Chemical ligation of DNA: the first non-enzymatic assembly of a biologically active gene.
An artificial gene comprising 183 base pairs has been assembled by template-directed condensation of 35- to 53-membered oligodeoxyribo nucleotides with cyanogen bromide as a condensing agent. The reaction is complete within several minutes at 0 degrees C in buffer. The resulting mini-gene was cloned and expressed in vivo and in vitro. We have also found that the polymerase inhibition technique (toe-printing) is a good way to ascertain that translation initiation complexes form in the case of single-stranded DNAs as well. Thus, along with the fully chemical assembly of synthetic genes, a rapid and sufficiently reliable method for determining their ribosome-binding properties was developed.
Shabarova
Z A
ZA
Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, USSR.
Merenkova
I N
IN
Oretskaya
T S
TS
Sokolova
N I
NI
Skripkin
E A
EA
Alexeyeva
E V
EV
Balakin
A G
AG
Bogdanov
A A
AA
D001483:Base Sequence; D003001:Cloning, Molecular; D003488:Cyanogen Bromide / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis*; D011089:Polydeoxyribonucleotides / Q000138:chemical synthesis*; D014176:Protein Biosynthesis / Q000502:physiology; D012270:Ribosomes / Q000378:metabolism; D013696:Temperature; D014158:Transcription, Genetic / Q000502:physiology
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D011089:Polydeoxyribonucleotides; D004259:DNA-Directed DNA Polymerase; D003488:Cyanogen Bromide