Technical Description

Oligonucleotide probes provide a useful method for the detection of target nucleic acids by the formation of a double-helical structure between complementary sequences based on stringent requirements of Watson-Crick base pairing, making hybridization extremely specific. First described by Kool in 1991,[1] the padlock probe or C-probe is a uniquely designed oligonucleotide probe containing three regions: two target complementary sequences located at the 5' and 3' termini and an interposed generic linker region.[2,3] Once the padlock probe hybridizes to a target, its 5' and 3' ends are juxtaposed (Fig. 1A) and a closed circular molecule is then generated by incubating the padlock probe-target complex with a DNA ligase. The resulting closed circular molecule is locked on its target due to the helical turns formed between the complementary sequences of the target and the padlock probe.[2] The permanently bound padlock probe allows for stringent washing to remove unbound components, thereby enhancing assay signal-to-noise ratios. Therefore, the advantage of the padlock probe is that it offers greater specificity and less background when compared to current conventional probes. Furthermore, once the padlock probe hybridizes to a specific target sequence, it can then serve as a template for linear (rolling circle amplification, RCA) or exponential (ramification amplification method, RAM) amplification and significantly increases the specificity and sensitivity of signal detection.

Recently, two interesting methods were presented for catenation of padlock probes to double-stranded DNA (dsDNA) sequences. Escude et al.[4] demonstrated the formation of a triplex complex between a dsDNA and a circularizable probe through the interaction between polypurine-polypyrimidine sequences in the dsDNA target and padlock probe. The advantage of the triplex padlock probe is that it allows for binding of the padlock probe to dsDNA without predenaturation. Kuhn and colleagues designed two peptide nucleic acid (PNA) probes that are complementary to the opposite strand of padlock probe binding site.[5] These PNA ''openers'' can invade the dsDNA and may facilitate binding of the padlock probe to target DNA. Furthermore, they prevent the padlock probe from shifting laterally, resulting in more specific localization of the signal intracellularly.

The padlock probe ranges from 70 to 125 nucleotides long with two target complementary regions, each ranging from 15 to 30 nucleotides, and a linker region ranging from 30 to 75 nucleotides.[2,3,6,7] The padlock probe can be readily synthesized with most DNA synthesizers. The 5' end of the probe must be phosphorylated and the 3' end must contain a hydroxyl group; both are required by DNA ligase. Addition of a phosphate group to the 5' end is usually achieved by incubation with a polynucleotide kinase in the presence of ATP or during chemical synthesis.[8]

Successful ligation of the two ends depends on two factors: both ends must bind adjacent to each other and no mismatch should exist at the ends, especially the first 1-3 nucleotides at its 5' and 3' ends (Fig. 1B).[2,8] A variety of ligases can be used for this reaction, such as T4 DNA ligase, Taq ligase, Ampligase, and T4 RNA ligase.[9] However, ligation efficiency can reach as high as 90% with T4 DNA ligase.[5,8] Furthermore, Nilsson et al.[2] demonstrated the ligation specificity of T4 DNA ligase depended on increasing the NaCl concentration. Whereas NaCl concentrations between 50 and 150 mM result in optimal ligation activity, at 250 mM NaCl T4 DNA ligase was shown to correctly distinguish a C/G match from notoriously difficult T/G mismatches, resulting in a greater than 1000-fold difference in ligation rates. Although T4 DNA ligase is efficient for ligation of DNA probe onto a DNA target, ligation of a DNA probe

Fig. 1 Schematic representation of the padlock probe. (A) A padlock probe hybridizes to its target through its complementary regions and helical turns to form a closed circular probe. This results in the interlocking of the padlock probe onto the target. (B) Only when the ends of the padlock probe perfectly match with its target can ligation occur, thus allowing detection of a single-nucleotide polymorphism.

Fig. 1 Schematic representation of the padlock probe. (A) A padlock probe hybridizes to its target through its complementary regions and helical turns to form a closed circular probe. This results in the interlocking of the padlock probe onto the target. (B) Only when the ends of the padlock probe perfectly match with its target can ligation occur, thus allowing detection of a single-nucleotide polymorphism.

onto an RNA target is very poor.[8] However, the ligation efficiency can reach 80% by addition of MnCl2, low NaCl concentration, low ATP concentration, and high concentration of T4 DNA ligase.[8,10] The stringent requirement of ligation allows detection of single nucleotide differences between target DNA and probe (Fig. 1B).

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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