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Solid-phase cloning for high-throughput assembly of single and multiple DNA parts.

Lundqvist M, Edfors F, Sivertsson Å, Hallström BM, Hudson EP, Tegel H, Holmberg A, Uhlén M, Rockberg J - Nucleic Acids Res. (2015)

Bottom Line: We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors.In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in.The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%.

View Article: PubMed Central - PubMed

Affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden.

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Success rate of RE-based experiments (blue bars, 1–46) grouped by insert characteristics (squares). The colored boxes indicate the magnitude of linguistic complexity content (green-red) or GC-content (yellow-blue) of inserts. Inserts with a low GC-content were unambiguously more successful compared to inserts with a high percentage of GC.
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Figure 3: Success rate of RE-based experiments (blue bars, 1–46) grouped by insert characteristics (squares). The colored boxes indicate the magnitude of linguistic complexity content (green-red) or GC-content (yellow-blue) of inserts. Inserts with a low GC-content were unambiguously more successful compared to inserts with a high percentage of GC.

Mentions: The effect of complexity on the cloning success rate was analyzed analogously by binning (Figure 2D). Here we found the most successful inserts having a high LC-value and a clear drop in success rate beginning with LC-values around 0.67. GC-content and complexity of a sequence is linked, hence these two characteristics were analyzed simultaneously in order to determine which one is of most importance (Figure 3). GC-content was found to be the most important with a distinct trend showing a decline in success rate with a higher percentage of GC while LC-values were more scattered. A logistic regression analysis test using GC content and linguistic complexity as independent variables and the cloning success status as a dichotomous outcome variable showed that both variables have a significant effect on the success rate (BGC = 49.0, P < 0.0001 and BLC = 25.7, P < 0.0001) and that there is significant interaction between them (BGC:CL = −57.6, P < 0.0001). Although both variables significantly affect the success rate, the coefficients indicate a greater effect from GC content than from linguistic complexity. Of the 6252 constructs failing the PCR-step, 4.5% had neither a successfully cloned construct amplified from the same transcript nor a template verified by deep sequencing of the RNA pools (31).


Solid-phase cloning for high-throughput assembly of single and multiple DNA parts.

Lundqvist M, Edfors F, Sivertsson Å, Hallström BM, Hudson EP, Tegel H, Holmberg A, Uhlén M, Rockberg J - Nucleic Acids Res. (2015)

Success rate of RE-based experiments (blue bars, 1–46) grouped by insert characteristics (squares). The colored boxes indicate the magnitude of linguistic complexity content (green-red) or GC-content (yellow-blue) of inserts. Inserts with a low GC-content were unambiguously more successful compared to inserts with a high percentage of GC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402512&req=5

Figure 3: Success rate of RE-based experiments (blue bars, 1–46) grouped by insert characteristics (squares). The colored boxes indicate the magnitude of linguistic complexity content (green-red) or GC-content (yellow-blue) of inserts. Inserts with a low GC-content were unambiguously more successful compared to inserts with a high percentage of GC.
Mentions: The effect of complexity on the cloning success rate was analyzed analogously by binning (Figure 2D). Here we found the most successful inserts having a high LC-value and a clear drop in success rate beginning with LC-values around 0.67. GC-content and complexity of a sequence is linked, hence these two characteristics were analyzed simultaneously in order to determine which one is of most importance (Figure 3). GC-content was found to be the most important with a distinct trend showing a decline in success rate with a higher percentage of GC while LC-values were more scattered. A logistic regression analysis test using GC content and linguistic complexity as independent variables and the cloning success status as a dichotomous outcome variable showed that both variables have a significant effect on the success rate (BGC = 49.0, P < 0.0001 and BLC = 25.7, P < 0.0001) and that there is significant interaction between them (BGC:CL = −57.6, P < 0.0001). Although both variables significantly affect the success rate, the coefficients indicate a greater effect from GC content than from linguistic complexity. Of the 6252 constructs failing the PCR-step, 4.5% had neither a successfully cloned construct amplified from the same transcript nor a template verified by deep sequencing of the RNA pools (31).

Bottom Line: We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors.In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in.The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%.

View Article: PubMed Central - PubMed

Affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden.

Show MeSH