GC-rich templates

It is important to realize that a random primer may anneal anywhere along the template so that on average the template size is halved after each duplication. Consequently, the probes would be very short if (G/Oj is closely spaced as in GC-rich templates. 

Fig. 2. Sets of overiap sequences for fusion PCR. Each set of primers, such as 15C and 15G, should be used together to amplify the two fragments to be fused, one at the 5 end and another at the 3 end. Type 1 sequence is used for fusions that allow mutations in 15C/15G region (see Note 2). Type 2 sequence is useful for in-frame fusion owing to its low mutation frequency. Type 3 sequence Is used for GC-rich templates that sometimes prevent amplification with GC-rich primers.

Low PCR amplification, or none at all, from certain GC-rich templates, such as mammalian and virus genes, was experienced with primers containing Type 1 and 2 sequences. This suggests that primer with a high GC content anneal to several places on the templates, resulting in weak amplification. For such templates, primers with Type 3 sequences are more effective for the conventional amplification and fusion PCR. 

Rho-independent termination occurs when the newly formed RNA folds back on itself to form a GC-rich hairpin loop closely followed by 6-8 U residues. These two structural features of the newly synthesized RNA promote dissociation of the RNA from the DNA template. This is the type of terminator shown in Figure 1-3-4. 

The promoters for RNA polymerase III have been clearly proven to be internal on the template. In one instance the 5 S gene promoter lies between 45 and 83, whereas in tRNA genes it is split into two separate locations, one between 8 and 30 and a second between 51 and 72. Termination in 5 S genes is caused by a sequence of four A residues situated between two GC-rich regions. tRNA precursors are converted into mature tRNAs by a series of alterations (see later). 

RNA Polymerase slows down, or pauses, when it reaches the first GC-rich segment, because the stability of G-C base pairs makes the template hard to unwind. In vitro, RNA polymerase does pause for several minutes at a GC-rich segment. 

The pausing gives time for the complementary GC-rich parts of the nascent transcript to base-pair with one another. In the process, the downstream GC-rich segment of the transcript is displaced from its template (or from that part of the enzyme molecule to which it is bound see Figure 26.8b). Hence, the ternary complex of RNA polymerase,... 

Template Loop Loops of more than approx 4 to 5 GC-rich regions will be flagged by an asterisk and shonld be avoided. Loops less than 4 bases should also be avoided if possible to reduce the likelihood of background. 

The termination that is not dependent on p, is controlled by the specific sequence in DNA termed termination sites (terminators). The terminators that signal the termination and release of RNA transcript, code for GC-rich inverted repeats punctuated by a nonrepeating segment between them just preceding the 3 polyU (U(j-g)-end of the transcript. Therefore a G C-rich stem/loop structure (hairpin) is formed between the inverted repeats preceding the 3 -polyU end of the transcript. The stem/loop structure causes a pause in the RNA polymerase and resulting dissociation of the RNA transcript form the DNA template. 

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