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Home » Oligo Synthesis Applications » Custom DNA Oligo Synthesis » Oligo Purification

Overview

GenScript oligonucleotides produced with state-of-the-art oligo-synthesizers are of high purity with minimum truncated sequences and salt residuals.

However, improperly synthesized oligonucleotides from incomplete synthesis cycles and protecting groups cleaved from the bases after synthesis may exist in the final oligonucleotide samples in addition to the full length products. There are different methods for removing these impurities and key considerations in selecting the proper purification approach include the evaluation of the probable degree of contamination and the possible effects of these contaminants for downstream applications and experiments.

Purification steps are used for

Number one
Separation of full-length n-mers from incomplete products (n-1 mer, n-2 mer, et al).
Number two
Removal of oligonucleotides resulting from incomplete deprotection, depurination, dimerization, branching, et al.
Number three
Removal of cleaved blocking groups from bases.
Oligo Synthesis

GenScript Purification Options

Desalt

Purification with reverse-phase silica gel column which can effectively remove salts. The purity can meet the needs of most molecular biology experiments.

Advantages

  • Fast
  • High throughput
  • Cost-effective

Applications

PCR amplification, gene synthesis, DNA sequencing, NGS capture probes, NGS Multiplex PCR oligos

HPLC

The purification of oligo with HPLC can achieve high purity and is mainly used for short chain (< 40 nt) and modified oligo.

Advantages

  • Purity can reach > 90%
  • Remove N-1 short fragment effectively
  • Purification for modified oligo with hydrophobic groups

Applications

Point mutation, subcloning, diagnostic PCR, protein binding gel migration electrophoresis analysis, modified primers with hydrophobic groups.

PAGE

Using denaturing polyacrylamide gel electrophoresis, oligo of different molecular weights migrate at different speeds during electrophoresis, allowing for separation. Finally, the target DNA is recovered from the gel.

Advantages

  • Purity can be > 90%
  • Effective for purification of long-stranded oligo( 151 – 200 nt )

Applications

Mutation libraries, protein binding gel migration electrophoresis analysis, modified oligos such as phosphate and biotin

ePAGE

Utilizing oligonucleotide purification columns and other methods to remove short sequences and impurities from the product. By using automated equipment, DNA purification and recovery can be completed efficiently in batches.

Advantages

  • Fast, cost-effective
  • Similar purity as PAGE within 40 nt

Applications

  • Multiplex PCR, point mutation, gene synthesis
HPLC+ (Special for MDx)

HPLC purification done in a clean room. Before purification of each oligo, columns are thoroughly cleared of residues by multiple special rinsing procedures, and different oligos are kept reasonably spaced apart and purified separately to avoid exogenous contamination.

Advantages

  • Strict control of exogenous contamination
  • No NTC peaks within 40 cycles guaranteed

Applications

  • TaqMan probes, qPCR oligos and commercial diagnostic oligo
  • Suitable for large-scale production for ＞100 nmol per sequence
PAGE+ (Special for MDx)

PAGE purification done in a clean room.The target sequences are separated by denaturing polyacrylamide gel electrophoresis, and each oligo is purified with an independent purification system and disposable consumables to avoid the possibility of mixing with other oligos.

Advantages

  • Cross-contamination rate as low as 0.01%

Applications

  • Commercial diagnostic oligos ( 151 – 200 nt )
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Purification Recommendations

Purification Methods Desalt ePAGE PAGE* HPLC PAGE+* HPLC+
NGS adapters Recommended
NGS capture probes Recommended
NGS multiplex PCR oligo Recommended
TaqMan probes, qPCR oligos Recommended
molecular biology experiments (PCR Primer/sequencing …) Recommended
<10 nt Suitable Not suitable Not suitable Suitable Not suitable Suitable
10-39 nt Recommended Recommended Not Suitable Recommended Not Suitable Recommended
40-59 nt Suitable Suitable Not Suitable Recommended Not Suitable Recommended
60-150 nt Suitable Not suitable Not suitable Recommended Not Suitable Recommended
151 – 200 nt Suitable Not suitable Recommended Suitable Recommended Suitable
Modifications dI, dU, C3/6/9/12/18 etc. dI; dU; LNA(ACGT); invent dT; dspacer ; spacer C3,C6,C9; S；5‘Biotin dI, dU, C3/6/9/12/18 P, S, dI, dU, LNA etc. dI, dU, C3/6/9/12/18 P, S, dI, dU, LNA CY5, BHQ, VIC etc. dI, dU, C3/6/9/12/18 P, S, dI, dU, LNA etc. dI, dU, C3/6/9/12/18 P, S, dI, dU, LNA CY5, BHQ, VIC etc.

# GenScript recommends PAGE/PAGE+ purification for primers of 151-200 nts. For PAGE-purified primers shorter than 150 nt, if you prioritize cost-effectiveness and faster turnaround times, you can choose ePAGE; if you prioritize higher purity, you can choose HPLC or HPLC+.

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