DNA targeting

The plaque assay is desirable because it is very sensitive and only detects infectious viral particles. However, there are viral agents which cannot be supported by cell lines. In these cases other methods must be used. The polymerase chain reaction (PGR), which amplifies DNA or RNA from viral agents, can be used to detect the presence and quantity of viral agents. The amount of RNA or DNA target in the initial sample can be determined by competitive PGR where the quantity of amplified product is compared to a control PGR product where the initial amount of target is known. Quantification is also possible by an end-point dilution method similar to that used to determine a tissue culture infections dose. PGR methods can be very sensitive however. 

Most sequence-specific regulatory proteins bind to their DNA targets by presenting an a helix or a pair of antiparallel p strands to the major groove of DNA. Recognition of the TATA box by TBP is therefore exceptional it utilizes a concave pleated sheet protein surface that interacts with the minor groove of DNA. Since the minor groove has very few sequence-specific... 

Belotserkovskii B.P., Zarling D.A. Peptide nucleic acid (PNA) facilitates multistranded hybrid formation between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded DNA probes. Biochemistry 2002 41 3686-3692. 

This protocol was extended to other inorganic colloids (e.g., ZnS, PbS), and it was pointed out that such extension paves the way to an electrochemical coding technology for the simultaneous detection of multiple DNA targets based on nanocrystal tags with diverse redox potentials [148]. 

Wang J, Liu G, Merkogi A (2003) Electrochemical coding technology for simultaneous detection of multiple DNA targets. J Am Chem Soc 125 3214-3215... 

Primers The primers are short (15-30) oligonucleotide sequences designed to base pair or anneal to complementary sequences that flank the DNA target sequence to be amplified. The primers are added at 0.1-1 qM in the assay. 

Kohn, K. W. Beyond DNA cross-linking history and prospects of DNA-targeted cancer treatment—Fifteenth Bruce F. Cain Memorial Award Lecture. Cancer Res. 1996, 56, 5533-5546. 

Doria, F. Richter, S.N. Nadai, M. Colloredo-Melz, S. Mella,M. Palumbo, M. Freccero, M. BINOL-amino acid conjugates as triggerable carriers of DNA-targeted potent photo-cytotoxic agents. J. Med. Chem. 2007, 50, 6570-6579. 

Many examples of mobile elements are found in bacteria, where they are called transpo-sons. Bacterial transposons have terminal repeat sequences that both code for the enzymes catalyzing the process of transposition (transposases) and physically interact with these enzymes to bring them to the DNA target site. At this site the DNA-bound transposase presumably catalyzes the endonucleolytic cleavage of the terminal repeat sequence of the trahsposon and also catalyzes a similar sequence in the target DNA. 

Multiplex PCR a PCR reaction where more than one primer set is included in the reaction pool allowing 2 or more different DNA targets to be amplified by PCR in a single reaction tube. 

Sulfhydryl groups also can be added to 5 -phosphate end of DNA probes (Chapter 27, Section 2.2). Biotinylation at these sites avoids disruption of base pairing with complementary DNA targets, since the point of modification is restricted to a single end position on the oligonucleotide. 

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