Part:BBa_K4347005

Backward Loop primer (BL)

The backward loop primer (BL) is one of the six primers needed to carry out Loop Mediated Isothermal Amplification (LAMP). This BL was designed to detect conserved 16s rRNA regions in STEC E.coli strains, campylobacter, shigella and salmonella.

Backward loop primer of LAMP reaction from Wong et, al (2017)^[1].

Usage and Biology

The loop-mediated isothermal amplification (LAMP) is a rapid method that allows for the specific DNA amplification through the utilization of multiple primers and three major steps^[2]. This method relies on 3 major components- Bst, a DNA polymerase, and two sets of primers, and the target DNA/RNA. The first step, referred to as the “starting material producing step”, composes the stem-looped DNA that is needed for the second stage. The forward and backward inward and outer primers bind to the complementary sequences within the target DNA sequence^[2]. The stem-looped DNA undergoes additional annealing within the target sequences and with the Bst polymerase, which produces stem-loop DNA which concludes the first step^[2]. The stem-loop DNA enters into step two- “cycling amplification step”, which uses the internal primers FIP and BIP in hybridization and synthesis reactions to produce an original and a repaired stem-loop DNA strands^[2]. For further amplification, loop primers can be inserted into the reactions^[3]. The third step is “elongation and recycling”, where the DNA products are recycled and elongated through a BIP-primed strand displacement reaction^[2].

Basic schematic of LAMP reaction from Wong et, al (2017)^[1].

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Wong, Y.-P., Othman, S., Lau, Y.-L., Radu, S., & Chee, H.-Y. (2017). Loop-mediated isothermal amplification (LAMP): A versatile technique for detection of micro-organisms. Journal of Applied Microbiology, 124(3), 626–643. https://doi.org/10.1111/jam.13647

2. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., & Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28(12), e63–e63. https://doi.org/10.1093/nar/28.12.e63

3. Niessen, L., & Vogel, R. F. (2010). Detection of Fusarium graminearum DNA using a loop-mediated isothermal amplification (LAMP) assay. International Journal of Food Microbiology, 140(2–3), 183–191. https://doi.org/10.1016/j.ijfoodmicro.2010.03.036