Advantages and disadvantages of common isothermal amplification techniques!
In recent years, with the rapid development of molecular biology technology, a large number of diagnostic methods based on nucleic acid detection have been established and widely used in laboratory detection of diseases, and isothermal amplification technology is one of them.
Compared with other nucleic acid amplification techniques, isothermal amplification has the advantages of fast, efficient, and specific, and does not require special equipment. Therefore, it has been considered by many scholars as a detection method that may be comparable to qPCR once it appeared.
Isothermal amplification technology is developing rapidly and its genres are complex. In order to facilitate everyone's learning and understanding, this article summarizes the current common isothermal nucleic acid amplification technologies: LAMP, NERA, NASBA, RCA, HDA, RPA and ERA. At the same time, the classical PCR technique and several constant temperature nucleic acid amplification techniques were compared.
In order to selectively develop and utilize this technology, the principles, characteristics and applications of these isothermal amplification technologies are briefly summarized.
Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) uses high-temperature-resistant DNA polymerase (Taq enzyme) to cycle template DNA, primers, deoxynucleoside triphosphate (dNTP) and buffer at different temperatures to achieve double-stranded DNA Separation, primer binding to the complementary region on the template, and finally deoxynucleoside triphosphates are added one by one to the newly synthesized DNA strand under the action of DNA polymerase.
PCR is to use DNA denaturation in vitro at a high temperature of 95°C to become a single strand. At a low temperature (often around 60°C), primers and single strands are combined according to the principle of complementary base pairing, and then the temperature is adjusted to the optimum for DNA polymerase. Reaction temperature (around 72°C), DNA polymerase synthesizes complementary strand along the direction from phosphoric acid to five-carbon sugar (5'-3'). The PCR instrument based on polymerase is actually a temperature control device, which can well control the denaturation temperature, renaturation temperature, and extension temperature.
Loop-mediated isothermal amplification (LAMP)
The amplification principle of loop-mediated isothermal amplification is based on the fact that DNA is in a state of dynamic equilibrium at about 65°C. When any primer performs base pairing extension to the complementary part of double-stranded DNA, the other strand will dissociate and become a single strand. Under this premise, 4 different specific primers are used to recognize 6 specific regions of the target gene. Under the action of strand displacement DNA polymerase, starting from the 3' end of the outer primer segment, it is combined with the template DNA Complementary sequences pair to initiate strand-displacing DNA synthesis.
Advantages of LAMP:
(1) The amplification efficiency is high, and it can effectively amplify 1-10 copies of the target gene within 1 hour, and the amplification efficiency is 10-100 times that of ordinary PCR.
(2) The reaction time is short, the specificity is strong, and no special equipment is required.
Disadvantages of LAMP:
(1) The requirements for primers are particularly high.
(2) The amplified product cannot be used for cloning and sequencing, but can only be used for judgment.
(3) Due to its strong sensitivity, it is particularly easy to form aerosols, causing false positives to affect the test results.
Nicking endonuclease constant temperature amplification technology (NEAR)
Nicking endonuclease constant temperature amplification technology (NEAR) is a kind of constant temperature nucleic acid amplification technology with the least research. It was developed and patented by researchers at Ionian Technologies in 2008 (Brain et al. 2009). Ionian is a California startup founded in 2000 and acquired by Alere in 2010. Then Alere was acquired by Abbott in 2017, and NEAR became Abbott's patent.
NEAR is a strand displacement amplification technology. Its principle is to use dNTPs as raw materials to polymerize and extend from the 3' end of the cleavage through the action of polymerase at the cleavage formed by the nicking endonuclease to replace the allele DNA. strand, thus forming a new complete DNA sequence containing the nicking enzyme recognition site. This double strand is recognized and cleaved by the nuclease endonuclease again, and then begins the cycle of "polymerization-nicking", generating a large number of displaced DNA single strands, forming an exponential amplification.
Advantages of NERA:
(1) Fast (positive results can be obtained within 5 minutes at the fastest).
(2) High sensitivity (only hundreds of virus copies are needed initially).
(3) Different from the LAMP technology with more complicated primer design, the NEAR technology has various reaction forms to meet different amplification requirements.
(4) Good reaction stability.
Disadvantages of NEAR:
(1) There is a high possibility of false positives in the design of short sequences.
(2) The reaction mechanism is complex and the product yield is easily limited by the amplification template, which makes the later amplification change from exponential growth to linear growth.
(3) The specificity and sensitivity of NEAR constant temperature amplification technology depend on molecular beacon primers and Nicking enzymes. The design of molecular beacon primers is difficult, and Nicking enzymes and other constant temperature amplification enzymes are generally better than traditional real-time fluorescence PCR enzymes are expensive, so NEAR constant temperature amplification technology is inferior to qPCR in terms of detection cost and application range.
