Highly parallel oligonucleotide purification and functionalization using reversible chemistry.
We have developed a cost-effective, highly parallel method for purification and functionalization of 5'-labeled oligonucleotides. The approach is based on 5'-hexa-His phase tag purification, followed by exchange of the hexa-His tag for a functional group using reversible reaction chemistry. These methods are suitable for large-scale (micromole to millimole) production of oligonucleotides and are amenable to highly parallel processing of many oligonucleotides individually or in high complexity pools. Examples of the preparation of 5'-biotin, 95-mer, oligonucleotide pools of >40K complexity at micromole scale are shown. These pools are prepared in up to ~16% yield and 90-99% purity. Approaches for using this method in other applications are also discussed.
Illumina, Inc., Advanced Research Group, San Diego, CA 92121, USA.
Kerri T
KT
York
Ryan C
RC
Smith
Rob
R
Yang
Peter C
PC
Melnyk
Melissa M
MM
Wiley
Casey M
CM
Turk
Mostafa
M
Ronaghi
Kevin L
KL
Gunderson
Frank J
FJ
Steemers
D019921:Biotinylation; D060326:Chemistry Techniques, Synthetic; D002853:Chromatography, Liquid; D006639:Histidine / Q000737:chemistry; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification*; D009842:Oligopeptides / Q000737:chemistry
C471213:His-His-His-His-His-His; D009841:Oligonucleotides; D009842:Oligopeptides; D006639:Histidine