Chain reaction cloning: a one-step method for directional ligation of multiple DNA fragments.
A novel DNA assembly method, chain reaction cloning (CRC), is described. CRC enables the ordered assembly of multiple DNA fragments in a single step. The power of the technique was demonstrated by the directed in vitro assembly of a plasmid comprised of six DNA fragments from a pool of 12 available fragments. The odds of obtaining the correct plasmid clone in a single step, using conventional techniques, is less than 1 in 191000000. Using CRC, the desired plasmid was recovered at a frequency of one in two. Ligation is no longer the rate limiting step in cloning, and limitless possibilities exist for the reconstruction of complex genomes.
Pachuk
C J
CJ
Wyeth Research, One Great Valley Parkway, Suite 30, Malvern, PA, USA. pachukc@labs.wyeth.com
Samuel
M
M
Zurawski
J A
JA
Snyder
L
L
Phillips
P
P
Satishchandran
C
C
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
D009841:Oligonucleotides; D004247:DNA; D004256:DNA Polymerase I; D011088:DNA Ligases