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Oligonucleotide quality requirements for mutagenesis protocols

For mutagenesis applications quality of the oligonucleotide primers is critical. Impure oligonucleotides can adversely affect reaction efficiency and can introduce additional, undesired mutations. IDT monitors every custom synthesis reaction on every synthesis platform and maintains a basecoupling efficiency that is higher than the industry standard. IDT has also pioneered the use of high-throughput quality control (QC) methods and is the only oligonucleotide manufacturer that offers 100% QC and purity guarantees. QC documents are even made available to customers.

IDT also evaluates product quality in comparison to competitor products—IDT oligonucleotides consistently rank as the purest. This exceptional oligonucleotide quality reduces downstream processing costs, such as assembly and sequencing, and lowers the overall cost of generating sequences that carry mutations.

In addition to comparing for purity, IDT tests its oligonucleotides against those from competitors in functional studies. A performance test examined primers used for site-directed mutagenesis (SDM). Four pairs of SDM primers were ordered from four different companies (including IDT). These primer sets were designed to introduce the following changes:

  • Set 1—Single base change C to G (40mers)
  • Set 2—Random 20 bp mutagenesis (60mers)
  • Set 3—Addition of a 20 bp section of the repetitive element GGT (60mers)
  • Set 4—Deletion of a 20 bp section (60mers)

These oligonucleotides were used in parallel SDM experiments, and resulting clones were screened by IDT scientists. The data from the cumulative cloning experiments show that, in every case, using IDT oligonucleotides led to better mutagenesis results (Table 1).

          Table 1. Correct Mutants From 8 Colonies Tested for Each Set of Oligos.

Free Mutagenesis Application Guide

The IDT Mutagenesis Application Guide contains an overview of various in vitro mutagenesis approaches, describes the use of long oligonucleotides called Ultramer™ Oligonucleotides to simplify mutagenesis experiments, gives two protocols for general site-directed mutagenesis, and provides an extensive troubleshooting section. Download in ePub or PDF format, or request a copy today.

Author: Jaime Sabel is a Scientific Writer at IDT.

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