sequencing

Experimental Objectives

To demonstrate the validity of Transcriptic’s Autoprotocol-implemented Sequencing Quick Instruction.

Methodology

pBabe-puro plasmid [1]  harboring human K-RAS (isoform B) cDNA was dideoxysequenced by using the Sequencing Quick Instruction.  Sequencing primer was generated using the Oligosynthesis Quick Instruction and designed to anneal immediately 3' of K-RAS, forming a complementary interaction with the sense strand. 

Figure 1 Sequencing primer designed to anneal 3' of the K-RAS cDNA sequence, binding the sense strand.

Figure 1 Sequencing primer designed to anneal 3' of the K-RAS cDNA sequence, binding the sense strand.

Results

Sequencing primer design enabled dideoxysequencing with high-quality chromatogram formation from template DNA.

Figure 2  Dideoxysequence chromatogram acquired using the Sequencing Quick Instruction.  Sequence coverage extended through the K-RAS cDNA sequence. The N-termus of K-RAS and upstream vector sequence is displayed (ATG start codon depicted by arrow).

Figure 2  Dideoxysequence chromatogram acquired using the Sequencing Quick Instruction.  Sequence coverage extended through the K-RAS cDNA sequence. The N-termus of K-RAS and upstream vector sequence is displayed (ATG start codon depicted by arrow).

Figure 3  Quality scores forthe coding sequence of K-RAS.  An average quality score of 58.1 was attained for the 527 bp of K-RAS starting at the 5’ ATG start site. A quality score of 20 is highlighted (red line). Sequencing primer annealed 587-618 bp downstream of the K-RAS start site.

Figure 3  Quality scores forthe coding sequence of K-RAS.  An average quality score of 58.1 was attained for the 527 bp of K-RAS starting at the 5’ ATG start site. A quality score of 20 is highlighted (red line). Sequencing primer annealed 587-618 bp downstream of the K-RAS start site.

 

[1]  Morgenstern JP, Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990;12:3587-96


VALIDATION DATE:  August 2015