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Oligonucleotide Purity and Purification Method

What type of oligonucleotide purity and purification method I need for my applications?

UltraPure Double Purification

By using a combination of an high quality synthesis and purification process developed in house and double purification provide purity beyond 95% for oligo between 8 to 150 bases.

 >95%
  • Gene synthesis > 80 bases
  • NMR, X-ray crystallography

PAGE Purification

Using the principal of molecular weight and charge group, denaturing acrylamide gel electrophoresis are used to provide higher degree of purity than HPLC (93-95%). this is recommended for purification of oligonucleotides longer than 80 bases.

 85-95%
  • Production of cloning linkers
  • Site-directed mutagenesis
  • Cloning and subcloning PCR
  • Gene synthesis < 80 bases
  • Gel-shift assay
  • Special modifications ( G-clamp)
  • mrRNA, siRNA and antisense

Ie-HPLC Purification

Typical yields a product of 80-90% purity. The capacity and resolving properties of HPLC columns are also much greater than cartridge devices, so HPLC is the method of choice for purifying larger quantities of oligos (i.e. >=1 umol). As with cartridges, reverse-phase HPLC is usually not recommended for purifying oligos longer than 60 bases.

 85-95%
  • First-strand cDNA synthesis
  • In situ hybridization
  • Real-Time PCR
  • Gel-shift assay
  • Non-radioactive labeling
  • Antisense
  • First-strand cDNA synthesis for generation of libaries

Reverse-Phase (RP) purification

By using the difference in hydrophobicity between the full-length product with a 5' DMT group and truncated sequences (without DMT groups). The RP-cartridge method is typically used for oligo up to 39 bases and RP-LP cartridge use for oligonucleotide between 40 and 80 bases. Thus giving 75-85% purity for oligos up to the 1 umole scales.

 75-85%
  • AFLP
  • OLA
  • Sensitive PCR (Diagnostic)
  • Classical modifications (modified bases, chemical linkers...)

Desalting

At Bio-Synthesis, every oligo is desalted free of charge. Desalting removes residual by-products from the synthesis, cleavage, and deprotection procedures. Desalting is fine for oligos <=30 bases; the overwhelming abundance of full length oligo outweighs any contributions from shorter oligos.

 70-75%
  • Isothermal sequencing
  • Cycle Sequencing
  • Routine PCR
  • Hybridization
  • DNA MicroArray
  • SNP Analysis
  • AFLP Analysis