Overview

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• Source: Microbioz India


• Date: 18 Apr,2024

In molecular biology, the LAMP (Loop-Mediated Isothermal Amplification) method is also known as Cue PCR or Loop Amplification. Particularly, it can be used for rapid amplification of nucleic acids which enables detection of specific DNA or RNA sequences under isothermal conditions having a constant temperature mainly 60-65°C. Very important applications including diagnostics, field-based testing and point-of-care testing have made Cue PCR famous due to its simplicity, speediness and versatility.

To know in details how Cue PCR works:

Principle of Cue PCR:

Cue PCR makes use of an isothermal amplification technique involving performance of DNA or RNA amplifications at the same temperature.This contrasts with traditional PCR that requires temperature cycling so that nucleic acids are amplified by CuePCR uses a strand-displacement DNA polymerase along with four to six primers that targeting multiple sites within the target sequence. The reaction goes through several strand-displacement and annealing steps resulting in typical amplification products commonly referred to as “loop” structures or “stem-loops.”

Key Components of Cue PCR:

1. DNA/RNA template: Nucleic acid template containing specific target sequence to be amplified.
2. Loop primers: Small-sized primers which identify particular parts within the target sequence enabling loop formation and increase.
3. Outer primers: Primers surrounding target sequence which begin its amplification.
4. Strand-displacement DNA polymerase: Enzyme involved in DNA synthesis during replication and displacement processes.
5. Reaction buffer: Includes salts and cofactors essential for an optimal enzyme activity and efficient
6. dNTPs (deoxynucleotide triphosphates): Blocks supplied by DNA polymerase used in synthesis of new strands.
7. Magnesium ions (Mg2+): Co-factor required for polymerases’ activities on DNAs.

Steps in Cue PCR:

1. Initiation: mixture has DNA/RNA template, outer primers, loop primers, dNTPs, DNA polymerase, Mg2+ and reaction buffer.
2. Primer annealing and loop formation: Loop primers hybridize with various regions within the target sequence leading to start of loop formation and synthesis of DNA.
3. Strand displacement and amplification: As new ones are synthesized by the DNA polymerase, the existing strands are displaced resulting in an amplification product for that target sequence.
4. Cycle repetition: Several cycles of amplification as a result exponential increase in target DNA or RNA during this process.

Advantages of Cue PCR:

1. Isothermal amplification: It has no need for equipment used in heat cycling since it is carried out at one temperature
2. Rapid amplification: this method gives fast results within 30-60 minutes maximum
3. High sensitivity and specificity: Very small amounts of some specific nucleic acid targets can be detected using this technique
4. Simplicity and versatility: Field-based testing or Point-of-Care Testing (POCT) does not demand many instruments or expert knowledge thus making it suitable for those settings.
5. Robustness : This type works well even when crude samples such as blood, saliva or plant extracts are used

Applications of Cue PCR:

1. Diagnostics – Identification of viral, bacterial and parasitic infections including infectious diseases.
2. Environmental monitoring – Pathogens screening from water samples, soil samples and foodstuffs.
3. Point-of-care testing – Rapid tests performed in low resource settings like remote villages
4. Forensic analysis–This method provides forensic identification from blood stains or saliva based on its detection methods
5. Research –genotyping gene expressions SNP detection molecular biology researches.