Loop-mediated isothermal amplification
Encyclopedia
Loop mediated isothermal amplification (LAMP) is a single tube technique for the amplification of DNA
. This may be of use in future as a low cost alternative to detect certain diseases. It may be combined with a reverse transcription step to allow the detection of RNA.
, a color change can be seen without equipment. Also in-tube detection of DNA amplification is possible using manganese loaded calcein which starts fluorescing upon complexation of manganes by pyrophosphate during in vitro DNA synthesis.
LAMP is a relatively new DNA amplification technique, which due to its simplicity, ruggedness, and low cost could provide major advantages. In LAMP, the target sequence is amplified at a constant temperature of 60 - 65 °C using either two or three sets of primers and a polymerase with high strand displacement activity in addition to a replication activity. Typically, 4 different primers are used to identify 6 distinct regions on the target gene, which adds highly to the specificity. Due to the specific nature of the action of these primers, the amount of DNA produced in LAMP is considerably higher than PCR based amplification. The corresponding release of pyrophosphate results in visible turbidity due to precipitation, which allows easy visualization by the naked eye, especially for larger reaction volumes or via simple detection approaches for smaller volumes. The reaction can be followed in real-time either by measuring the turbidity or the signals from DNA produced via fluorescent dyes that intercalate or directly label the DNA, and in turn can be correlated to the number of copies initially present. Hence, LAMP can also be quantitative. While LAMP is widely being studied for detecting infectious diseases such as tuberculosis, malaria, and sleeping sickness in developing regions, it has yet to be extensively validated for other common pathogens.
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
. This may be of use in future as a low cost alternative to detect certain diseases. It may be combined with a reverse transcription step to allow the detection of RNA.
Technique
LAMP is a novel approach to nucleic acid amplification which uses a single temperature incubation thereby obviating the need for expensive thermal cyclers. Detection of amplification product can be by photometry for turbidity caused by increasing quantity of Magnesium pyrophosphate in solution or with addition of SYBR greenSYBR Green
SYBR Green I is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. SYBR Green I binds to DNA. The resulting DNA-dye-complex absorbs blue light and emits green light . The stain preferentially binds to double-stranded DNA, but will stain single-stranded DNA with lower...
, a color change can be seen without equipment. Also in-tube detection of DNA amplification is possible using manganese loaded calcein which starts fluorescing upon complexation of manganes by pyrophosphate during in vitro DNA synthesis.
Uses and benefits
LAMP has the potential to be used as a simple screening assay in the field or at the point of care by clinicians. As previously mentioned, LAMP is isothermal which eradicates the need for expensive thermocyclers used in conventional PCR, it may be a particularly useful method for infectious disease diagnosis in low and middle income countries. <LAMP is a relatively new DNA amplification technique, which due to its simplicity, ruggedness, and low cost could provide major advantages. In LAMP, the target sequence is amplified at a constant temperature of 60 - 65 °C using either two or three sets of primers and a polymerase with high strand displacement activity in addition to a replication activity. Typically, 4 different primers are used to identify 6 distinct regions on the target gene, which adds highly to the specificity. Due to the specific nature of the action of these primers, the amount of DNA produced in LAMP is considerably higher than PCR based amplification. The corresponding release of pyrophosphate results in visible turbidity due to precipitation, which allows easy visualization by the naked eye, especially for larger reaction volumes or via simple detection approaches for smaller volumes. The reaction can be followed in real-time either by measuring the turbidity or the signals from DNA produced via fluorescent dyes that intercalate or directly label the DNA, and in turn can be correlated to the number of copies initially present. Hence, LAMP can also be quantitative. While LAMP is widely being studied for detecting infectious diseases such as tuberculosis, malaria, and sleeping sickness in developing regions, it has yet to be extensively validated for other common pathogens.