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Isothermal amplification methods for the detection of nucleic acids in microfluidic devices.

Zanoli LM, Spoto G - Biosensors (Basel) (2012)

Bottom Line: The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device.In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features.Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed.

View Article: PubMed Central - PubMed

Affiliation: Istituto Biostrutture e Bioimmagini, CNR, Viale A. Doria 6, Catania, Italy; E-Mail: lzanoli@unict.it.

ABSTRACT
Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed.

No MeSH data available.


Schematic representation of helicase-dependent amplification (HDA) amplification process.
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biosensors-03-00018-f002: Schematic representation of helicase-dependent amplification (HDA) amplification process.

Mentions: A scheme of HDA mechanism is shown in Figure 2. It mimics the replication fork and enables DNA synthesis to occur by using chemical energy [61]. The helicase enzyme in the presence of ATP loads on to the dsDNA template and traverses along the target DNA, disrupting the hydrogen bonds linking the two strands. The formed single strand (ss) DNAs are then coated by single-stranded binding proteins (SSBs; Figure 2, step 1). Two sequence-specific primers hybridize to the 3'-end of each ssDNA template (Figure 2, step 2). DNA polymerases extend primers annealed to the target by producing dsDNA (Figure 2, step 3). The two newly synthesized dsDNA products act then as substrates for DNA helicases in the next round of the reaction (Figure 2, step 4), resulting in an exponential amplification of the selected target sequence.


Isothermal amplification methods for the detection of nucleic acids in microfluidic devices.

Zanoli LM, Spoto G - Biosensors (Basel) (2012)

Schematic representation of helicase-dependent amplification (HDA) amplification process.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263587&req=5

biosensors-03-00018-f002: Schematic representation of helicase-dependent amplification (HDA) amplification process.
Mentions: A scheme of HDA mechanism is shown in Figure 2. It mimics the replication fork and enables DNA synthesis to occur by using chemical energy [61]. The helicase enzyme in the presence of ATP loads on to the dsDNA template and traverses along the target DNA, disrupting the hydrogen bonds linking the two strands. The formed single strand (ss) DNAs are then coated by single-stranded binding proteins (SSBs; Figure 2, step 1). Two sequence-specific primers hybridize to the 3'-end of each ssDNA template (Figure 2, step 2). DNA polymerases extend primers annealed to the target by producing dsDNA (Figure 2, step 3). The two newly synthesized dsDNA products act then as substrates for DNA helicases in the next round of the reaction (Figure 2, step 4), resulting in an exponential amplification of the selected target sequence.

Bottom Line: The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device.In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features.Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed.

View Article: PubMed Central - PubMed

Affiliation: Istituto Biostrutture e Bioimmagini, CNR, Viale A. Doria 6, Catania, Italy; E-Mail: lzanoli@unict.it.

ABSTRACT
Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed.

No MeSH data available.