Solid-phase genetic engineering with DNA immobilized on a gold surface.
A novel method for immobilizing large DNA fragments on a solid surface was developed. A mixed self-assembled monolayer of thiolated single-stranded DNA with inert alkanethiol was generated on a gold (Au) surface through the Au-S reaction. Surface-tethered DNA generated by this method was compatible with various genetic engineering techniques, including hybridization, polymerization, restriction enzyme digestion and ligation. Kinetic control of surface coverage of immobilized DNA was critical for optimizing genetic engineering techniques on solid-phase. Multi-step reaction schemes utilizing various genetic engineering techniques described above were employed for solid-phase gene assembly. We were able to immobilize DNA fragments of up to 1180 bp on a solid surface. Furthermore, we showed that these immobilized genes can be regenerated by PCR. The present work suggests that these types of assembled genes can be used to store and regenerate genes on solid-phase.
Kim
Jeong Hee
JH
Department of Oral Biochemistry, Kyung Hee University, Seoul 130-701, South Korea. jhkimh@khu.ac.kr
Hong
Jung-A
JA
Yoon
Myungok
M
Yoon
Moon Young
MY
Jeong
Han-Seung
HS
Hwang
Hyun Jin
HJ
D001483:Base Sequence / Q000502:physiology; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000493:pharmacokinetics; D005818:Genetic Engineering / Q000379:methods*; D006046:Gold / Q000737:chemistry*; D008956:Models, Chemical; D009841:Oligonucleotides / Q000378:metabolism*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013499:Surface Properties
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D006046:Gold