Direct patterning of modified oligonucleotides on metals and insulators by dip-pen nanolithography

The use of direct-write dip-pen nanolithography (DPN) to generate covalently anchored, nanoscale patterns of oligonucleotides on both metallic and insulating substrates is described. Modification of DNA with hexanethiol groups allowed patterning on gold, and oligonucleotides bearing 5'-terminal acrylamide groups could be patterned on derivatized silica. Feature sizes ranging from many micrometers to less than 100 nanometers were achieved, and the resulting patterns exhibited the sequence-specific binding properties of the DNA from which they were composed. The patterns can be used to direct the assembly of individual oligonucleotide-modified particles on a surface, and the deposition of multiple DNA sequences in a single array is demonstrated.     

Recognition of thymine adenine.base pairs by guanine in a pyrimidine triple helix motif

Oligonucleotide recognition offers a powerful chemical approach for the sequence-specific binding of double-helical DNA. In the pyrimidine-Hoogsteen model, a binding size of greater than 15 homopurine base pairs affords greater than 30 discrete sequence-specific hydrogen bonds to duplex DNA. Because pyrimidine oligonucleotides limit triple helix formation to homopurine tracts, it is desirable to determine whether oligonucleotides can be used to bind all four base pairs of DNA. A general solution would allow targeting of oligonucleotides (or their analogs) to any given sequence in the human genome. A study of 20 base triplets reveals that the triple helix can be extended from homopurine to mixed sequences. Guanine contained within a pyrimidine oligonucleotide specifically recognizes thymine.adenine base pairs in duplex DNA. Such specificity allows binding at mixed sites in DNA from simian virus 40 and human immunodeficiency virus.     

用聚赖氨酸包被国产载玻片制作DNA芯片

用多聚赖氨酸对国产载玻片进行包被处理,在不同的固定条件下,比较其与进口多聚赖氨酸载玻片对大片段DNA和3′端氨基修饰寡核苷酸的固定效果.结果表明,在DNA固定率上,自行包被处理的国产载玻片与进口多聚赖氨酸载玻片没有显著差异;紫外交联对大片段DNA固定率影响显著,但对氨基修饰的寡核苷酸影响不显著;3种点样液中,进口点样液MSS点样效果最好,其次为3×SSC,第三是水.氨基修饰的寡核苷酸可以很好地固定在聚赖氨酸包被的载玻片上.     

Specific block of calcium channel expression by a fragment of dihydropyridine receptor cDNA

Although the structure of rabbit skeletal muscle dihydropyridine (DHP) receptor, deduced from cDNA sequence, indicates that this protein is the channel-forming subunit of voltage-dependent calcium channel (VDCC), no functional proof for this prediction has been presented. Two DNA oligonucleotides complementary to DHP-receptor RNA sequences coding for putative membrane-spanning regions of the DHP receptor specifically suppress the expression of the DHP-sensitive VDCC from rabbit and rat heart in Xenopus oocytes. However, these oligonucleotides do not suppress the expression of the DHP-insensitive VDCC and of voltage-dependent sodium and potassium channels. Thus, the gene for DHP receptor of rabbit skeletal muscle is closely related, or identical to, a gene expressed in heart that encodes a component of the DHP-sensitive VDCC. The DHP-sensitive and DHP-insensitive VDCCs are distinct molecular entities.     

预测酵母内含子中的转录调控元件*

 基于酵母基因非编码序列中寡核苷酸的出现频率，拟用期望频率法从转录效率较高的内含子序列中提取到一些过表达的寡核苷酸。将提取的寡核苷酸与其它方法获得的结果和试验证实的转录因子结合位点相比较，结果显示这些寡核苷酸可能是潜在的转录因子结合位点。这些推测有待试验的进一步证实。     

预测酵母内含子中的转录调控元件

基于酵母基因非编码序列中寡核苷酸的出现频率 ,拟用期望频率法从转录效率较高的内含子序列中提取到一些过表达的寡核苷酸。将提取的寡核苷酸与其它方法获得的结果和试验证实的转录因子结合位点相比较 ,结果显示这些寡核苷酸可能是潜在的转录因子结合位点。这些推测有待试验的进一步证实     

Translating biomolecular recognition into nanomechanics

We report the specific transduction, via surface stress changes, of DNA hybridization and receptor-ligand binding into a direct nanomechanical response of microfabricated cantilevers. Cantilevers in an array were functionalized with a selection of biomolecules. The differential deflection of the cantilevers was found to provide a true molecular recognition signal despite large nonspecific responses of individual cantilevers. Hybridization of complementary oligonucleotides shows that a single base mismatch between two 12-mer oligonucleotides is clearly detectable. Similar experiments on protein A-immunoglobulin interactions demonstrate the wide-ranging applicability of nanomechanical transduction to detect biomolecular recognition.     

基于CRISPR/Cas9技术构建猪miR-136基因编辑载体

为了阐明microRNA对猪产仔数的调节机制,采用CRISPR/Cas9技术构建针对猪miR-136的基因编辑表达载体。通过实验,在miR-136基因成熟区及其下游设计两个单链引导RNA(sgRNA),合成4条磷酸化的单链DNA寡核苷酸,复性后形成两条带粘性末端的双链寡核苷酸片段,插入线性化的载体pX462的BpiI酶切位点处,转化感受态大肠杆菌DH5α,经测序确认,得到两个编辑表达载体pX462-miR-136-1和pX462-miR-136-2。构建了两个编辑miR-136基因的表达载体,为猪繁殖力的调控研究奠定了技术基础。     

Genetic variation in the human insulin gene

Four recombinant lambda phages containing nucleotide sequences complementary to a cloned human preproinsulin DNA probe have been isolated from human DNA. Restriction analyses in conjunction with Southern hybridizations reveal two types of gene sequences. One isolate of each type was subjected to complete nucleotide sequence determination. The sequences contain the entire preproinsulin messenger RNA region, two intervening sequence. 260 nucleotides upstream from the messenger RNA capping site, and 35 nucleotides beyond the polyadenylate attachment site. Our results strongly suggest that these two gene types are allelic variants of a single insulin gene.     

D-alanine in the frog skin peptide dermorphin is derived from L-alanine in the precursor

A D-alanine-containing peptide termed dermorphin, with potent opiate-like activity, has been isolated from skin of the frog Phyllomedusa sauvagei. Complementary DNA (cDNA) libraries were constructed from frog skin messenger RNA and screened with a mixture of oligonucleotides that contained the codons complementary to five amino acids of dermorphin. Clones were detected with inserts coding for different dermorphin precursors. The predicted amino acid sequences of these precursors contained homologous repeats of 35 amino acids that included one copy of the heptapeptide dermorphin. In these cloned cDNAs, the alanine codon GCG occurred at the position where D-alanine is present in the end product. This suggests the existence of a novel post-translational reaction for the conversion of an L-amino acid to its D-isomer.     

Integration and stable germ line transmission of genes injected into mouse pronuclei

Genetic material has been successfully transferred into the genomes of newborn mice by injection of that material into pronuclei of fertilized eggs. Initial results indicated two patterns of processing the injected DNA: one in which the material was not integrated into the host genome, and another in which the injected genes became associated with high molecular weight DNA. These patterns are maintained through further development to adulthood. The evidence presented indicates the covalent association of injected DNA with host sequences, and transmission of such linked sequences in a Mendelian distribution to two succeeding generations of progeny.     

DNA-templated self-assembly of protein arrays and highly conductive nanowires

A DNA nanostructure consisting of four four-arm junctions oriented with a square aspect ratio was designed and constructed. Programmable self-assembly of 4 x 4 tiles resulted in two distinct lattice morphologies: uniform-width nanoribbons and two-dimensional nanogrids, which both display periodic square cavities. Periodic protein arrays were achieved by templated self-assembly of streptavidin onto the DNA nanogrids containing biotinylated oligonucleotides. On the basis of a two-step metallization procedure, the 4 x 4 nanoribbons acted as an excellent scaffold for the production of highly conductive, uniform-width, silver nanowires.     

cDNA Cloning of Goat DNA Methyltransferase 1,Screening of shRNA Vectors and Influences to Development of Nuclear Transfer Embryos

This study was designed to clone cDNA of goat DNA methyltransferase 1（DNMT1） gene,to screen an effective shRNAproducing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells.In this study,PCR primers were designed against regions of high homology between bovine and sheep sequences and then used to amplify the larger portions of the coding regions.Next,3 RNAi oligonucleotides were designed based on the cloned sequences and inserted into pRNAT-U6.1/Neo vector,acquiring 3 new vectors,respectively termed pRNAD1,pRNAD2 and pRNAD3.Then the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic sequencing.Finally,the developmental rates of nuclear transfer（NT） embryos generated using donor cells with and without the effective shRNA vector respectively,as well as in vitro fertilization（IVF） embryos were observed and recorded.The results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned（GenBank no.FJ617538）.Furthermore,an effective interfering shRNA（pRNAD2） was obtained,with its interference effect being 47.88%.Finally,NT embryos with shRNA vector harbored better developmental competence during morula and blastocyst stage compared to controls（P 〈 0.05）,reaching the similar rates to IVF embryos（P 〉 0.05）.In conclusion,goat DNA methyltransferase 1 gene cDNA was cloned and sequenced,an effective shRNA vector responsible for inhibiting DNA methyltransferase 1 expression was developed and the developmental competence of goat nuclear transfer morulae and blastcysts was significantly improved,which provided a feasible pathway for improving goat nuclear transfer embryo development competence by decreasing the methylation level in donor cells through RNAi-mediated manner.     

聚合酶链式反应(PCR)技术检测牛奶中金黄色葡萄球菌

根据金黄色葡萄球菌特异性STAA-Aul基因序列,设计、合成了一对引物,运用聚合酶链式反应(PCR)技术从10头患有奶牛乳房炎的奶牛奶样中扩增得到8个大小为420 bp的DNA产物;阴性样品中未扩增到产物。测定了其中2个DNA片段的核酸序列,确定为金黄色葡萄球菌的STAA-Aul基因。     

Specific mutations induced by triplex-forming oligonucleotides in mice

Triplex-forming oligonucleotides (TFOs) recognize and bind to specific duplex DNA sequences and have been used extensively to modify gene function in cells. Although germ line mutations can be incorporated by means of embryonic stem cell technology, little progress has been made toward introducing mutations in somatic cells of living organisms. Here we demonstrate that TFOs can induce mutations at specific genomic sites in somatic cells of adult mice. Mutation detection was facilitated by the use of transgenic mice bearing chromosomal copies of the supF and cII reporter genes. Mice treated with a supF-targeted TFO displayed about fivefold greater mutation frequencies in the supF gene compared with mice treated with a scrambled sequence control oligomer. No mutagenesis was detected in the control gene (cII) with either oligonucleotide. These results demonstrate that site-specific, TFO-directed genome modification can be accomplished in intact animals.     

Identifying global centers of genetic diversity for grain sorghum with the use of rice DNA markers

STS and InDel markers developed on the basis of DNA sequences of certain rice genes are used for a comparative study of grain sorghum accessions. The main sorghum genetic diversity centers are revealed, two in Africa and a third in regions of Central, East, and South Asia.