Visualization of circular DNA molecules labeled with colloidal gold spheres using atomic force microscopy

We have imaged gold‐labeled DNA molecules with the atomic force microscope(AFM). Circular plasmid DNA was labeled at internal positions by nick‐translation using biotinylated dUTP. For visualization, the biotinylated DNA was linked to streptavidin‐coated colloidal gold spheres (nominally 5 nm diam) prior to AFM imaging. Reproducible images of the labeled DNA were obtained both in dry air and under propanol. Height measurements of the DNA and colloidal gold made under both conditions are presented. The stability of the DNA‐streptavidin colloidal gold complexes observed even under propanol suggests that this labeling procedure could be exploited to map regions of interest in chromosomal DNA

Atomic force microscopy of oriented linear DNA molecules labeled with 5nm gold spheres

The atomic force microscope (AFM;1) can image DNA and RNA in air and under solutions at resolution comparable to that obtained by electron microscopy (EM) (2–7). We have developed a method for depositing and imaging linear DNA molecules to which 5nm gold spheres have been attached. The gold spheres facilitate orientation of the DNA molecules on the mica surface to which they are adsorbed and are potentially useful as internal height standards and as high resolution gene or sequence specific tags. We show that by modulating their adhesion to the mica surface, the gold spheres can be moved with some degree of control with the scanning tip.This article is from Nucleic Acids Research 21 (1993): 99, doi: 10.1093/nar/21.1.99. Posted with permission.</p

The replication advantage of a free linear rRNA gene is restored by somatic recombination in Tetrahymena thermophila

The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10 per nucleus. A mutant in which the replication properties of this molecule were altered was isolated and characterized. This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmml mutation (D. L. Larson, E. H. Blackburn, P. C. Yaeger, and E. Orias, Cell 47:229-240, 1986). The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion. This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmml. Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4IB-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion. In somatic nuclei in which this rare recombinational event had occurred, all 104 copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA. The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage. We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T. thermophila.This article is from Molecular and Cellular Biology 9 (1989): 452, doi: 10.1128/MCB.9.2.452. Posted with permission.</p

The replication advantage of a free linear rRNA gene is restored by somatic recombination in Tetrahymena thermophila.

The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10(4) per nucleus. A mutant in which the replication properties of this molecule were altered was isolated and characterized. This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmm1 mutation (D. L. Larson, E. H. Blackburn, P. C. Yaeger, and E. Orias, Cell 47:229-240, 1986). The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion. This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmm1. Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4/B-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion. In somatic nuclei in which this rare recombinational event had occurred, all 10(4) copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA. The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage. We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T. thermophilia

Atomic force microscopy of DNA-colloidal gold and DNA-protein complexes

The atomic force microscope (AFM)1 is capable of imaging and manipulating nucleic acids in solution and in air29' 13 We are developing methods for random and site-specific labeling of individual DNA molecules to facilitate manipulation of fragments excised in the AFM and for localization of specific DNA domains, such as protein binding sites and origins of replication. One successful method was to incorporate biotinylated nucleotides at random internal locations or specifically at the ends of linearized DNA molecules in vitro. Following complex formation with Snm diameter streptavidin-gold conjugates, chromatographic purification and passive adsorption of the complexes to mica, the biotinylated domains were easily localized in the AFM by virtue of the distinctive size and shape of the streptavidin-gold complex. In many cases unconjugated streptavidin (i.e., lacking gold) was also observed attached to the biotinylated DNA. A second approach to site-specific labeling of DNA for imaging in the AFM was to react DNA with restriction enzymes having sequence-specific binding properties. Like the unconjugated streptavidin-DNA complexes, these enzyme-DNA complexes were visible without attached colloidal gold. Efforts to image DNA labeled in vivo using bromodeoxyuridine (BrdU) and anti-BrdU antibodies are ongoing. Luming Niu ; Wenling Shaiu ; James Vesenka ; Drena D. Larson ; Eric Henderson; Atomic force microscopy of DNA-colloidal gold and DNA-protein complexes. Proc. SPIE 1891 (1993); doi:10.1117/12.146706.</p

Visualization of circular DNA molecules labeled with colloidal gold spheres using atomic force microscopy

We have imaged gold‐labeled DNA molecules with the atomic force microscope(AFM). Circular plasmid DNA was labeled at internal positions by nick‐translation using biotinylated dUTP. For visualization, the biotinylated DNA was linked to streptavidin‐coated colloidal gold spheres (nominally 5 nm diam) prior to AFM imaging. Reproducible images of the labeled DNA were obtained both in dry air and under propanol. Height measurements of the DNA and colloidal gold made under both conditions are presented. The stability of the DNA‐streptavidin colloidal gold complexes observed even under propanol suggests that this labeling procedure could be exploited to map regions of interest in chromosomal DNA.This article is from Journal of Vacuum Science & Technology A 11 (1993): 820, doi: 10.1116/1.578311. Posted with permission.</p