Blunt-end and single-strand ligations by Escherichia coli ligase: influence on an in vitro amplification scheme.
A ligase-based, in vitro DNA amplification system (LAR) has been described by Wu and Wallace [Genomics 4 (1989) 560-569]. This strategy is based on the ability of a DNA ligase to join the 5' phosphate of one DNA molecule to the 3' hydroxyl of a second during a nick-closing reaction. Escherichia coli DNA ligase has been used in place of the T4 DNA ligase in our study in order to limit template-independent ligation activities, which lower the sensitivity of this amplification procedure. The results of this study indicate that E. coli ligase also joins blunt-ended DNA molecules and some single-stranded oligodeoxyribonucleotides, in the absence of a complementary template, with an efficiency which is sensitive to both the concentrations of DNA substrate and enzyme.
Salk Institute Biotechnology/Industrial Associates, Inc., San Diego, CA 92138.
K J
KJ
Barringer
L
L
Orgel
G
G
Wahl
T R
TR
Gingeras
D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D015345:Oligonucleotide Probes; D011118:Polynucleotide Ligases / Q000378:metabolism*; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology
D015345:Oligonucleotide Probes; D011088:DNA Ligases; D011118:Polynucleotide Ligases