Solid-phase nested deletion: a new subcloning-less method for generating nested deletions.
We have developed a new subcloning-less method for generating nested deletions which we have termed Solid-Phase Nested Deletion. The basic procedure for this method is as follows. The target DNA fragment is cloned in the multiple cloning site of a cloning vector, pUC or its derivatives, and amplified by PCR using a set of primers, one of which is 5'-biotinylated. The amplified DNA is partially digested by a restriction enzyme with a 4-base recognition sequence. The digested DNA is ligated with a synthetic adapter DNA. Monodiverse beads coupled with streptavidin (Dynabeads M-280 streptavidin) are added to the mixture and the biotinylated DNA fragments are separated by applying magnetic field. The unidirectionally deleted DNA fragments are recovered by PCR from the magnetic beads, and size-fractionated by agarose gel electrophoresis. The DNA fragments are amplified by PCR and used for sequencing. We demonstrate the potential of this method using a 4878-bp EcoRI fragment of lambda phage DNA.
Yohda
M
M
Chemical Engineering Laboratory, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Kato
N
N
Endo
I
I
D010582:Bacteriophage lambda / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D015246:Deoxyribonuclease EcoRI / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D008280:Magnetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA / Q000379:methods*; D017384:Sequence Deletion
D015246:Deoxyribonuclease EcoRI