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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 the loop-mediated isothermal amplification (LAMP) amplification process. Adapted with permission from [40].
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biosensors-03-00018-f001: Schematic representation of the loop-mediated isothermal amplification (LAMP) amplification process. Adapted with permission from [40].

Mentions: LAMP reaction is initiated by a forward inner primer (FIP), containing sequences of the sense strand of the target DNA, which hybridizes to F2c in the target and initiates complementary strand synthesis (Figure 1). Then, the outer primer F3 hybridizes to F3c portion in the target sequence leading to the displacement of the just synthesized strand that is release as a single-stranded DNA with a loop out structure at one end. The FIP-linked complementary strand acts as the template for a new DNA synthesis primed by backward inner (BIP) and outer (B3) primers that hybridize to the other end of the target, leading to the production of a dumb-bell form DNA which produces a stem–loop DNA structure as a consequence of self-primed DNA synthesis.


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

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

Schematic representation of the loop-mediated isothermal amplification (LAMP) amplification process. Adapted with permission from [40].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

biosensors-03-00018-f001: Schematic representation of the loop-mediated isothermal amplification (LAMP) amplification process. Adapted with permission from [40].
Mentions: LAMP reaction is initiated by a forward inner primer (FIP), containing sequences of the sense strand of the target DNA, which hybridizes to F2c in the target and initiates complementary strand synthesis (Figure 1). Then, the outer primer F3 hybridizes to F3c portion in the target sequence leading to the displacement of the just synthesized strand that is release as a single-stranded DNA with a loop out structure at one end. The FIP-linked complementary strand acts as the template for a new DNA synthesis primed by backward inner (BIP) and outer (B3) primers that hybridize to the other end of the target, leading to the production of a dumb-bell form DNA which produces a stem–loop DNA structure as a consequence of self-primed DNA synthesis.

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.