Adduct-containing Plasmids
Adduct-containing Plasmids - Related Videos
11:46
Single Molecule Analysis of Laser Localized Psoralen Adducts
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2017
Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.
07:52
Removal of Branched and Cyclic Compounds by Urea Adduction for Uk'37 Paleothermometry
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2023
Source: Laboratory of Jeff Salacup - University of Massachusetts Amherst As mentioned in previous videos, the product of an organic solvent extraction, a total lipid extract (TLE), is often a complex mixture of hundreds, if not thousands, of different compounds. The researcher is often only interested in a handful of compounds. In the case of our two organic paleothermometers (Uk'37 and MBT/CBT), the interest is in only 6 compounds (2 alkenones and 4 isoprenoidal glycerol dialkyl glycerol...
08:16
Plasmid Purification
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2023
Plasmid purification is a technique used to isolate and purify plasmid DNA from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is a small, circular, double-stranded DNA that is used as a carrier of specific DNA molecules. When introduced into a host organism via transformation, a plasmid will be replicated, creating numerous copies of the DNA fragment under study. In this video, a step-by-step generalized procedure is described for how to perform plasmid purification.
01:28
Plasmids
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2025
Plasmids are extrachromosomal DNA molecules found in bacteria, archaea, and some eukaryotic microbes like yeast. These small, circular DNA structures typically contain fewer than 30 genes, although some may exist linearly. Plasmids vary in their number within a cell, known as copy number. Single-copy plasmids are present in one copy per cell and multi-copy plasmids are present in multiple copies, reaching over 100 copies per cell.Plasmids usually replicate independently of the chromosomal DNA...
07:11
Homemade Site Directed Mutagenesis of Whole Plasmids
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Cited by 92 •
2009
Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.
FAQs
The adduct can interfere with copying by changing a nucleotide or distorting the DNA helix. If cellular machinery cannot copy past the lesion, replication may stall; if a polymerase copies across it inaccurately, the event can generate a characteristic mutation. Thus, the same experimental system can reveal both blocking and mutagenic consequences of DNA damage.
Nucleotide excision repair and translesion synthesis address the lesion in different ways. Nucleotide excision repair recognizes and processes damaged DNA, whereas translesion synthesis allows replication to proceed across a lesion. Tracking these responses in cells carrying the plasmid helps distinguish removal of the adduct from tolerance of it during copying, clarifying how each pathway contributes to the observed genetic outcome.
Because the plasmid carries a defined lesion, researchers can examine cellular responses to a known DNA alteration rather than relying only on damage produced broadly by an exposure. Following the same construct in living cells connects lesion recognition, repair processing, replication behavior, and mutation formation, making the relationship between a specific adduct and its genetic consequences easier to evaluate.
Researchers first use a circular plasmid carrying the defined DNA adduct, introduce it into cells, and then follow how the cell handles the lesion. Measurements can focus on repair processing, replication across the damaged site, or mutations associated with copying. This workflow links a chemically specified DNA change to a cellular response in a living genetic system.
They can show whether the lesion is recognized and processed, whether replication is blocked or continues through lesion bypass, and whether copying produces characteristic mutations. These outcomes address complementary questions: repair efficiency indicates how effectively cells remove or process damage, while bypass and mutation patterns indicate how the lesion affects replication and genome stability.
An adduct can serve as a defined molecular consequence of exposure to an environmental or therapeutic agent. Introducing the modified plasmid into cells allows researchers to examine how repair and replication pathways respond to that type of damage and whether characteristic mutations arise. The results help connect DNA lesion processing with broader questions about genome stability and chemical carcinogenesis.