mouse and to identify the effect of the adenoviral vector,AdHIF-1α-shRNA-EGFP,on the expression of HIF-1α in cultured mouse embryonic neural stem cells,and provide a basis for investigating the contributiou of HIF-1 signaling pathway in neural stem cell biology.Methods The HIF-1α template DNA sequence was designed by using bioinformatics.DNA recombinant technique was used to construct recombinant adenoviral vector,AdHIF-1α-shRNA-EGFP.Polymerase chain reaction(PCR) and sequencing were performed.The neural stem cells from frontal cortex of the mouse were transfected with AdHIF-1α-shRNA-EGFP in vitro.The efficiency of transfection was detected by flow cytometry.The transfected cells were cultured under hypoxia condition.Double immunofluorescent staining of nestin and HIF-α was performed,and the expression of HIF-1α was quantified by reverse transcription - polymerase chain reaction (RT-PCR) and Western blot analysis.Results The sequence cloned into the adenoviral vector was identical with the one synthesized chemically.The neural stem cells were transfeeted effectively by AdHIF-1α-shRNA-EGFP.The results from RT-PCR and Western blot analysis showed that the expression of HIF-1α gene and protein in the neural stem cells transfected with AdHIF-1α-shRNA-EGFP decreased by 52.05％ and 67.27％ under hypoxia condition,respectively.Conclusion AdHIF-1α -shRNA-EGFP could effectively inhibit hypoxia - induced expression of HIF-1α gene and protein in the mouse neural stem cells in vitro. Key words: Neural stem cells; Mouse; Hypoxic inducible factor-1α; Small interference RNA; Adenoviral vector

Ag K-Edge EXAFS Analysis of DNA-Templated Fluorescent Silver Nanoclusters: Insight into the Structural Origins of Emission Tuning by DNA Sequence Variations

DNA-templated silver nanoclusters are promising biological fluorescence probes due to their useful fluorescence properties, including tunability of emission wavelength through DNA template sequence variations. Ag K-edge EXAFS analysis of DNA-templated silver nanoclusters has been used to obtain insight into silver nanocluster bonding, size, and structural correlations to fluorescence. The results indicate the presence of small silver nanoclusters (<30 silver atoms) containing Ag-Ag bonds and Ag-N/O ligations to DNA. The DNA sequence used leads to differences in silver-DNA ligation as well as silver nanocluster size. The results support a model in which cooperative effects of both Ag-DNA ligation and variations in cluster size lead to the tuning of the fluorescence emission of DNA-templated silver nanoclusters.

Direct Detection and Sequencing of Damaged DNA Bases

Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications.

Identifying and Removing Artificial Replicates from 454 Pyrosequencing Data

An intrinsic artifact of 454-based pyrosequencing leads to artificial overrepresentation of >10% of the original DNA sequencing templates. This artificial amplification of sequences is unbiased with regard to position on the pyrosequencing plate or sequence identity, and it occurs in all currently available 454 technologies. The amplified sequences start at the same position and are identical (duplicates), or vary in length, or contain a sequencing discrepancy. If the abundance of any sequence in a data set is going to be enumerated, either for comparative community analysis, transcriptional analysis or other applications, it is important to remove these artificial replicates before analysis. A web-based tool that incorporates the clustering algorithm cd-hit was developed to identify and remove artificially replicated sequences in 454-based pyrosequencing data sets. This tool cannot be used for data sets that have an initial amplification step before the standard pyrosequencing procedure, because artificial replicates cannot be distinguished from expected replication due to polymerase chain reaction (PCR) amplification, e.g., in sequencing of amplified gene "tags." This protocol provides details on how to use the replicate filter and obtain a file of unique sequences for use in metagenomic or transcriptomic analyses.

Identification of sister chromatids by DNA template strand sequences

It is generally assumed that sister chromatids are genetically and functionally identical and that segregation to daughter cells is a random process. However, functional differences between sister chromatids regulate daughter cell fate in yeast and sister chromatid segregation is not random in Escherichia coli. Differentiated sister chromatids, coupled with non-random segregation, have been proposed to regulate cell fate during the development of multicellular organisms. This hypothesis has not been tested because molecular features to reliably distinguish between sister chromatids are not obvious. Here we show that parental 'Watson' and 'Crick' DNA template strands can be identified in sister chromatids of murine metaphase chromosomes using CO-FISH (chromosome orientation fluorescence in situ hybridization) with unidirectional probes specific for centromeric and telomeric repeats. All chromosomes were found to have a uniform orientation with the 5' end of the short arm on the same strand as T-rich major satellite repeats. The invariable orientation of repetitive DNA was used to differentially label sister chromatids and directly study mitotic segregation patterns in different cell types. Whereas sister chromatids appeared to be randomly distributed between daughter cells in cultured lung fibroblasts and embryonic stem cells, significant non-random sister chromatid segregation was observed in a subset of colon crypt epithelial cells, including cells outside positions reported for colon stem cells. Our results establish that DNA template sequences can be used to distinguish sister chromatids and follow their mitotic segregation in vivo.

Magnetic beads based rolling circle amplification–electrochemiluminescence assay for highly sensitive detection of point mutation

The identification of point mutations is particularly essential in the fields of medical diagnosis and prognosis of many pathogenic and genetic diseases. In this study, an rolling circle amplification (RCA) based electrochemiluminescence (ECL) assay for highly sensitive point mutation detection was developed. In the assay, an allele-discriminating padlock probe was designed for targeting the sequence in the p53 oncogene locus. A circular template generated by enzymatic ligation upon the recognition of a point mutation (CGT to CAT) on the oncogene could be amplified isothermally by Phi29 DNA polymerase. The elongated products, containing hundreds of copies of the circular DNA template sequence, were hybridized with Ru(bpy)32+ (TBR)-tagged probes and then captured onto streptavidin-coated paramagnetic beads. The resulting products were analyzed by magnetic bead based ECL platform. As low as 2amol of mutated strands was detected by this assay, which could be attributed to the high amplification efficiency of Phi29 DNA polymerase and current magnetic bead based ECL detection platform. In addition, the positive mutation detection was achieved with a wild-type to mutant ratio of 10000:1, due to the high fidelity of DNA ligase in differentiating mismatched bases at the ligation site. It is demonstrated that this proposed method provides a highly sensitive and specific approach for point mutation detection.

Construction of conditionally replicative adenovirus expressing short interference RNA targeting the β-actin and GAPDH genes with hTERT promoter regulation and control

Objective To construct conditionally replicative adenovirus (CRAds) expressing short interference RNA (siRNA) targeting the β-actin and GAPDH genes with hTERT promoter regulation and control. Methods β-actin and GAPDH siRNA template DNA sequences were inserted into pGPH1/GFP/Neo plasmid, respectively. Two pairs of specific primers were designed and used to get the expression cassettes of β-actin-siRNA and GAPDH-siRNA which were inserted into pZHTERT and pZD55 plasmid to construct the recombinant plasmid pZHTERT-β-actin and pZD55-GAPDH, respectively. The pZHTERT-β-actin plasmid was cut with Kpn Ⅰ and Pvu Ⅰ to get the expression cassette of β-actin which was cloned in-to pZD55-GAPDH to form pTD-GAPDH-β-actin plasmid. The plasmid pTD-GAPDH-β-actin was transfect-ed into 293 cells together with plasmid pBHGE3 to obtain the recombinant CRAds,TD-GAPDH-β-actin. The viral plaques appeared 9-12 days after infection. The extracted DNA of recombinant adenoviruses was verified by PCR. Viruses were plaque purified, propagated on HEK293 cells and purified by CsCI gradient according to standard techniques, and functional PFU titers were determined by plaque assay on 293 cells. Results The conditionally replicative adenovirus expressing siRNA targeting the GAPDH and β-actin genes with hTERT promoter regulation and control had been constructed successfully. Viral PFU titers were 2 × 1011 PFU/ml. Conclusion The conditionally replicative adenovirus will lay a good foundation for fur-ther study on cancer gene therapy. Key words: GAPDH; Promoter; Conditionally rephcative adenovirus; RNA interference

Synthesis and characterization of deoxyuridine triphosphates labeled with pyrene

Deoxyuridine triphosphate derivatives modified with pyrene was synthesized to functionalize DNA with fluorescent molecules based on the template DNA sequence. Incorporation of pyrene-labeled deoxyuridine triphosphates into DNA by DNA polymerase was investigated by using reverse-phase HPLC and polyacrylamide gel electrophoresis. The fluorescent properties of functionalized DNA were characterized by the steady-state fluorescence measurements.

Conformational Changes during Nucleotide Selection by Sulfolobus solfataricus DNA Polymerase Dpo4

The mechanism of nucleotide selection by Y-family DNA polymerases has been the subject of intense study, but significant structural contacts and/or conformational changes that relate to polymerase fidelity have been difficult to identify. Here we report on the conformational dynamics of a model Y-family polymerase Dpo4 from Sulfolobus solfataricus. Hydrogen-deuterium exchange in tandem with mass spectrometry was used to monitor changes in Dpo4 structure as a function of time and the presence or absence of specific substrates and ligands. Analysis of the data revealed previously unrecognized structural changes that accompany steps in the catalytic cycle leading up to phosphoryl transfer. For example, the solvent accessibility of the alphaB-loop-alphaC region in the finger domain decreased in the presence of all four dNTP insertion events, but the rate of deuterium exchange, an indicator of conformational flexibility, only decreased during an accurate insertion event. Of particular note is a change in the region surrounding the H-helix of the thumb domain. Upon binding DNA and Mg2+, the H-helix showed a decrease in solvent accessibility and flexibility that was relaxed only upon addition of dCTP, which forms a Watson-Crick base pair with template dG and not during mispairing events. The current study expands upon a previous report from our group that used a fluorescent probe located near the thumb domain to measure the kinetic properties of Dpo4 conformational changes. We now present a model for nucleotide selection by Dpo4 that arises from a synthesis of both structural and kinetic data.

Aquifex aeolicus tRNA (N2,N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA

Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m(2)(2)G26) in tRNA. In the reaction, N2-guanine at position 26 (m(2)G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNA(Cys) has an m(2)(2)G26m(2)G27 or m(2)(2)G26m(2)(2)G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m(2)G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.

Cost effective DNA sequencing and template preparation from bacterial colonies and plasmids

Cost-effective DNA sequencing and template preparation were evaluated for high-throughput screening of bacterial colonies. The rolling-circle amplification to generate template for DNA sequencing was carried out using 10-fold smaller amount of phi29 DNA polymerase and excluding two other enzymes (yeast pyrophosphatase and calf intestine alkaline phosphatase) used previously. Then, a 1/40 volume of the fluorescent terminator mix recommended by the manufacturer gave a usable sequencing result although a 1/26.7 volume of the mix was used to compare the protocol more accurately with manufacturer's protocol.

Effect on the proliferation of bovine corneal endothelial cells by small hairpin RNA interference targeting p27Kip1

To clarify the proliferation of bovine corneal endothelial cells (bCEC) by interference with the recombinant plasmid of short hairpin RNA (shRNA) against p27Kip1, a kind of cyclin-dependent kinase inhibitor (CKI). It was an experimental study. Three p27Kip1-shRNA template DNA sequences containing small hairpin structure were designed and synthesized as experimental groups. Plasmid expressing irrelevant shRNA with a random combination was used as negative shRNA. The products were inserted into the Pgensil-1 plasmid and the recombinant plasmid of Pgenesil-P1, Pgenesil-P2, Pgenesil-P3 and Pgenesil-HK were constructed. The recombinant plasmids were transfected into bCEC cells with liposome and a blank group. The expression of mRNA and protein of p27Kip1 was detected by RT-PCR and Western blot after stable transfection, and the plasmid with the best inhibitory effect was selected. The growth of the experimental group, Pgenesil-HK group and blank group were assessed by MTT. The influence of shRNA-p27Kip1 on bCEC cell cycle was deteceted by flow cytometry (FCM). All statistical analyses were performed using one-way ANOVA. Restrictive enzyme digestion and sequence analysis showed that four recombinant plamids were constructed successfully and the aim sequence was obtained. The expression of p27Kip1 mRNA and p27Kip1 protein of Pgenesil-P1 group, Pgenesil-P2 group and Pgenesil-P3 group were all lower than that in the control group, including blank group and negative siRNA group. The inhibitive rate of mRNA reached 32.71%, 67.76% and 80.28% (F = 453.102, P = 0.000 in each group) and the inhibitive rate of protein reached 29.27%, 64.73% and 76.13% (F = 75.385, P = 0.000 in each group) compared with the blank group. As the lowest expression among the three positive shRNA group, Pgenesil-P3 was selected for the next steps. There was no significant difference between blank group and negative Pgenesil-HK of the expression of p27Kip1 protein (P = 0.356) and the express of p27Kip1 mRNA (P = 0.246). Compared with the control group and the blank group, the growth of the bCEC transfected by Pgenesil-P3 was significantly promoted with increased cell percent of S-phrase (F = 334.957, P = 0.000) and decreased cell percent of G1-phrase (F = 134.224, P = 0.000). shRNA-p27Kip1 can down-regulate the expression of bCEC effectively and increase the growth of bCEC. shRNA-p27Kip1 RNA interference may be an effective method to promote the proliferation of CEC.

Real-time DNA Synthesis Dynamics Of Single f29 DNA Polymerase Molecules With Base Pair Resolution

Real-time observation of uninterrupted DNA synthesis by individual f29 DNA polymerase molecules is presented. Four spectrally distinct fluorophores were used to label the four nucleotides via the terminal phosphate moiety, enabling identification of nucleotide incorporation events into the growing DNA strand with base-pair resolution, and correlating these events to the DNA template sequence. Immobilization of polymerase molecules inside zero-mode waveguide nanostructures allowed detection of incorporation events at 100-500 nM nucleotide concentrations, resulting in polymerization rates of ∼1-5 bases/s, and lasting for thousands of bases synthesized. Many aspects of the underlying DNA polymerase dynamics are directly observable, such as nucleotide binding, catalysis, pausing, persistence of distinct kinetic states, DNA template sequence context effects, and switching between polymerization and exonuclease activities.