oligosynthesis

EXPERIMENTAL OBJECTIVES

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

methodology

Mutagenesis oligonucleotides were designed and ordered from IDT (www.idtdna.com) using the Oligosynthesis Quick Instruction at a 25 nm scale with standard purification.  Oligonucleotides were designed to anneal to the cDNA sequence of human K-RAS (isoform B) and to mutate amino acid 71 in the coding sequence (T211A: Y71N).  Plasmids harboring K-RAS cDNA underwent sequence-independent, liagation-indpendent cloning (SLIC) [1]  using the designed oligonucleotide primers to generate a single basepair mutation and create viable plasmid constructs.  Subsequent to cloning, plasmids were transformed into competent bacteria which were then cultured before plasmid DNA extraction and dideoxysequencing. 

primer design

Figure 1 The Oligosynthesis Quick Instruction was performed in order to create sequencespecific mutagenesis primers complementary to the sense and antisense strands of K-RAS cDNA, incorporating a single base-pair substitution at position 211.

Figure 1 The Oligosynthesis Quick Instruction was performed in order to create sequencespecific mutagenesis primers complementary to the sense and antisense strands of K-RAS cDNA, incorporating a single base-pair substitution at position 211.

results

The SLIC protocol dictated the use of oligonucleotides for long-range PCR (~2.5 Kbp) and subsequent to T4 polymerase treatment (formation of sticky ends) then annealing, viable mutant plasmids were created.  The Oligosynthesis Quick Instruction instigated the generation of high quality (mass spectroscopy and capillary electrophoresis-verified) oligonucleotides.

Figure 2   Sequence validation of successful mutagenesis.  Top: in silico predicted human K-RAS sequence (ensembl.org).  Middle: control (non-mutated) K-RAS cDNA sequence.  Bottom: primer-derived mutagenesis of K-RAS (highlighted in red).

Figure 2   Sequence validation of successful mutagenesis.  Top: in silico predicted human K-RAS sequence (ensembl.org).  Middle: control (non-mutated) K-RAS cDNA sequence.  Bottom: primer-derived mutagenesis of K-RAS (highlighted in red).

 

[1]  Li MZ, Elledge SJ. SLIC: a method for sequence- and ligation-independent cloning. Methods Mol Biol.2012;852:51-9


VALIDATION DATE:  August 2015