Overlapping sequencing scheme

FIGURE A.5 A general scheme for standard PCR mutagenesis which employs two mutagenic primers (forward and reverse, m( and m respectively) and two universal (or amplification) primers (Uf and u,). The two PCR products are joined by annealing at the overlap sequences and are further extended and PCR amplified. The overlap-extension strategy produces a homoduplex mutant cassette which is then inserted into a suitable expression vector. REl and RE2 indicate two available (natural or engineered) restriction sites. 

Figure Al.6.30. (a) Two pulse sequence used in the Tannor-Rice pump-dump scheme, (b) The Husuni time-frequency distribution corresponding to the two pump sequence in (a), constmcted by taking the overlap of the pulse sequence with a two-parameter family of Gaussians, characterized by different centres in time and carrier frequency, and plotting the overlap as a fiinction of these two parameters. Note that the Husimi distribution allows one to visualize both the time delay and the frequency offset of pump and dump simultaneously (after [52a]).

As in the case of the HN(CO)CA-TROSY scheme, the HN(CO)CANH-TROSY experiment can be readily expanded to a four-dimensional HN(CO)CANH-TROSY experiment without increasing the overall length of the pulse sequence. This can be accomplished by labelling the 13C (f) chemical shift during additional incremented time delay, implemented into the 13C — 13C INEPT delay. As a result, a well-dispersed 13C (i- 1), 13C (i- 1), 15N(i), Hn(0 correlation map is obtained with minimal resonance overlap albeit with the inherent sensitivity loss by a factor of y/2. 

These reactions do not have to occur in perfect sequence, and the phases may have some overlap (e.g., phases 2, 3, and 4 may overlap, and possibly even 5 and 6). However, the present evidence, such as intermediate products formed by starch mutants of C. reinhardtii and of higher plants, supports the sequence of reactions shown in Fig. 1 for amylopectin and amylose biosynthesis. Further experiments are required to test this hypothetic scheme, and attempts to purify and characterize the debranching enzyme, crucial to this hypothesis, are under way. 

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