Targeting Sequences

PCR can also be used to modify DNA sequences using primers differing at one or several positions from the target sequence. This is possible because PCR does not require perfect complementarity of a primer to the sequence flanking the target. Since all of the PCR products contain the primer sequence, an insertion or deletion can thus be incorporated into the product by modifying a primer. It is also possible to add new sequences to the 5 -ends of the primers. Modified or additional genetic information may thus be multiplied and transr ported. 

An effective therapeutic agent must also have the abiUty to reach its target sequence m vivo. BioavailabiUty requires that the antisense oligonucleotide be able to pass through the cell membrane, and that it have a low affinity for nontarget cellular compartments and, in animal systems, nontarget organs. 

An enzyme-amplified detection scheme, based on tire coupling of a streptavidin-alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalysed the hydrolysis of the elecn oiiractive a-naphthyl phosphate to a-naphtlrol this product is elecU oactive and has been detected by means of differential... 

All current comparative modeling methods consist of four sequential steps (Fig. 2) [5,6]. The first step is to identify the proteins with known 3D structures that are related to the target sequence. The second step is to align them with the target sequence and pick those known structures that will be used as templates. The third step is to build the model... 

A. Identifying Known Protein Structures Related to the Target Sequence... 

B. Aligning the Target Sequence with the Template Structures... 

Because modeling by satisfaction of spatial restraints can use many different types of information about the target sequence, it is perhaps the most promising of all comparative modeling techniques. One of the strengths of modeling by satisfaction of spatial restraints... 

For each fold one searches for the best alignment of the target sequence that would be compatible with the fold the core should comprise hydrophobic residues and polar residues should be on the outside, predicted helical and strand regions should be aligned to corresponding secondary structure elements in the fold, and so on. In order to match a sequence alignment to a fold, Eisenberg developed a rapid method called the 3D profile method. The environment of each residue position in the known 3D structure is characterized on the basis of three properties (1) the area of the side chain that is buried by other protein atoms, (2) the fraction of side chain area that is covered by polar atoms, and (3) the secondary stmcture, which is classified in three states helix, sheet, and coil. The residue positions are rather arbitrarily divided into six classes by properties 1 and 2, which in combination with property 3 yields 18 environmental classes. This classification of environments enables a protein structure to be coded by a sequence in an 18-letter alphabet, in which each letter represents the environmental class of a residue position. 

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