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MGC premier cDNA Clones

The importance of full insert sequencing when working with cDNA and ORFs

All 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 sequencing

It 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 anemia

Single 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. 

Visual validation of pTCP and pTCN expression. TurboGFP was inserted into each expression vector in place of the cDNA.  HEK293T cells were transfected with pTCN expressing tGFP (A), pTCP expressing TurboGFP (B) and a previously validated TurboGFP expression vector (C).

pTCP and pTCN Validation

pTCN and pTCP expression was visually assessed using the fluorescent marker TurboGFP.  TurboGFP was inserted into each expression vector in place of the cDNA.  Cells were treated following the OMNIfect transfection protocol and fluorescence was visualized after 48 hrs.  Expression was compared to a known TurboGFP expression vector.  Representative fields from the biological replicates of each vector are shown in adjacent figure.  All vectors show robust expression.