Ligation-LAMP to Detect miR-221 Cancer Biomarker

Abstract

Breast cancer is the second most common cancer in women and can be fatal. Current breast cancer diagnostic tools such as mammograms, MRIs, and biopsies are invasive, costly, time-consuming, and inefficient. Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), can be designed to detect miRNA cancer biomarkers in blood samples. The focus of our project is to detect miRNA-221, a breast cancer biomarker, using a ligation-LAMP assay with high specificity and sensitivity. During ligation LAMP, miR-221 is reverse transcribed to cDNA. The cDNA acts as a splint to ligate two hairpins that form the dumbbell needed for LAMP amplification by Bst Polymerase. LAMP amplification can be detected through colorimetric methods, using phenol red and HNB. Intercalating fluorescent dye can also be used, or a CRISPR system can be used and yield a fluorescent output as well. Our primary findings include successful tests using colorimetric LAMP. We have seen nonspecific amplification causing false positives, but by adding betaine, a reagent that hinders nucleic acid amplification rate, to our reactions, the positive control amplifies sooner than the negative. Using the colorimetric and fluorescent methods described, ligation LAMP can be used to efficiently detect breast cancer biomarker miR-221.