Ligation products

LI ligase s large intrinsic flexibility was revealed by the recent crystal structure of the ligation product of a reduced size variant with two vastly different conformers, differing by reorientation of one of the stems by around 80 A, that were resolved in the same asymmetric cell [106], Based on the presence/absence of specific contacts between distant conserved parts including the ligation site and a totally conserved residue, U38, one of the conformers was postulated to represent the catalytically active or on conformation, the other the inactive or off one [106],... 

The ligation product is a copy of the template, so this represents a self-replication procedure for Pl-Cys-P2. Initial rates depend on the square root of the template concentration (a common observation, readily explained where the template is active as the monomer but present mostly as the dimer), but rates fall off, as expected, as the product accumulates. 

Fig. 36 Plot of the free energy change for metathesis reactions versus the chain length of the ligation product with a central imine bond (filled square) and with an imine bond off-center (filled diamond) for imine-containing mPE oligomers. The magnitude of the equilibrium shifting was referenced to the free energy change for dimer formation...

Entry Reactants Ligation product Approx, extent of helicity Jfeq (294 K) AAG (kcal moL ) ... 

Transform the ligation product into E. coli cells and screen minipreps for inserts by digesting with EcoRI and SacII. Correctly ligated clones show an 850 bp band for the insert and a 3.4 kb vector band. Inversely oriented inserts produce 150 bp and 4 kb bands. Clones without insert show only the 3.4 kb vector band. Use M13 forward and M13 reverse primers for sequence confirmation. Prepare Maxipreps for one confirmed clone and test the functionality of the lox-STOP-lox cassette as described below. 

Fig. 8.2.2 Principle of genomic quantification by multiplex ligation-dependent probe amplification (MLPA). Synthetic oligonuleotides are designed to bind to exon 1 (oligo 1a and 1b) and 2 (2a and 2b). After specific hybridisation, the oligos are ligated. The ligation products undergo quantitative polymerase chain reaction using universal primers X and Y...

Figure 17. The ligation products of antiparallel double crossover molecules. Ligations of the DAE, DAO, and DAE+J molecules, respectively, from the top to the bottom of the drawing. One domain has been capped by hairpins. Ligation of the DAE molecule leads to a reporter strand, which is drawn more darkly. Ligation of the DAE+J molecule also leads to a reporter strand, similar to the one in DAE. However, ligation of the DAO molecule produces to a polycatenated structure.

Purification of the foll-length-labeled RNA can be performed by PAGE. Even small amount of the ligated product can be detected by fluorescent scanners, and detection of fluorescence from both dyes can be used to identify the correct band.4... 

N-terminal fragment C-terminal fragment ligation product... 

The high chemoselectivity of NCL relies upon the distinct reactivity of an N-terminal cysteine. The requirement for cysteine at the ligation, however, restricts the applicability of NCL. It is possible to subject the ligation product to desulfurization, resulting in the net formation of a more commonly found X-Ala bond [29]. The presence of cysteines other than that needed for ligation is not tolerated, however, since desulfurization would occur at both protected and unprotected cysteine thiols. 

The possibilities of N-(dialkylphosphoryl)amino acids for the prebiotic syntheses of peptides and polynucleotides have been studied in a series of papers [24,116-122], However, it must be emphasized that the phosphoryl group does not behave as an amino-activating group, the hydrolysis of which would be coupled to peptide bond formation. Actually, further peptide elongation requires the subsequent hydrolysis of the N-terminal phosphoryl group of the ligated product. In the presence of an amino acid ester, dipeptide esters 16 with an unreacted N-phosphoryl protection are formed, support-... 

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