Capture probes

Probes that mediate capture of the target nucleic acid are termed capture extenders. These probes are approximately 50 bases, one portion (20 to 40 bases) of which is complementary to the target, while the second portion (approximately 20 bases) binds the probe-target complex to a capture probe that is coupled to the surface of a microtiter plate well. 

An alkylamine linker is incorporated into either the 5 or 3 end of the capture probe and the probe is activated with ethylene glycol bis-succinimidylsuccinate. The activated capture probe is bound to polystyrene microtiter wells precoated with poly(Lys-HBr, Phe). Nearly all of the solid-phase probe coupled to the polystyrene by this procedure is available for hybridization (Running and Urdea, 1990). 

ED Targel probes which bind to the capture probes 1 I T argel probes which bind to the preamp probes... 

The molecular sensitivities of the first and second generations of the bDNA assays were limited by nonspecific hybridization between the amplification probes and other nucleic acids. Short regions of hybridization between any of the probes constituting the amplification system, (preamplifier, amplifier, and labeled probe) and any nontarget nucleic acid sequence leads to amplification of the background signal. Capture probes, capture extenders, and sample nucleic acid are all sources of this background hybridization (Collins et al 1997). 

Indeed, a bDNA assay for diagnosis of African trypanosomiasis was developed and compared with buffy coat microscopy for detection of T brucei in human blood samples (Harris etal., 1996). Two repetitive DNA sequences found only in the T. brucei complex, a 177-bp satellite repeat and the ribosomal mobile element, were selected as targets in the bDNA assay. The assay used the standard bDNA components capture probes, target probes, amplifier molecules, and alkaline phosphatase-labeled probes. Various blood fractions and sample preparation methods were examined. Ultimately, buffy coat samples resulted in the highest sensitivity. Although typanosomes do not infect leukocytes, they cosediment with them. 

Preliminary results of DNA sequence detection using the OFRR are thoroughly described by Suter, et al.32 The experimental setup described above for tracking the resonant mode shift is used. An OFRR with RI sensitivity of about 7 nm/RIU was produced and used for these experiments. The OFRR surface was functionalized with 3APS and then DMA was used as an amine-amine crosslinker. The single-stranded oligonucleotide capture probe was synthesized with an amine functional group connected to the 5 end by a 6-carbon linker, and has a 25 base-pair sequence. ... 

All DNA hybridization assays are subject to cross-hybridization, in which an oligonucleotide that is not a perfect sequence match hybridizes with the capture probe. The cross-hybridization in the OFRR was investigated using samples of oligonucleotides with either 0-, 1-, 2-, 5-, or 25-base mismatches when compared with the 25 base-pair biorecognition capture probe. The resulting resonant mode spectral shifts for the respective mismatch are plotted in Fig. 14.7b. The measurements show a difference of 1.3 pm and 2.8 pm for one and two base-pair... 

Fig. 14.7 (a) Measured resonant mode spectral shift for increasing concentrations of target oligonucleotides. The line fit is a Michaelis Menton curve with a Kd of 2.9 nM and a maximum resonant wavelength shift of 5.1 pm. (b) Measured resonant mode shift for oligonucleotides of increasing number of base pair mismatches with the capture probe. Reprinted from Ref. 32 with permission. 2008 Elsevier... 

Degree to which capture probes Can be localized to the point of maximal electromagnetic intensity... 

Fig. 16.10 Reaction scheme for immobilization of DNA onto functionalized Si02 substrates. Plasma treated Si02 substrates are denoted as Si OH, APTMS functionalized substrates are denoted as Si NH2, dendrimer functionalized substrates are denoted as Si G(4.5)COOH, and substrates to which DNA capture probes have been immobilized are denoted as Si DNA. Inset Repeat unit of PAMAM dendrimer possessing terminal carboxylic acid functionality...

Hybridization can also be performed in solution phase. Since the capture probe is in solution, the kinetics of hybridization is faster than when the capture probe is immobilized. In the solution phase hybridization format, the capture probe is labeled with an affinity label such as biotin that captures the sample target sequence. The labeled probe then binds to the sample target sequence to form the sandwich. After the hybridization is complete, the sandwich is transferred to an affinity support such as avidin or streptavidin, which will capture the sandwich through the biotin-labeled capture probe. Sandwich hybridization performed in solution followed by capture on an affinity support has been referred to as affinity-based hybrid collection (Kl). 

Site-Selective Attachment of Capture Probes onto Chemical Templates. .. 107... 

SAMs of thiols (and thiol-modified ONDs) can be highly ordered and densely packed, improving the availability of active surface groups (and capture probes) for binding and hybridization. However, dense packing may not always be advantageous in DNA hybrid capture. Steric hindrance effects can occur if the immobilized capture probes are too close together. 

Nitrocellulose and nylon membranes have been widely used in the production of macroarrays (arrays with probe sites of diameter 0.5 to 1 mm), but not so much in the production of microarrays (feature size of 25 to 200 xm) because of a lack of spot resolution (see Sect. 5.2, Spotting of Capture Probes). These membranes exhibit lateral wicking characteristics and the probe therefore tends to spread out from the point of apphcation. Casting of these membranes onto the surface of glass slides is a solution to this problem [28]. 

Membranes have been developed that possess improved characteristics with respect to their lateral wicking and spot resolution. The best example of this type of material is anodically oxidized alumina whose structure consists of pores, with very little material forming the walls, possessing a surface area ratio of approximately 500 f. The benefits of this material are its higher sensitivity (more immobilized capture probe) and higher probe densities. 

A multiplexed immrmoassay has likewise been introduced based upon the ArrayPlate format in which antibody-oligonucleotide conjugates are assembled by hybridization to complementary capture probes. Beckman Coulter introduced the A MicroArray System in March 2004. The A squared or A (array of arrays) approach involves the printing of a capture oligonucleotide "zip code" in the bottom of a rounded square 96-well... 

Figure 3.5 Comparison of adsorptive and covalent attachment of capture probes. (From Belosludtsev, Y. et al.. Anal. Biochem., 292, 250-256, 2001. With permission.)...

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