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1.
A highly efficient directional cDNA cloning method utilizing an asymmetrically tailed linker-primer plasmid.
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A new procedure using an asymmetrically tailed linker-primer plasmid has been developed to prepare extremely high complexity cDNA libraries. This procedure yields plasmid primed libraries with a final form equivalent to those made by the procedure of Okayama and Berg. However, the number of steps involved in library preparation is decreased. The form of the vector is such that one end of the linearized linker-primer plasmid has a 3' terminal extension of 40 deoxythymidylate residues (the dT end). The other end has a 3' terminal extension of 10 deoxycytidylate residues (the dC end). The dC end of the plasmid is blocked to further 3' extension by a 3' phosphate group. This configuration enables one to prime first strand cDNA synthesis at the dT end, tail the 3' end of the cDNA with deoxyguanylate residues without tailing the dC end (due to the 3' phosphate block). The plasmid primed cDNA can then be self-annealed and the 3' phosphate blocking group removed during the synthesis of double stranded cDNA. The efficiency of this procedure is significantly higher than other methods (including phage based libraries): linker-primer libraries have 15 to 900-fold higher complexity than libraries prepared by other methods. A cloning efficiency of 9 x 10(8) colonies per microgram of linker-primer DNA was achieved. This method should be useful for the cloning of cDNAs corresponding to extremely rare mRNAs. 相似文献
2.
Kawachi R Koike Y Watanabe Y Nishio T Sakuda S Nagasawa H Oku T 《Molecular biotechnology》2004,27(3):179-186
This article describes a simple method for accurate rapid amplification of complementary deoxyribonucleic acid (cDNA) ends
(RACE), the distinctive feature being that only a gene-specific primer is used, without an anchor or adapter primer. Under
these conditions, Thermus aquaticus (Taq) polymerase synthesizes cDNA ends exactly, so that amplified products obtain a characteristic structure: a terminal inverted
repeat composed of a gene-specific primer and occasionally several nucleotides from its 3′ flanking sequence. These structures
suggest a hypothetical mechanism of cDNA end synthesis in which Taq DNA polymerase synthesizes a sequence complementary to the gene-specific primer at the 3′ end of the daughter strand by switching
the template to the 5′ terminal region through circularization of the DNA. As a result, the targeted cDNA will be efficiently
amplified with only a single gene-specific primer. This technique, which provides highly specific amplification of the 5′
and 3′ ends of a cDNA, is especially useful for isolation of cDNA when the corresponding messenger ribonucleic acid is scarce. 相似文献
3.
Many studies have been performed on venomous peptides derived from animals. However, little of this research has focused on peptides from centipede venoms. Here, a venom gland cDNA library was suc-cessfully constructed for the centipede Scolopendra subspinipes mutilans. A new cDNA encoding the precursor of a venom peptide, named SsmTx, was cloned from the venomous gland cDNA library of the centipede S. subspinipes mutilans. The full-length SsmTx cDNA sequence is 465 nt, including a 249 nt ORF, a 45 nt 5′ UTR and a 171 nt 3′ UTR. There is a signal tail AATAAA 31 nt upstream of the poly (A) tail. The precursor nucleotide sequence of SsmTx encodes a signal peptide of 25 residues and a mature peptide of 57 residues, which is bridged by two pairs of disulfide bonds. SsmTx displays a unique cysteine motif that is completely different from that of other venomous animal toxins. This is the first reported cDNA sequence encoding a venom peptide from the centipede S. subspinipes mutilans. 相似文献
4.
PCR-based cDNA library construction: general cDNA libraries at the level of a few cells. 总被引:36,自引:7,他引:29
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A procedure for the construction of general cDNA libraries is described which is based on the amplification of total cDNA in vitro. The first cDNA strand is synthesized from total RNA using an oligo(dT)-containing primer. After oligo(dG) tailing the total cDNA is amplified by PCR using two primers complementary to oligo(dA) and oligo(dG) ends of the cDNA. For insertion of the cDNA into a vector a controlled trimming of the 3' ends of the cDNA by Klenow enzyme was used. Starting from 10 J558L micron3 myeloma cells, total cDNA was synthesized and amplified approximately 10(5) fold. A library containing 10(6) clones was established from 1/6 of the amplified cDNA. Screening of the library with probes for three genes expressed in these cells revealed a number of corresponding clones in each case. The longest obtained clones contained inserts of 1.5 kb length. No sequences originating from carriers or from rRNA was found in 14 randomly picked clones. 相似文献
5.
Directional cloning of complementary DNA (cDNA) primed by oligo(dT) is commonly achieved by appending a restriction site to the primer, whereas the second strand is synthesized through the combined action of RNase H and Escherichia coli DNA polymerase I (PolI). Although random primers provide more uniform and complete coverage, directional cloning with the same strategy is highly inefficient. We report that phosphorothioate linkages protect the tail sequence appended to random primers from the 5′ → 3′ exonuclease activity of PolI. We present a simple strategy for constructing a random-primed cDNA library using the efficient, size-independent, and seamless In-Fusion cloning method instead of restriction enzymes. 相似文献
6.
本实验用不同方法研究鲤鱼垂体mRNA反转录为互补DNA的活性。用硫氰酸胍法,从垂体中提取总RNA,经过Oligo(dT)-纤维素柱层析,得到poly(A)~+RNA。 以mRNA为模板,30%反转录成单链cDNA。利用RNaseH-DNA聚合酶Ⅰ-E.coli DNA连接酶合成双链cDNA。单链cDNA拷贝成双链cDNA。经放射自显影分析,证明合成了全长cDNA。用水解法合成双链cDNA,大多数为不完整的双链cDNA。平末端的双链cDNA连接上Eco RI-linker经Sepharose-4B分离,收集大片段cDNA,与pUC 19载体相连接,经转化构建成10~5克隆/μg mRNA的cDNA文库。 相似文献
7.
8.
Tian Yuan Ji-Rui Gu Wen-Bo Gu Jiang Wu Shao-Rong Ge Heng Xu 《Molecular biology reports》2011,38(3):2059-2065
Adenylosuccinate lyase (ADSL) is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling.
In the present study, we have constructed a grass carp (Ctenopharyngodon idella) intestinal cDNA library that has over 2.3 × 105 primary clones. An expressed sequence tag (EST) of grass carp adenylosuccinate lyase (gcADSL) gene was screened from this
library. Both 5′-RACE and 3′-RACE were carried out in order to obtain the complete cDNA sequence, which contains a 1,446 bp
open reading frame encoding 482 amino acids about 54.552 kDa. The deduced amino acid sequence shares high homology with its
vertebrate counterparts, which shares 94% similarity with zebrafish, 81% with African clawed frog as well as chicken, 77%
with human and 76% with mouse. This gcADSL genomic sequence, consisted of 13 exons and 12 introns, is 8,557 bp in size. Real-time
quantitative PCR analysis revealed that the highest expression level of gcADSL was detected in muscle and the lowest in gill.
In western blotting analysis, His6-tagged gcADSL protein expressed in Escherichia coli could be recognized not only by an anti-His6-tag monoclonal antibody but also by an anti-human ADSL polyclonal antibody, indicating immunological crossreactivity occurs
between grass carp and human ADSL protein. 1,082 bp 5′-flanking region sequence was cloned and analyzed. 相似文献
9.
Aurora Daniele Fiorella Altruda Marina Ferrone Lorenzo Silengo Laura Chiarantini Marzia Bianchi Vilberto Stocchi Mauro Magnani 《Molecular and cellular biochemistry》1991,107(2):87-94
Summary A 1,820bp full-length clone encoding for a new human protein was isolated from a gt11 placental cDNA library using anti-human hexokinase antibodies. The cDNA complete sequence includes a 12 by 5 noncoding region, a single open reading frame encoding a protein of 55 KDa (HP-10) and a 177 by non-coding with two putative polyadenylation signals upstream of 3poly(A)tail. The deduced amino acid sequence reveals a sequence of 492 amino acids that contains a stretch of 7 glutamic acid from position 169 and one potential glycosylation site at position 274. Although antibodies against hexokinase recognize the fusion protein and antibodies against the fusion protein recognize hexokinase, HP-10 is not human hexokinase, by a number of criteria including the alignment of determined amino acid sequences.In searching for a possible functional role of HP-10 its cDNA was inserted into a procaryotic vector which allows the expression of the non-fused protein. Bacteria expressing the HP-10 encoded protein were isolated and found to have a dramatic increase in endogenous phosphorylated proteins. Since HP-10 does not have a protein kinase activity per se it should be considered a new regulatory phosphorylation protein which is active in E. coli
Abbreviations HK
Hexokinase (EC 2.7.1.1) 相似文献
10.
11.
The Arabidopsis
AHL gene encodes a 3′(2′),5′-bisphosphate nucleotidase (BPNTase) involved in the reductive sulfate activation pathway. A bacterial
expression vector containing AHL cDNA was randomly mutagenized with hydroxylamine and transformed into the E. coli cysteine auxotrophic mutant cysQ. Bacterial colonies that did not show evidence of complementation, i.e. those that exhibited slower growth on cysteine-free
medium, were selected for further study. Sequencing of the AHL cDNA in one such clone revealed the conversion of cytosine 635 (C635) to thymine, resulting in an Alanine (A212) to Valine substitution. This microbial complementation procedure is useful in BPNTase structure-activity studies for biotechnological
applications. 相似文献
12.
柔嫩艾美耳球虫孢子化卵囊cDNA文库的构建 总被引:2,自引:0,他引:2
用建立表达性文库的方法 ,构建了Eimeriatenella孢子化卵囊噬菌体ZAP表达性cDNA文库。首先用TRIzol试剂盒从E .tenella孢子化卵囊中提取总RNA ,再用Oligo(dT)12纤维素柱从总RNA中分离mRNA ,以mRNA为模板 ,Oligo(dT)18Linker Primer为引物 ,反转录合成cDNA第一链 ,再在DNA聚合酶Ⅰ作用下置换合成第二链cDNA。cDNA第二链合成后用PfuDNA聚合酶补平XhoⅠ位点 ,再与EcoRⅠAdapters连接 ,经XhoⅠ酶切后 ,凝胶电泳回收 500bp~4.0kp之间的cDNA片段 ,纯化后的双链cDNA与载体ZAPExpressvector连接。体外包装后得到E .tenella孢子化卵囊的cDNA表达性文库 ,经测定该文库的容量为 6×106 ,扩增后文库的滴度为 1×1011 Pfu mL ,经PCR测定 ,该文库的重组率为 96%。 相似文献
13.
Mo L Hellmich HL Fong P Wood T Embesi J Wills NK 《The Journal of membrane biology》1999,168(3):253-264
Loss of function mutations of the renal chloride channel, ClC-5, have been implicated in Dent's disease, a genetic disorder
characterized by low weight proteinuria, hypercalciuria, nephrolithasis and, in some cases, eventual renal failure. Recently,
our laboratory used an RT-PCR/RACE cloning strategy to isolate an amphibian cDNA from the renal epithelial cell line A6 that
had high homology to human ClC-5. We now report a full-length native ClC-5 clone (xClC-5, containing 5′ and 3′ untranslated
regions) isolated by screening a cDNA library from A6 cells that was successfully expressed in Xenopus oocytes. In addition, we compared the properties of xClC-5 and hClC-5 using isogenic constructs of xClC-5 and hClC-5 consisting
of the open reading frame subcloned into an optimized Xenopus expression vector. Expression of the full-length ``native'
xClC-5 clone resulted in large, strongly rectifying, outward currents that were not significantly affected by the chloride
channel blockers DIDS, DPC, and 9AC. The anion conductivity sequence was NO−
3 > Cl−= I− > HCO−
3 >> glutamate for xClC-5 and NO−
3 > Cl− > HCO−
3 > I− >> glutamate for hClC-5. Reduction of the extracellular pH (pH
o
) from 7.5 to 5.7 inhibited outward ClC-5 currents by 27 ± 9% for xClC-5 and 39 ± 7% for hClC-5. The results indicate that
amphibian and mammalian ClC-5 have highly similar functional properties. Unlike hClC-5 and most other ClC channels, expression
of xClC-5 in oocytes does not require the removal of its untranslated 5′ and 3′ regions. Acidic solutions inhibited both amphibian
and human ClC-5 currents, opposite to the stimulatory effects of low external pH on other ClC channels, suggesting a possibly
distinct regulatory mechanism for ClC-5 channels.
Received: 28 August 1998/Revised: 13 January 1999 相似文献
14.
15.
为了进一步分离人尿道(阴茎)鳞癌组织特异性表达基因和鳞癌特异性相关基因,采用SMART技术,构建了人尿道 (阴茎)鳞癌上皮细胞cDNA文库,从人尿道(阴茎)鳞癌上皮细胞中分离总RNA并纯化mRNA,利用经修饰的oligo(dT)引物 合成cDNA第一链,利用SMART核苷酸作为cDNA第一链在mRNA5′端延伸出去的模板,采用LD-PCR合成双链cDNA,双链 cDNA经酶切和过柱分级分离后,克隆入λTriplEx2载体后经体外包装而成cDNA文库。结果表明原始人尿道(阴茎)鳞癌上 皮cDNA文库获得1.57×107个重组子,重组率达到98%。文库扩增后,滴度达到4.0×109pfu/ml,插入cDNA平均长度为2.5kb。 构建的人尿道(阴茎)鳞癌上皮cDNA文库具有良好的质量,该cDNA文库为进一步筛选鳞癌抑癌基因及鳞癌特异性表达基因 奠定了基础。 相似文献
16.
Maurice Durand Nguyen Thanh Thuong Jean Claude Maurizot 《Journal of biomolecular structure & dynamics》2013,31(6):1191-1202
Abstract The interaction of berenii molecule, a minor groove binding drug, with T-A-T triple helix and A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by an oligonucleotide (dA)12?x-(dT)12?x-(dT)12, where x is a hexaethylene glycol chain bridged between the 3′ phosphate of one strand and the 5′ phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Circular dichroism spectroscopy demonstrates that berenil can bind to the triple helical structure. Spectral analysis shows that in the same ionic strength the drug bound to a double-stranded structure exhibits a conformation and an environment close to those observed in triple-stranded structure. The influence of the ionic strength on the interaction between the berenil molecule and the 36-mer is clearly demonstrated. We showed that when no NaCl salt is added in the buffer the triplex form of (dA)12?x-(dT)12-x-(dT)12 is stabilized by berenil whereas it is destabilized slightly by the dye when NaCl concentration is 1 M. 相似文献
17.
An expressed sequence tag (EST) is simply a segment of a sequence over 150 bp from a randomly selected cDNA. EST helps to
quickly identify functions of expressed genes and to understand the complexity of gene expression with database comparison.
Sequencing of random cDNA clones can be applicable to discovery of new genes, mapping of the genome, identification of coding
regions in genomic sequences, and antisense method. To accomplish these goals, in this research, randomly selected cDNA sequencing
was performed with watermelon plant. Among 30 clones picked up and analyzed, all clones had an insert length over 0.5 kb.
The average size of insert was about 1.3 kb. The size range of most cDNA insert was 1.0–2.0 kb. For sequence comparison, data
from the coding region at 5′ end of selected cDNA should be much more informative than those from the untranslated 3′ tail.
Thirty clones were sequenced from one end (5′ end). Of these, 29 had no poly (A) tail in this direction, while only one was
inverted. Thus, this library is over 96% unidirectional. Two clones had homologies to ribulose bisphosphate carboxylase/oxigenase
(Rubisco) small subunit precursor gene. Thirteen cDNAs had high degree of sequence similarity to genes from other organisms.
The remaining cDNA clones seem to be new genes not only in watermelon but also in all organisms. 相似文献
18.
A simple and versatile method for the preparation of vector-primers by adapter-end-primer ligation 总被引:2,自引:0,他引:2
A group of efficient cDNA cloning strategies employs vector-primers where cDNA synthesis starts from the oligo(dT)-primer tail, which is conventionally attached to cloning vectors by use of terminal deoxynucleotidyl transferase. An alternative, efficient and more versatile method of vector-primer preparation is to directly ligate, by use of T4 DNA ligase, a double-digested vector, e.g., pTZ18R/Pst I/Bam HI, to a synthetic (Bam HI)-adapter-end-primer, 5'-pGATCC-Tn or 5'-pGATCC-site-specific sequence. The use of a utility-vector containing a sizable spacer between the two selected restriction sites enables unambiguous separation on agarose gels of the double-digested vector precursors from single-digested ones, further simplifying the vector preparation. The adapter-end-primer ligation method can be applied to any suitable vectors with multiple cloning sites for the preparation of not only oligo(dT)-tailed, but also site-specific sequence-tailed vectors. Thus, the method enables the cDNA cloning of total poly (A+)-mRNAs, as well as specific RNA or mRNA species with or without poly(A)-tail. 相似文献
19.
François Barrieu Dominique Thomas Danièle Marty-Mazars Maryse Charbonnier Francis Marty 《Planta》1998,204(3):335-344
The vacuolar membrane (tonoplast) of plant cells contains aquaporins, protein channels that facilitate the selective transport
of water. These tonoplast intrinsic proteins (TIPs) of 23–29 kDa belong to the ancient major intrinsic protein (MIP) family.
A monospecific polyclonal antiserum directed against a 26 kDa intrinsic protein from the tonoplast of meristematic cells from
cauliflower (Brassica oleracea L. var. botrytis) was used to screen a cDNA library. Two distinct cDNAs have been isolated. Both clones, c26-1 and c26-2, encode closely related TIPs. The c26-1 insert, consisting of 933 bp upstream of the poly(A) tail, is a full-length cDNA with an open reading frame encoding a protein
of 251 amino acids with a calculated Mr of 25 500. The c26-2 insert is a 5′ truncated cDNA. The two cDNAs share 90.5% sequence identity within their overlapping coding regions but only
35% sequence identity in the 3′␣untranslated regions, indicating that highly related TIP-encoding genes are expressed in meristematic
cells. Although TIPs have previously been found in a variety of cell types, they have not been found in meristems. The derived
amino acid sequences (BobTIP26-1 and BobTIP26-2, respectively) closely resemble the aquaporin γ-TIP from Arabidopsis thaliana. Northern blot analysis and in situ hybridization show that BobTIP26 mRNAs preferentially accumulate in highly meristematic cells, mostly before and during cell enlargement, and in the living
cells of the xylem. This differential pattern of expression is also found by immunodetection of BobTIP26 polypeptides. The
gene expression patterns are discussed with respect to the probable function of the gene products.
Received: 27 March 1997 / Accepted: 20 May 1997 相似文献
20.
Graciela E. Santillán Marisa J. Sandoval Yuti Chernajovsky Patricia L. Orchansky 《Molecular and cellular biochemistry》1992,110(2):181-191
A number of cell-surface proteins are anchored in plasma membranes by a glycosylated phosphatidylinositol (PI) moiety that
is covalently attached to the carboxyl-terminal amino acid of the mature protein. We have previously reported the construction
of a cDNA clone of a truncated Platelet-derived growth factor (PDGF) receptor that consists of the extracellular domain without
the transmembrane and cytoplasmic domains. In the construction of the vector, a sequence of 51 base pairs (bp) from the 3′-untranslated
region of the receptor cDNA was linked in frame with the external domain coding sequence. The truncated receptor protein with
the peptide VTSGHCHEERVDRHDGE fused to its carboxyl terminus was covalently attached to the membrane by a PI linkage and it
was released by phosphatidylinositol specific-phospholipase C (PI-PLC). When the 51 bp sequence was deleted, the external
domain receptor protein was secreted into the media. To determine whether the PI linkage of the protein was due to the 17
amino acids added, the peptide was fused to the carboxyl terminus of the secreted protein human Interferon-β (hu-IFN-β). Chinese
hamster ovary (CHO) cells transfected with the hu-IFN-β cDNA secreted the protein to theconditioned media, whereas CHO cells
transfected with the carboxyl terminus modified-hu-IFN-β cDNA did not secrete detectable levels of protein. CHO cells expressing
the carboxyl terminus modified-hu-IFN-β were treated with PI-PLC, the media and cell lysates were analyzed by SDS-PAGE after
immunoprecipitation with antibodies against hu-IFN-β. The modified protein is anchored to the plasma membrane by a PI linkage
and it is specifically released by PI-PLC, whereas a control preparation of CHO cells expressing wild type hu-IFN-β does not
show the same pattern. The 17 amino acid peptide fused to the carboxyl terminus of IFN-β directs attachment of a PI anchor
and targets the fusion protein to the plasma membrane. 相似文献