Light-directed 5'-->3' synthesis of complex oligonucleotide microarrays.
Light-directed synthesis of high-density microarrays is currently performed in the 3'-->5' direction due to constraints in existing synthesis chemistry. This results in the probes being unavailable for many common types of enzymatic modification. Arrays that are synthesized in the 5'-->3' direction could be utilized to perform parallel genotyping and resequencing directly on the array surface, dramatically increasing the throughput and reducing the cost relative to existing techniques. In this report we demonstrate the use of photoprotected phosphoramidite monomers for light-directed array synthesis in the 5'-->3' direction, using maskless array synthesis technology. These arrays have a dynamic range of >2.5 orders of magnitude, sensitivity below 1 pM and a coefficient of variance of <10% across the array surface. Arrays containing >150,000 probe sequences were hybridized to labeled mouse cRNA producing highly concordant data (average R(2) = 0.998). We have also shown that the 3' ends of array probes are available for sequence-specific primer extension and ligation reactions.
Albert
Thomas J
TJ
NimbleGen Systems Inc., One Science Court, Madison, WI 53711, USA. talbert@nimblegen.com
Norton
Jason
J
Ott
Markus
M
Richmond
Todd
T
Nuwaysir
Kate
K
Nuwaysir
Emile F
EF
Stengele
Klaus-Peter
KP
Green
Roland D
RD
D015394:Molecular Structure; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000528:radiation effects*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D014466:Ultraviolet Rays
D009841:Oligonucleotides