A protocol for the construction of microsatellite enriched genomic library

An improved protocol for constructing microsatellite-enriched libraries was developed. The procedure depends on digesting genomic DNA with a restriction enzyme that generates blunt-ends, and on ligating linkers that, when dimerized, create a restriction site for a different blunt-end producing restriction enzyme. Efficient ligation of linkers to the genomic DNA fragments is achieved by including restriction enzymes in the ligation reaction that eliminate unwanted ligation products. After ligation, the reaction mixture is subjected to subtractive hybridization without purification. DNA fragments containing microsatellites are captured by biotin-labeled oligonucleotide repeats and recovered using streptavidin-coated beads. The recovered fragments are amplified by PCR using the linker sequence as primer, and cloned directly into a plasmid vector. The linker has the sequence GTTT on the 5′ end, which promotes efficient adenylation of the 3′ ends of the PCR products. Consequently, the amplified fragments could be cloned into vectors without purification. This procedure enables efficient enrichment and cloning of microsatellite sequences, resulting in a library with a low level of redundancy.     

Preparing a human membrane and secreted protein-enriched cDNA library using PCR primers derived from a genomic database

We describe here a strategy for preparing a human membrane and secreted protein (MSP)-enriched cDNA library based on human MSP- and non-MSP-encoding cDNA sequences in the databases. The signal peptide parts of the MSP-encoding cDNA sequences, which currently comprise about half of the estimated total number in humans, were analyzed for common patterns. These patterns form a ‘minimal’ set of polymerase chain reaction primer candidates of length varying from 9 to 21 nt. The products stemming from each primer candidate were determined and the results allowed us to obtain an ‘optimal’ mixed-length primer set. Ninety-six percent of the primers in this set were predicted to yield ≤10% undesired products, and the desired MSP-cDNA products could be easily separated by gel electrophoresis. The present analysis establishes a methodology for preparing a cDNA library that enables the analysis of individual MSPs. This methodology may also help identify new MSPs. As many cell regulatory processes are mediated by secreted proteins and their membrane-bound receptors, the preparation of a MSP-enriched cDNA library should benefit research on MSPs.     

Solid-phase cDNA library construction, a versatile approach.

A rapid and versatile method for cDNA library construction was developed. It is based on conventional cDNA library synthesis including all enzymatic steps usually required, but is performed on a solid support. The cDNA is immobilised via a biotin residue to streptavidin coupled magnetic beads, which allows rapid and easy to perform changes of buffers and enzymes. Therefore, it combines speed (library construction within a single day) with high quality libraries, making it ideally suited for most purposes.     

一种存放大基因组文库的新方法

用野生一粒小麦为材料,以细菌人工染色体（pECBAC1)为载体构建了细菌人工染色体克隆混合池（Bacterial artificial chromosome pool）,每池100个克隆。经初步验证,池中靶克隆经12h的培养后仍稳定存活。克隆之间的竞争实验仍在进行当中。     

A novel immobilization strategy using oligonucleotide as linker for small molecule microarrays construction

A novel immobilization method based on oligonucleotide as linker has been developed for small molecule microarrays (SMMs) construction. The oligonucleotide tail was employed as a linker in solid-phase synthesis. Small molecules could be easily conjugated at the 5′ end of the oligonucleotide, previously modified with a functional group. Being a reactive species, the oligonucleotide was activated by UV irradiation, for the attachment of the conjugate to the slide surface. The method was successfully applied to structurally distinct small molecules, including biotin, antibiotic and drug. This immobilization strategy showed high efficiency, 1.1 fmol of small molecules in the spotting solution per spot gave a detectable signal (mean S/N = 10.9). The results suggest that it is very promising for exploring interaction between small molecules and proteins, and high throughput detecting the chemical compounds.     

A short, novel, and cheaper procedure for oligonucleotide synthesis using automated solid phase synthesizer

Dimethylthiuram disulfide (DTD) has been developed as an efficient thiolation reagent during automated synthesis of oligonucleotides using phosphoramidite chemistry. Simultaneous thiolation and capping was accomplished by mixing DTD with capping solution B, which saved 20% of solvent consumption and compressed the four-step synthesis cycle to three. Large-scale (1 mmol) synthesis of phosphorothioate oligonucleotides has been demonstrated with excellent yield and purity.     

A simplified and efficient vector-primer cDNA cloning system

A simplified, efficient, and versatile vector-primer cDNA cloning system is presented. The dimer-primer system is a modification of the method of Okayama and Berg (1982) with the following features: (i) the vector-primer molecules are more rapidly and reliably prepared by virtue of the elimination of an endonuclease digestion and the agarose gel purification step from the original method, and (ii) the final cDNA products contain polylinkers at both cDNA-vector junctions, simplifying the size analysis, subcloning, and sequencing of inserts. The system is highly efficient, yielding greater than 10(5) transformants using 1 microgram mRNA and 1 pmol of vector-primer ends, with 75% or more of the transformants having inserts. The ability of the system to produce clones of full-length or near full-length is demonstrated by the analysis of 32 ribulose-1,5-bisphosphate carboxylase small subunit cDNA clones from tomato.     

cDNA library construction from meristematic tissue of finger millet panicle

A protocol to obtain full-length cDNA using a SuperScript® Full-Length cDNA Library Construction Kit II (Invitrogen, United States) was developed, and a high quality cDNA library of meristem tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was constructed. The titer of the constructed cDNA library was 3.01 × 10⁵ CFU/mL, the average length of the insert was approximately 1070 base pairs, and the average efficiency of insertion of cDNA fragments was 99.5%. The sequencing of randomly selected clones created cDNA library was carried out. The cDNA sequences of clones were identified by BLAST search. The cDNA library analysis and selective sequencing indicate good functionality and full size of cDNA inserts of the clones. The constructed cDNA library from meristematic tissue of finger millet panicle is a good and reliable source for isolation and identification of key genes of metabolism and development of meristem as well for creation of new genetic markers for genetic research and molecular selection.     

Identification of a new gene encoding 5-enolpyruvylshikimate-3-phosphate synthase using genomic library construction strategy

Applying the genomic library construction strategy and colony screening, a new aroA gene encoding 5-enolpyruvylshikimate-3-phosphate synthase has been identified, cloned and overexpressed in Escherichia coli, and the enzyme was purified to homogeneity. Kinetic analysis of the AroA _{P.fluorescens} indicated that the full-length enzyme exhibits 10-fold increased IC50 and an approximately 38-fold increased K _i for glyphosate compared to those of the AroA _E.coli , while retaining high affinity for the substrate phosphoenolpyruvate. Furthermore, we have transformed the new aroA _{P.fluorescens} gene into Arabidopsis thaliana via a floral dip method, and demonstrated that transgenic A. thaliana plants exhibit significant glyphosate resistance when compared with the wild type.     

Cloning of PCR-amplified total cDNA: construction of a mouse oocyte cDNA library.

We describe a general method for the synthesis and cloning of cDNA, applicable to cases in which the availability of biological material for mRNA extraction is extremely limited. A protocol allowing amplification of a heterogeneous mixture of cDNAs by the polymerase chain reaction has been devised and applied successfully to the construction of an apparently representative cDNA library, using as a model of a scarce RNA source 50 mouse ovulated eggs that can yield a maximum of 1.75 ng of poly(A)+ RNA. However, about 5% of the material obtained after amplification was adequate for cloning. Using the cloned sequences, we have derived a preliminary indirect measurement of the sequence complexity of the maternal poly(A)+ RNA in this mammalian oocyte.