IDT
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Full length, sequence-verified cDNA clonesMGC premier cDNA clones from transOMIC technologies provide the highest sequence quality and confidence when purchasing pre-made full length cDNA clones. Based on the Mammalian Gene Collection (MGC) developed by the National Institutes of Health (NIH) with rigorous sequence analysis resulting in less than 1 error in 50,000 bp (MGC Project Team 2009). All MGC premier cDNA clones are backed by a 100% guarantee to be an exact match to the published sequence.MGC premier cDNA clones have the following advantages: • Ideal for native protein expression • Full insert sequence easily available • Best value on the market today Full length, sequence-verified cDNA clones are available for human, mouse, rat, bovine, Xenopus and zebrafish genomes. These collections are available as individual cDNA clones, rearrays of cDNA clones for gene families and pathways as well as genome libraries. MGC premier ORF clones Over the course of the MGC project there was a transition to add functionality to the clones. One example is the production of Open Reading Frame (ORF) clones. These ORF clones lack the untranslated regions (UTRs) and have their native stop codon removed to allow the addition of peptide tags at the C- or N- terminus of the protein and are flanked by att sites for easy transfer to a destination expression vector via Gateway® reaction. While these clones were created as part of the original cDNA clone project that produced the MGC premier cDNA clones they are designated MGC premier ORF clones in our gene search results and product literature to call out their unique value. Additional technical information can be found in the technical manual on the documents tab of this section. All MGC premier ORF clones are clonally isolated and end-sequenced to confirm their identity before shipment. Turnaround time for shipment of these clones is 5 days. Sequence verification service MGC premier cDNA clone identities can also be confirmed by end-sequencing prior to shipping. This risk-free option ensures the correct identity of the cDNA clone ordered.
Use the Fetch my Gene™ search tool to find and purchase cDNA or ORF clones representing your gene of interest. Gateway® is a registered trandemark of Life Technologies Save time and money with pre-made MGC premier cDNA clonesCreating cDNA clones from biological material requires time, labor and money. While cDNA clone production is a well-established process it requires three major steps: synthesis, cloning and validation. These steps can delay a research project due to a number of variables including availability of biological material expressing the gene of interest, expression level of the gene, reverse transcription efficiency, amplification efficiency and ability to sequence verify the cDNA clone once it is created.Pre-made MGC premier cDNA clone collections offer a convenient solution that decreases the time required to start an experiment and increases the chances of success. Rather than spending time on cloning, only a simple gene search using Fetch my Gene search tool is required. Find and purchase your MGC premier cDNA clone today! The importance of full insert sequencing when working with cDNA and ORFsAll MGC premier cDNA clones are fully sequenced and have been verified to include the full coding sequence (CDS). This is significant because even a single nucleotide polymorphism (SNP) can have a profound impact on a gene product’s function (Ingram 1957).The sequencing pipeline resulting in the MGC premier cDNA collections excluded clones with different types of aberrant sequences including premature stop codons and retained introns eliminating approximately 45% of possible full length clones. The remaining clones were then checked for full coding sequence (MGC Project Team 2009). Fig. 2 Alternative splicing of tyrosine kinase receptor Mst1r regulates cell mobility. Importance of full insert sequencingIt is thought that over ~95% of multiexon human genes are alternately spliced (Pan et al 2008). A retained intron can alter protein function but remain undetected in a clone unless it has under gone full insert sequence.Mstr1 is a member of the scatter protein family whose signaling elicits normal cell growth and protection from apoptosis. An alternatively spliced isoform, excluding exon 11, is constitutively active however and over expression is associated with invasive cancer-like behavior (Ghigna et al 2005). In the context of the over 4500 bp clone insert for the Mstr1 transcript, a 147 bp difference would easily be missed without full insert sequence even when tested by restriction digest (figure 2). Figure 1 depicts the abnormal phenotype caused by a common human SNP resulting in sickle cell anemia. Common human SNP results in sickle cell anemiaSingle nucleotide polymorphisms (SNPs) and larger splice variant changes in cloned sequences can be difficult to detect without full insert sequencing, but can significantly impact experimental results. A single nucleotide dramatically alters cellular phenotype as well as a patient’s life span. If left unchecked a cDNA clone bearing this mutation would dramatically alter the outcome an experiment. The biological impact of a single nucleotide change was shown in sickle cell anemia patients (Ingram 1957). A single nucleotide mutation causes dramatic morphological change in cell structure and significantly affects the life span of patients with sickle cell anemia. A point mutation in the β-globin chain of hemoglobin, causes the amino acid glutamic acid to be replaced with acid valine at the sixth position (Ghigna et al 2005) This change could go unnoticed in clones without full insert sequencing.ReferencesPan et al. (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nature Genetics, 40: 1413 - 1415 Ingram VM. Nature. (1957) Gene mutations in human haemoglobin: the chemical difference between normal and sickle cell haemoglobin 180(4581): 326-8.Ghigna et al. (2005) Cell Motility Is Controlled by SF2/ASF through Alternative Splicing of the Ron Protooncogene. Molecular Cell: 20: 881–890. MGC-premier-cDNA-and-ORF-Technical-manual.pdf (723,747kb) MGC-premier-cDNA-and-ORF-Brochure.pdf (466,274kb) |
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