cDNA Library Construction

As a pioneer biotechnology company in the world, Creative Biogene can provide high quality cDNA libraries construction service to customers worldwide. Creative Biogene’s advanced technologies and highly experienced staffs are available to provide you a range of cDNA libraries construction service, including standard cDNA library, subtractive cDNA library, normalized cDNA library, full length cDNA library, yeast two-hybrid cDNA library and SSH cDNA library. Creative Biogene’s goal is to provide you with the most affordable and high-quality cDNA libraries construction service to ensure your satisfaction in a timely and professional manner

An improved protocol for small RNA library construction using High Definition adapters

Next generation sequencing of small RNA (sRNA) libraries is widely used for studying sRNAs in various biological systems. However, cDNA libraries of sRNAs are biased for molecules that are ligated to adapters more or less efficiently than other molecules. One approach to reduce this ligation bias is to use a pool of adapters instead of a single adapter sequence, which allows many sRNAs to be ligated efficiently. We previously developed High Definition (HD) adapters for the Illumina sequencing platform, which contain degenerate nucleotides at the ligating ends of the adapters. However, the current commercial kits produced a large amount of 5’ adapter – 3’ adapter ligation product without the cDNA insert when HD adapters were used to replace the kit adapters. Here, we report a protocol to generate sRNA libraries using HD adapters with dramatically reduced adapter-adapter product. This protocol was developed from the procedure invented by Vaidyanathan et al. The libraries can be completed within two days and can be used for various biological and clinical samples. As examples for using this protocol, we constructed sRNA libraries using total RNA extracted from cultured mammalian cells and plant leaf tissue. The PCR products contained a very small amount of adapter-adapter product. Bioinformatic analysis of the sequencing data revealed sRNAs with diverse sequences and many different miRNA families

SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones

BACKGROUND: cDNA libraries are widely used to identify genes and splice variants, and as a physical resource for full-length clones. Conventionally-generated cDNA libraries contain a high percentage of 5'-truncated clones. Current library construction methods that enrich for full-length mRNA are laborious, and involve several enzymatic steps performed on mRNA, which renders them sensitive to RNA degradation. The SMART technique for full-length enrichment is robust but results in limited cDNA insert size of the library. RESULTS: We describe a method to construct SMART full-length enriched cDNA libraries with large insert sizes. Sub-libraries were generated from size-fractionated cDNA with an average insert size of up to seven kb. The percentage of full-length clones was calculated for different size ranges from BLAST results of over 12,000 5'ESTs. CONCLUSIONS: The presented technique is suitable to generate full-length enriched cDNA libraries with large average insert sizes in a straightforward and robust way. The representation of full-coding clones is high also for large cDNAs (70%, 4–10 kb), when high-quality starting mRNA is used

Comparison between Normalised and Unnormalised 454-Sequencing Libraries for Small-Scale RNA-Seq Studies

Next-generation sequencing of transcriptomes (RNA-Seq) is being used increasingly in studies of nonmodel organisms. Here, we evaluate the effectiveness of normalising cDNA libraries prior to sequencing in a small-scale study of the zebra finch. We find that assemblies produced from normalised libraries had a larger number of contigs but used fewer reads compared to unnormalised libraries. Considerably more genes were also detected using the contigs produced from normalised cDNA, and microsatellite discovery was up to 73% more efficient in these. There was a positive correlation between the detected expression level of genes in normalised and unnormalised cDNA, and there was no difference in the number of genes identified as being differentially expressed between blood and spleen for the normalised and unnormalised libraries. We conclude that normalised cDNA libraries are preferable for many applications of RNA-Seq and that these can also be used in quantitative gene expression studies

A cDNA subtraction approach to isolate male-specific genes from Ceratitis capitata : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University

The Mediterranean fruit fly, Ceraritis capitata (medfly), is a significant world wide agricultural pest. Sterile Insect Technique (SIT) is a biological method that has been used to control medfly successfully in several parts of the world for around two decades. SIT involves the release of sterile insects into wild populations which, due to sterile matings, lead to a reduction in the size of the wild population. The effectiveness of this technique is significantly increased when only sterile males are released. This can be achieved by using sexing strains, but these strains are prone to breakdown as a result of recombination leading to the disassociation of the selection gene from the Y chromosome. An alternative system, that could be more robust, would involve the control of the expression of the male determining gene. The aim of this thesis was to identify the male determining gene of medfly by creating subtracted cDNA libraries enriched for male-specific transcripts. Subtracted libraries were made by subtractive suppression PCR, using the ClonTech cDNA subtraction kit. The libraries were screened by Southern hybridisation analysis using male and female total cDNA probes. Only one clone appeared to display a bias toward male-specific hybridisation, but this was found to be a result of unequal transfer of DNA. A selection of clones were individually used to probe membrane bound genomic DNA. These hybridisation analyses indicated a general lack of male-specific enrichment. In addition to this, sequence analysis of a selection of clones revealed a number of mitochondrial gene fragments, showing that there had been insufficient subtraction. As results indicated that the creation of subtracted, male-specifically enriched libraries had been unsuccessful another approach to the identification of the male determining gene was attempted. Genomic DNA was screened with an Sxl probe, under low stringency hybridisation conditions, to identify Y-linked encoding RNA binding proteins distantly related Sxl, which could represent the male determining gene. This screen showed that there were no male-specific RNA binding proteins, of the SXL family in medfly

Cloning and Expression Studies of Starch Phosphorylase From Solanum tuberosum

The aim of this project was to study starch phosphorylase gene expression in potato tubers during different physiological conditions when starch was mobilised, like wound healing and sprouting. One of the objectives was to clone plastidic potato tuber starch phosphorylase cDNA. cDNA libraries were constructed using mRNA from wounded and unwounded tuber tissue as templates for cDNA synthesis. Based on the amino acid sequence (Nakano & Fukui, 1986) of specific regions of potato tuber starch phosphorylase, two different sets of mixed oligonucleotide probes (called SP1 and SP2) were prepared and subsequently used to screen the cDNA libraries for starch phosphorylase recombinants

Next-Generation cDNA Screening for Oncogene and Resistance Phenotypes

There is a pressing need for methods to define the functional relevance of genetic alterations identified by next-generation sequencing of cancer specimens. We developed new approaches to efficiently construct full-length cDNA libraries from small amounts of total RNA, screen for transforming and resistance phenotypes, and deconvolute by next-generation sequencing. Using this platform, we screened a panel of cDNA libraries from primary specimens and cell lines in cytokine-dependent murine Ba/F3 cells. We demonstrate that cDNA library-based screening can efficiently identify DNA and RNA alterations that confer either cytokine-independent proliferation or resistance to targeted inhibitors, including RNA alterations and intergenic fusions. Using barcoded next-generation sequencing, we simultaneously deconvoluted cytokine-independent clones recovered after transduction of 21 cDNA libraries. This approach identified multiple gain-of-function alleles, including KRAS G12D, NRAS Q61K and an activating splice variant of ERBB2. This approach has broad applicability for identifying transcripts that confer proliferation, resistance and other phenotypes in vitro and potentially in vivo

Analysis of expressed sequence tags from Musa acuminata ssp. burmannicoides, var. Calcutta 4 (AA) leaves submitted to temperature stresses.

In order to discover genes expressed in leaves of Musa acuminata ssp. burmannicoides var. Calcutta 4 (AA), from plants submitted to temperature stress, we produced and characterized two full-length enriched cDNA libraries. Total RNA from plants subjected to temperatures ranging from 5°C to 25°C and from 25°C to 45°C was used to produce a COLD and a HOT cDNA library, respectively. We sequenced 1,440 clones from each library. Following quality analysis and vector trimming, we assembled 2,286 sequences from both libraries into 1,019 putative transcripts, consisting of 217 clusters and 802 singletons, which we denoted Musa acuminata assembled expressed sequence tagged (EST) sequences (MaAES). Of these MaAES, 22.87% showed no matches with existing sequences in public databases. A global analysis of the MaAES data set indicated that 10% of the sequenced cDNAs are present in both cDNA libraries, while 42% and 48% are present only in the COLD or in the HOT libraries, respectively. Annotation of the MaAES data set categorized them into 22 functional classes. Of the 2,286 high-quality sequences, 715 (31.28%) originated from full-length cDNA clones and resulted in a set of 149 genes

Global expression mapping of mammalian genomes

he aim of genome projects is to decipher all the information contained within the DNA of an organism and to study the way this information is processed in physiological processes. It is believed that more than 95% of the information content of the mammalian genome is represented in the protein coding sequences that make up only approximately 2% of the DNA sequence. Consequently much effort is being invested in the study of coding sequences in the form of cDNA analysis. This thesis is concerned with the development of a new strategy for a highly parallel approach to analyse entire cDNA libraries. The strategy is based upon generating sufficient sequence information to identify uniquely more than 100,000 cDNA clones by hybridisation with short oligonucleotides, typically 7 - 10 mers. Each oligonucleotide is hybridised to all cDNA clones in parallel and under stringent conditions positively identifies a subset (3 - 10%) of clones. Oligonucleotides are designed in such a way that each will positively identify a different subset of clones and statistical simulations estimate that approximately 200 such hybridisation events are required to identify uniquely upto 100,000 cDNA sequences. Such a fingerprint can be generated from many cDNA libraries constructed from different tissue mRNAs and will not only lead to the identification of most sequecnes expressed from the genome but also indicate the level of expression by determining the number of times any given sequence is represented across different cDNA libraries. A human foetal brain cDNA library has been constructed and 100,000 clones arrayed into microtitre plates and on nylon membranes. All the required technological developments have been carried out successfully and are presented. In excess of 200 oligonucleotide hybridisations have been performed on a subset of 32,000 cDNA clones and 1,000 sequenced control clones. A detailed analysis of the data on the control clones is presented and the implications for cDNA fingerprinting discussed

In vitro recombination cloning of entire cDNA libraries in Arabidopsis thaliana and its application to the yeast two-hybrid system

In the postgenomic era many experiments rely on the availability of transcript sequence for cloning. As these clones usually originate from cDNA libraries, the quality of these libraries is crucial. If a good library is generated it is desirable to use a versatile cloning system suitable for many different kinds of applications. The cloning systems based on in vitro recombination proves fitting for this task. However, the use of this method for shuttling entire cDNA libraries between different vectors has not yet been studied in great detail. Here we describe the construction of four cDNA libraries from different tissues of Arabidopsis thaliana, the shuttling of the libraries into expression vectors, and evaluation of this method as well as its suitability for downstream applications. Libraries were constructed from seedlings, hormone treated seedlings, flowers, developing seeds and primary leaves in the ldquoentry vectorrdquo of the Gatewaytrade cloning system. After initial characterization of the libraries, they were shuttled into an expression vector (a yeast two-hybrid prey vector). To monitor for a size bias generally assumed to be inherent to in vitro recombination methods, the libraries were characterized before and after the transfer into the expression vector. However no significant difference could be detected. The functionality of the in vitro recombination system for the shuttling of entire libraries was then further tested by protein-protein interaction screens. The results of the library characterization and of the yeast two-hybrid screens and their implications for large-scale proteomic approaches are discussed