Generating tandem repeats by cloning with double initiator fragments.
The ability to generate tandem repeats of a DNA sequence has proven important for a large variety of studies of DNA structure and function. The most commonly used method to produce tandem repeats involves cloning of an oligomerized monomer sequence that contains asymmetric overlapping ends, but, in practice, this approach is inefficient because of the circularization of oligomers before they ligate into vector. Described here is a method that circumvents this problem by the use of two separate oligomerization reactions, each containing an initiator fragment onto which monomer polymerizes without circularization. Subsequent mixing of the two reactions permits circularization, generating a viable plasmid containing the sum of the added repeats from each reaction. A variation of this method is also demonstrated that permits the synthesis of constructs with a defined number of repeats.
Radlinska
M
M
Henry Ford Hospital, One Ford Place 5D, Detroit, MI 48202-3450, USA.
Drabik
C E
CE
Tong
W S
WS
Lutter
L C
LC
D003001:Cloning, Molecular / Q000379:methods*; D015252:Deoxyribonucleases, Type II Site-Specific; D005820:Genetic Testing; D005822:Genetic Vectors; D010957:Plasmids; D020080:Tandem Repeat Sequences / Q000235:genetics*
C053688:endodeoxyribonuclease AlwNI; C028678:CYCGRG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific