Nucleic Acids Research, 2001, Vol. 29, No. 4 996-1004
© 2001 Oxford University Press
Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes
1Department of Chemistry, University of Illinois at Chicago, IL 60607, USA and 2Tm Bioscience Corporation, Toronto, Ontario, Canada
A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T2-biotin·dT-T2 loop. The third base was a biotinylated uracil (UB) necessary for coupling to avidin coated microtiter wells. The capture region of the hairpin was a 3' dangling end composed of either 16 or 32 bases. Fundamental parameters of the system, such as probe density and avidin adsorption capacity of the plates were characterized. The target DNA consisted of 65 bases whose 3' end was complementary to the dangling end of the hairpin or to the linear probe sequence. The assay system was employed to measure the time dependence and thermodynamic stability of target hybridization with hairpin and linear probes. Target molecules were labeled with either a 5'-FITC, or radiolabeled with [
-33P]ATP and captured by either linear or hairpin probes affixed to the solid support. Over the range of target concentrations from 10 to 640 pmol hybridization rates increased with increasing target concentration, but varied for the different probes examined. Hairpin probes displayed higher rates of hybridization and larger equilibrium amounts of captured targets than linear probes. At 25 and 45°C, rates of hybridization were better than twice as great for the hairpin compared with the linear capture probes. Hairpin–target complexes were also more thermodynamically stable. Binding free energies were evaluated from the observed equilibrium constants for complex formation. Results showed the order of stability of the probes to be: hairpins with 32 base dangling ends > hairpin probes with l6 base dangling ends > 16 base linear probes > 32 base linear probes. The physical characteristics of hairpins could offer substantial advantages as nucleic acid capture moieties in solid support based hybridization systems.
* To whom correspondence should be addressed at: Department of Chemistry, Room 4500, University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607-7061, USA. Tel: +1 312 996 0774; Fax: +1 312 996 0431; Email: abenight{at}uic.edu
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  B. C. Satterfield, M. R. Caplan, and J. A. A. West Tentacle probe sandwich assay in porous polymer monolith improves specificity, sensitivity and kinetics Nucleic Acids Res., November 1, 2008; 36(19): e129 - e129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wei, P. F. Kuan, S. Tian, C. Yang, J. Nie, S. Sengupta, V. Ruotti, G. A. Jonsdottir, S. Keles, J. A. Thomson, et al. A study of the relationships between oligonucleotide properties and hybridization signal intensities from NimbleGen microarray datasets Nucleic Acids Res., May 1, 2008; 36(9): 2926 - 2938. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Chen, W. Wang, Z. Wang, F. Wei, and X. S. Zhao Influence of secondary structure on kinetics and reaction mechanism of DNA hybridization Nucleic Acids Res., May 14, 2007; 35(9): 2875 - 2884. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. A. Sekar, W. Bloch, and P. M. St John Comparative study of sequence-dependent hybridization kinetics in solution and on microspheres Nucleic Acids Res., January 14, 2005; 33(1): 366 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Peplies, F. O. Glockner, and R. Amann Optimization Strategies for DNA Microarray-Based Detection of Bacteria with 16S rRNA-Targeting Oligonucleotide Probes Appl. Envir. Microbiol., March 1, 2003; 69(3): 1397 - 1407. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. V. Riccelli, K. E. Mandell, and A. S. Benight Melting studies of dangling-ended DNA hairpins: effects of end length, loop sequence and biotinylation of loop bases Nucleic Acids Res., September 15, 2002; 30(18): 4088 - 4093. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. W. Peterson, R. J. Heaton, and R. M. Georgiadis The effect of surface probe density on DNA hybridization Nucleic Acids Res., December 15, 2001; 29(24): 5163 - 5168. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. E. Broude, K. Woodward, R. Cavallo, C. R. Cantor, and D. Englert DNA microarrays with stem-loop DNA probes: preparation and applications Nucleic Acids Res., October 1, 2001; 29(19): e92 - e92. [Abstract] [Full Text] [PDF]
|
|