Slic primer design

Sequence- and Ligation-Independent Cloning (SLIC)

The SLIC cloning method¹ is based on annealing of single-stranded complementary overhangs on the target vector and an insert. This tool implements this basic logic to design primers which generate the appropriate PCR fragments where the vector has overhangs complementary to the insert or vice versa. Single-stranded overhangs can subsequently be generated by using the 3'->5'-exonuclease activity of the T4 DNA polymerase. After that it's just annealing, transformation, and you're done. It's super easy and it works like a charm!

For the full protocol, just click here. Further information and on the cloning procedure can also be found on this EMBL page and in the original paper¹.

Melting Temperature Calculations

The script is designed so that it extends the primer until the desired T_m is reached. We implemented two differnt methods for calculation of the T_m:

(1) Rule of thumb:

For less than 14 nucleotides, we use the classic Marmur formula²:

T_m = 4(G + C) + 2(A + T)

and for 14 or more nucleotides, the Wallace rule³ is applied:

T_m = 64.9 + 41(G + C - 16.4) / (A + T + G + C)

where G, C, A and T are the occurrences of the respective bases in the sequence. Both equations assume the standard conditions of 50 nM primer, 50 mM Na⁺, and pH 7.0.

(2) Base stacking:

This is a thermodynamical approach to approximate the T_m, which also takes salt concentrations into account. The underlying script is essentially a JavaScript version of the PHP script used here^4,5.

References

(1) Li, M. Z., Elledge, S. J. (2012). SLIC: A method for sequence- and ligation-independent cloning. Methods in Molecular Biology, 852, 51–59.

(2) Marmur, J., Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. Journal of Molecular Biology, 5(1), 109–118.

(3) Wallace, R. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T., Itakura, K. (1979). Hybridization of synthetic oligodeoxyribonucleotides to phi chi174DNA:the effect of single base pair mismatch. Nucleic Acids Research, 6(11), 3543–3557.

(4) SantaLucia, J. (1998). A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proceedings of the National Academy of Sciences of the United States of America, 95(4), 1460–5.

(5) Von Ahsen, N., Wittwer, C. T., Schütz, E. (2001). Oligonucleotide melting temperatures under PCR conditions: Nearest-neighbor corrections for MG2+, deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations with comparison to alternative empirical formulas. Clinical Chemistry, 47(11), 1956–1961.