PMID- 27403566
OWN - NLM
STAT- MEDLINE
DCOM- 20170811
LR  - 20181202
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 112
DP  - 2016 Jun 23
TI  - Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein 
      DNA Binding Peptide.
LID - 10.3791/54141 [doi]
LID - 54141
AB  - Large DNA molecules tethered on the functionalized glass surface have been 
      utilized in polymer physics and biochemistry particularly for investigating 
      interactions between DNA and its binding proteins. Here, we report a method that 
      uses fluorescent microscopy for visualizing large DNA molecules tethered on the 
      surface. First, glass coverslips are biotinylated and passivated by coating with 
      biotinylated polyethylene glycol, which specifically binds biotinylated DNA via 
      avidin protein linkers and significantly reduces undesirable binding from 
      non-specific interactions of proteins or DNA molecules on the surface. Second, 
      the DNA molecules are biotinylated by two different methods depending on their 
      terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' 
      hydroxyl terminus, by terminal transferase, while the sticky ended DNA is 
      hybridized with biotinylated complimentary oligonucleotides by DNA ligase. 
      Finally, a microfluidic shear flow makes single DNA molecules stretch to their 
      full contour lengths after being stained with fluorescent protein-DNA binding 
      peptide (FP-DBP).
FAU - Lee, Seonghyun
AU  - Lee S
AD  - Department of Chemistry and Interdisciplinary Program of Integrated 
      Biotechnology, Sogang University.
FAU - Jo, Kyubong
AU  - Jo K
AD  - Department of Chemistry and Interdisciplinary Program of Integrated 
      Biotechnology, Sogang University; jokyubong@sogang.ac.kr.
LA  - eng
PT  - Journal Article
PT  - Video-Audio Media
DEP - 20160623
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (Peptides)
RN  - 1405-69-2 (Avidin)
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - Avidin
MH  - Biophysical Phenomena
MH  - DNA/*analysis
MH  - Peptides
MH  - Protein Binding
PMC - PMC4993258
EDAT- 2016/07/13 06:00
MHDA- 2017/08/12 06:00
PMCR- 2018/06/23
CRDT- 2016/07/13 06:00
PHST- 2016/07/13 06:00 [entrez]
PHST- 2016/07/13 06:00 [pubmed]
PHST- 2017/08/12 06:00 [medline]
PHST- 2018/06/23 00:00 [pmc-release]
AID - 54141 [pii]
AID - 10.3791/54141 [doi]
PST - epublish
SO  - J Vis Exp. 2016 Jun 23;(112):54141. doi: 10.3791/54141.