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Seite: 65, Zeilen: 8-31
|Quelle: McIntyre and Fanning 2006|
Seite(n): 1, 2, 3, 4, Zeilen: 1: abstract: 2ff, r.col: 5ff; 2: l.col: 1ff; 3: l.col: letzte Zeile; 4: r.col: 21ff
|Moreover, McIntyre and Fanning (2006) revealed that shRNA vector construction can be disturbed by high mutation rates and ensuing sequencing is often problematic. shRNA expression vectors are constructed by one of three different methods. The most common method requires the synthesis, annealing and ligation of two complementary oligonucleotides into an expression vector. The frequency of false positives determined by sequencing is high, about 20-40% high (Miyagishi et al, 2004). The unreliability of this method is in part due to the difficulty in synthesizing long oligonucleotides (> 35 bases) and this method requires two long oligonucleotides then the chance of mutation is doubled. The second strategy is a PCR approach in which a promoter sequence serves as the template. Although it is advantageous that only single long oligonucleotides is required, the strong secondary structure predicted to form within this primer can lead to the amplification of false products (Castanotto et al., 2005). The third method comprises several techniques relating to primer extension. Each is based on the principle of a polymerase extending the 3 end of overlapping oligonucleotides. Nevertheless, this method reduces the cost of oligonucleotides and does not need purification but may cause off-set by a high rate of polymerase-induced mutations in both the initial extension and repeated cycling steps. In order to reduce mutations, conducting all reactions as single-step extensions and replacing Taq polymerase with an enzyme better able to counter the secondary structure of the hairpin template were adopted. Another reported strategy to alleviate sequencing difficulties is to include mismatched bases within the shRNA stem (Yu et al., 2003).||A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. [...]
[...] In a survey of more than 100 papers applying expressed shRNA in mammalian systems we determined that shRNA expression vectors are constructed by one of three different methods (see Additional file 1).
The most common method for making shRNA constructs (74 % of surveyed studies) requires the synthesis, annealing and ligation of two complementary oligonucleotides (oligos) into an expression vector (Fig. 1b and Additional file 2). While this cloning method is quick, the oligo synthesis cost is nearly double that of other methods and the frequency of false positives determined by sequencing is high (typically 20 – 40 %) . The unreliability of this method is in part due to the difficulty in the synthesis of long oligos (length > 35 bases) . As this method requires two long oligos the chance of mutation due to synthesis error is doubled.
The second strategy (employed in 22 % of studies) is a PCR approach in which a promoter sequence serves as the template (Fig. 1c). [...] Although it is advantageous that only a single long oligo is required, the strong secondary structure predicted to form within this primer can lead to the amplification of false products. [...] 
The third method (applied in 4 % of studies) encompasses several techniques relating to primer extension. Each is based on the principle of a polymerase extending the 3' end of overlapping oligos . [...] This technique is the cheapest of all the construction methods discussed as it both halves the cost of unique oligos (compared to the annealed oligo method) and does not need costly oligo purification (compared to the promoter based PCR method). However, this saving may be off-set by a high rate of polymerase-induced mutation in either the initial extension step or by repeated cycling .
Our first step to reduce mutations was to remove the possibility of cycling-induced errors by conducting all reactions as single-step extensions. [...]
To improve upon these results, we substituted Taq polymerase with an enzyme better able to counter the secondary structure of the hairpin template.
Another reported strategy to alleviate sequencing difficulties is to include mismatched bases within the shRNA stem [3,11].
3. Miyagishi M, Sumimoto H, Miyoshi H, Kawakami Y, Taira K: Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells. J Gene Med 2004, 6:715-723.
4. Paddison PJ, Cleary M, Silva JM, Chang K, Sheth N, Sachidanandam R, Hannon GJ: Cloning of short hairpin RNAs for gene knockdown in mammalian cells. Nat Methods 2004, 1:163-167.
6. Castanotto D, Scherer L: Targeting Cellular Genes with PCR Cassettes Expressing Short Interfering RNAs. Methods Enzymol 2005, 392:173-185.
7. Rossi JJ, Kierzek R, Huang T, Walker PA, Itakura K: An alternate method for synthesis of double-stranded DNA segments. J Biol Chem 1982, 257:9226-9229.
11. Yu JY, Taylor J, DeRuiter SL, Vojtek AB, Turner DL: Simultaneous inhibition of GSK3alpha and GSK3beta using hairpin siRNA expression vectors. Mol Ther 2003, 7:228-236.
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