2 research outputs found
Combining M-FISH and Quantum Dot technology for fast chromosomal assignment of transgenic insertions
<p>Abstract</p> <p>Background</p> <p>Physical mapping of transgenic insertions by Fluorescence in situ Hybridization (FISH) is a reliable and cost-effective technique. Chromosomal assignment is commonly achieved either by concurrent G-banding or by a multi-color FISH approach consisting of iteratively co-hybridizing the transgenic sequence of interest with one or more chromosome-specific probes at a time, until the location of the transgenic insertion is identified.</p> <p>Results</p> <p>Here we report a technical development for fast chromosomal assignment of transgenic insertions at the single cell level in mouse and rat models. This comprises a simplified 'single denaturation mixed hybridization' procedure that combines multi-color karyotyping by Multiplex FISH (M-FISH), for simultaneous and unambiguous identification of all chromosomes at once, and the use of a Quantum Dot (QD) conjugate for the transgene detection.</p> <p>Conclusions</p> <p>Although the exploitation of the unique optical properties of QD nanocrystals, such as photo-stability and brightness, to improve FISH performance generally has been previously investigated, to our knowledge this is the first report of a purpose-designed molecular cytogenetic protocol in which the combined use of QDs and standard organic fluorophores is specifically tailored to assist gene transfer technology.</p
RNA Polymerase Pausing during Initial Transcription
In bacteria, RNA polymerase (RNAP) initiates transcription
by synthesizing short transcripts that are
either released or extended to allow RNAP to escape
from the promoter. The mechanism of initial transcription
is unclear due to the presence of transient
intermediates and molecular heterogeneity. Here,
we studied initial transcription on a lac promoter
using single-molecule fluorescence observations
of DNA scrunching on immobilized transcription
complexes. Our work revealed a long pause (‘‘initiation
pause,’’ �20 s) after synthesis of a 6-mer RNA;
such pauses can serve as regulatory checkpoints.
Region sigma 3.2, which contains a loop blocking
the RNA exit channel, was a major pausing determinant.
We also obtained evidence for RNA backtracking
during abortive initial transcription and for
additional pausing prior to escape. We summarized
our work in a model for initial transcription, in which
pausing is controlled by a complex set of determinants
that modulate the transition from a 6- to a
7-nt RNA