Single molecule transcription profiling with AFM

Abstract

Established techniques for global gene expression profiling, such as microarrays, face fundamental sensitivity constraints. Due to greatly increasing interest in examining minute samples from microdissected tissues, including single cells, unorthodox approaches, including molecular nanotechnologies, are being explored in this application. Here, we examine the use of single molecule, ordered restriction mapping, combined with AFM, to measure gene transcription levels from very low abundance samples. We frame the problem mathematically, using coding theory, and present an analysis of the critical error sources that may serve as a guide to designing future studies. We follow with experiments detailing the construction of high density, single molecule, ordered restriction maps from plasmids and from cDNA molecules, using two different enzymes, a result not previously reported. We discuss these results in the context of our calculations.

Image Analysis and Length Estimation of Biomolecules Using AFM

“…While fragment distinction is beyond the scope of this paper, we analyze the feasibility of using a coding scheme to do this in [8] and we make this the center of our discussion in [34]. …”

“…A natural choice is to use AFM to perform single-molecule analysis. Reed et al [8] developed such an analysis that classifies each mRNA by the following three steps: 1) synthesize a complementary DNA (cDNA) copy of each mature mRNA, 2) multiply cleave the cDNAs with a restriction enzyme, and 3) construct each cDNA classification label from ratios of the lengths of its resulting fragments. Thus, they showed the transcription profiling problem reduces to making high-precision measurements of cDNA backbone lengths—correct to within 20–25 bp (6–7.5 nm).…”

AFM Imaging?Reliable or Not?: Validation and Verification of Images in Atomic Force Microscopy

“…An another interesting use of coding theory and sequence detection application is in the use of cantilever probes for sequencing DNA [28].…”

Optimization of specimen preparation of thin cell section for AFM observation

“…Furthermore, recent work has shown that a combination of AFM imaging and manipulation allows for the precise and controlled manipulation of biological systems, from whole cells to the individual molecules [14][15][16][17][18][19][20][21][22][23][24], including DNA cutting and isolation [17,21,22], protein folding and unfolding [15,18], chromosomal dissection [14,15,19], single molecule's reaction [23,24], and single cell pushing, pulling, dragging and vibrating [16,20]. Our group has extended earlier work on the manipulation of single molecules of DNA by AFM [17], achieving positional isolation and biochemical analysis of single DNA molecules based on nanomanipulation and single-molecule PCR [22].…”