Enhancing the "Barcode" Readability of Color-Labeled Molecular Tags by Linker Eng

Michigan Technological University, Houghton MI

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Abstract

DESCRIPTION (provided by applicant): Gene mutation affects 60% of all humans throughout their lifetime, indicating the importance of the direct study of genomic variation. However, mutation detection is still problematic, especially in clinical laboratories with limited resources and inadequate access to the latest detection technology. A precise, cost-effective, high-throughput method is required for large-scale mutation detection that involves analysis of large numbers of single nucleotide polymorphisms (SNPs) across many individuals. Fluorescence resonance energy transfer is a simple procedure for genetic analysis. However, current approaches are limited to single pairs of donor and acceptor dyes for each reaction. In this project we will utilize energy transfer principles and linker engineering strategy to construct a large number of molecular tags with unique fluorescent signatures. This will enable us to obtain a higher order of magnitude throughput in many of genomic approaches. Our proposed approach includes utilizing one, two or more dyes, disposed at varying molecular distances from each other, to generate many alternative discrete signatures. The rationale is based on the energy-transfer efficiency and fluorescent signature being highly dependant on the distance and the conformational behavior of the linkers that separate the donor and acceptor. The "barcode" fluorescent signature of these molecular tags, therefore, can be tuned by adjusting their orientation and distance via linker engineering. The key features of our designed fluorescent tags are follows: (i) Multiple unique fluorescent signatures can be easily achieved by simply linker engineering;(ii) Although with completely different linker engineering strategies, the fluorophore labeling energy transfer system can be readily constructed on a DNA/peptide synthesizer following the standard protocols;(iii) This synthetic protocol exhibits very high versatility. This means the synthetic fluorophore-labeled monomers can be attached to any position on the linker backbone in order to yield distinguishable fluorescent signatures;(iv) As all these fluorescent tags can be excited at a single wavelength and detected simultaneously by a simple optical system. These novel fluorescence molecular tags are expected to offer a valuable tool for multiplex genetic mutation analysis and high-throughput multicolor biological analyses. Beyond genetic analysis, these novel fluorescent tags are anticipated to find broader application in all areas where high sensitivity and simultaneous spectroscopic discrimination of several fluorescent tags are required. PUBLIC HEALTH RELEVANCE: Our long-term goal is to develop individualized medicine tailored to patient's genotypes. However, identifying and genotyping a vast number of genetic polymorphisms in large populations pose a great challenge. The objective of this project is to develop a library of novel "tunable" molecular tags with unique fluorescent signatures to facilitate multiplex genetic analysis. This project has the potential to offer a precise and cost-effective method for large-scale mutation detection involving analysis of a large numbers of genetic polymorphisms across many individuals.