Reagents Remove Fluorescent Groups and Terminators to Restore Polynucleotides to 3’-OH Form
Tris(3‐hydroxypropyl)phosphine [THPP or THP(15-6375)C] CAS number4706-17-6 and Tris(2‐carboxyethyl)phosphine hydrochloride [TCEP (15-7400)] CAS number 4706-17-6are water soluble, neutral, transparent compoun4706-17-6ds. They can be used as reagents in certain automated 2nd generation massive parallel gene sequencing machines for reversible terminator chemistry.1
In such machines, DNA fragments are immobilized on supports and amplified by surface-bound polymerase chain reaction (PCR) chemistry. They are then denatured and primed to form support-bound bundles of identical single strand DNA molecules. Millions of bundles, each from a different fragment, form within the channels of a flow cell.
Replication is conducted one nucleotide unit at a time by first flowing a mixture of all four nucleotides. Each nucleotide is labelled with a different fluorophore, as modified 3′-O-azidomethyl reversible terminators, containing cleavable 1-azido-1- alkoxy-acyclic units at specific locations. The location and identity of each added nucleotide unit is determined by recording the emission and wavelength of each fluorophore bundle. The fluorescent groups and terminators are then removed to restore the polynucleotides to their active 3’-OH form. This is accomplished by azide cleavage via Staudinger reduction with Tris(3‐hydroxypropyl)phosphine (15-6375) or Tris(2‐carboxyethyl)phosphine (15-7400), forming phosphine oxide and transient amine intermediates. The DNA bundles are now ready for the addition of a second nucleotide unit.2-4
Disulfide reducing agents for molecular diagnostics (THPP, TCEP) in gene chips
1. WO 2004018493, (2004) to Solexa, Ltd
2. M. L. Metzker, Nature Reviews, Genetics. 11, 51(2010).
3. A. N. Egan, J. Schlueter, D. M. Spooner, American Journal of Botany 99 (2): 175–185 (2012).
4. For Staudinger reduction, see Wikipedia entry, or Staudinger, Helv. Chim. Acta 2 635 (1919).
Products mentioned in this blog:
15-6375: Tris(3-hydroxypropyl)phosphine, min. 80%, [4706-17-6]
15-7400: Tris(2-carboxyethyl)phosphine, hydrochloride, 99% TCEP, [51805-45-9]