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Light-directed 5'-->3' synthesis of complex oligonucleotide microarrays
Thomas J Albert1, Jason Norton, Markus Ott
1NimbleGen Systems Inc., One Science Court, Madison, WI 53711, USA. talbert@nimblegen.com
Nucleic Acids Research
|March 26, 2003
Summary
This study introduces 5'-->3' array synthesis for enhanced enzymatic modification. This advancement enables high-throughput genotyping and resequencing directly on microarrays, reducing costs and increasing efficiency.
Area of Science:
- Biotechnology
- Molecular Biology
- Genomics
Background:
- Current light-directed microarray synthesis (3'-->5') limits enzymatic probe modification.
- This limitation hinders applications like parallel genotyping and resequencing on arrays.
Purpose of the Study:
- To demonstrate 5'-->3' light-directed microarray synthesis using photoprotected phosphoramidite monomers.
- To enable direct enzymatic modifications on array probes for advanced genomic applications.
Main Methods:
- Utilized maskless array synthesis technology with photoprotected phosphoramidite monomers.
- Synthesized high-density microarrays in the 5'-->3' direction.
- Performed hybridization experiments with labeled mouse cRNA.
Main Results:
- Achieved high-density microarrays (>150,000 probes) synthesized in the 5'-->3' direction.
- Demonstrated array performance: dynamic range >2.5 orders of magnitude, sensitivity <1 pM, CV <10%.
- Confirmed 3' probe end availability for sequence-specific primer extension and ligation reactions.
Conclusions:
- The developed 5'-->3' synthesis method overcomes limitations of traditional 3'-->5' synthesis.
- Enables direct enzymatic modifications on array probes, facilitating high-throughput genotyping and resequencing.
- Offers a cost-effective and efficient platform for genomic analysis.