Oligo Purification
VH Bio provides several purification options:
- Desalting. This is used as a standard purification for 1.0µmole syntheses.
- Reverse Phase Cartridge. RPC removes salt and failure sequences from the synthesised oligo. The oligo is synthesised with the final trityl group on so that the full-length oligo retains the trityl group. The full-length oligo remains on the column as the failure sequences flow through. The trityl group is then removed and the full length product eluted. We recommend this purification for oligos up to 50 bases.
- HPLC Purification. Reverse Phase High Performance Liquid Chromatography is used to remove failure sequences and unincorporated label in a similar way to RPC. Full-length product with a trityl group attached is retained on the column while failure sequences or unlabelled primers flow through. Full length product is then eluted by slowly increasing the solvent concentration. An analysis sheet showing the oligo peak is supplied for each oligo. HPLC purification is recommended for oligos up to 60 bases.
- Gel Purification. The efficiency of HPLC decreases rapidly for oligos over 60 bases in length. As a result of our co-operation with Elchrom Scientific AG, we are able to offer gel purification using proprietary gels based on Elchrom’s renowned Spreadex Gels. The resulting ultra-purified oligos are, we believe, the highest quality product available. We recommend this purification for all oligos longer than 60 bases.
Modifications
VH Bio offers a wide range of modifications, the most commonly used of which are listed below. If you do not see your desired modification, please contact our Customer Services on +44 (0)191 495 8213.
Amino Modifiers
Amino modifiers are used to introduce a primary amino group into an oligo. Several modifiers are available for selection according to individual requirements. VH Bio uses Amino Modifier C6 for standard 5’-labelling. This modification contains a primary amino group at the end of a six-carbon spacer. For 3’-labelling we use a branched seven-carbon spacer.
Biotin
Biotin is used to link an oligo to streptavidin-protein conjugates, labelled streptavidin or streptavidin affinity columns.
Degenerate Oligonucleotides
Mixed bases (wobbles) which are inserted internally on an equimolar basis are provided at no extra charge. Wobbles at the 3’ end, or doped oligos with wobbles present in non-equimolar proportions are available for an extra cost.
Deoxyuridine
Deoxyuridine contains the base uracil, which behaves very much like thymine. Uracil-N-glycosylase can specifically remove uracil, creating base-less sites at the dU positions. This property can be used to create specific strand breaks in a DNA structure.
Digoxygenin
Digoxygenin labelling is offered at any position on the oligonucleotide, via the appropriate amino linkage. We recommend gel purification for this modification. Digoxygenin-labelled oligos are used as hybridisation probes for diagnostics, sequencing, blot applications and in situ hybridisation.
Fluorescent Dyes
Fluorescent dye-labelled oligos are used routinely in automated DNA sequencing with fluorescent detection. They are also useful in PCR quantification and as probes for in situ hybridisation. Fluorescent dyes are normally attached at the 5’ end, although we can also provide 3’ attachment as well. They are routinely available on the 40nmole and 200nmole synthesis scales. We recommend HPLC purification for these modifications.
The following dyes are routinely available:
| Dye |
Excitation (nm) |
Emission (nm) |
| FITC |
494 |
518 |
| FAM |
492 |
515 |
| HEX |
535 |
556 |
| TET |
521 |
536 |
| Cy3 |
552 |
570 |
| Cy5 |
643 |
667 |
| IRD700 |
685 |
705 |
| IRD800 |
787 |
807 |
| TAMRA |
544 |
576 |
| Rhodamine |
505 |
527 |
| Texas Red |
583 |
603 |
Fluorogenic Quenching Probes
Fluorogenic Quenching Probes are oligonucleotides labelled with a 5’-FAM, HEX or TET and a TAMRA attached to phosphorylated nucleotide at the 3’–terminus. The probe is phosphorylated to prevent probe extension during a PCR reaction.
If the target of interest is present, the probe anneals between the forward and reverse primer sites. Taq DNA polymerase will cleave double-stranded DNA, but not free probe. During a PCR, therefore, the probe is digested, the shortened probe dissociates from the target and polymerisation continues. This cleavage results in an increase in the fluorescence of the 5’-dye which is proportional to the amount of product accumulated.
Specificity is maintained by the fact that the cleavage only occurs if the probe hybridises with the target sequence, which is subsequently amplified in the PCR. No signal is generated by non-specific amplification.
Fluorogenic Quenching Probes are available HPLC purified on the 40nmole, 0.2µmole and 1.0µmole scales.
Inosine
Inosine is a deoxynucleoside which contains the base hypoxanthine. When used in a hybridisation probe, deoxyinosine can form weak base pairs with dA, dC, dG or T residues. It can, therefore, be used instead of wobbles, having the advantage that the hybridisation probe is not diluted by the non-pairing components of the wobble.
Molecular Beacons
Molecular Beacons are hairpin-shaped molecules labelled with a 3’-dabcyl, which act as a quencher, and a 5’-fluorophore which acts as reporter. The most common fluorophores are Fluorescein, TAMRA or Texas Red. The probes become fluorescent when bound to their targets due to the separation of the fluorophore and quencher.
Molecular Beacons can be used to measure the quantity of target sequences and to distinguish single nucleotide differences in their targets. These probes are useful in quantitative PCR as well as in allele discrimination.
They are available HPLC purified on the 0.2µmole and 1.0µmole scales.
Phosphate
Oligonucleotides require a 5’-phosphate for ligation by a ligase to occur. Phosphorylated oligos can also be used to alter the susceptibility of a sequence to hydrolysis by an exonuclease. VH Bio offers phosphate modifications at either the 5’- or 3’-terminus on the 40nmole, 0.2µmole or 1.0µmole scale.
Phosphorothioate DNA
A phosphorothioate group is a modified phosphate in which one of the oxygen atoms is replaced by sulphur. In a phosphorothioated oligo, some, or all, of the internucleotide phosphates are replaced by phosphorothioate groups. The modified backbone of the phosphorothioated DNA is resistant to the action of most nucleases. Such oligos are used in anti-sense studies because of their enhanced stability.
VH Bio offers phosphorothioate sites between all residues or at specific locations within a sequence. A useful option is to insert them between the last few residues at each end of the oligo. This produces an oligo which is resistant to exonucleases but which has a natural DNA centre.
The oligos are provided HPLC purified on the 0.2µmole and 1.0µmole scales.
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