A PCR-mediated method for cloning spot DNA on restriction landmark genomic scanning (RLGS) gel.
A PCR-mediated direct cloning for target spot DNA from RLGS gel has been established. The method consists of PCR amplification of adaptor-ligated spot DNA fragments without excluding similar-sized DNA fragments co-localized on RLGS gel, and following selective ligation with the NotI-dT vector. Applying this method, we have successfully cloned several DNA fragments derived from target spots whose intensities change developmentally due to DNA methylation in the telencephalon of C3H/HeN mice. Since only a few micrograms of total DNA is sufficient for our spot cloning, our method may be highly useful when the total DNA sample prepared for cloning is limited.
Suzuki
H
H
Shionogi Institute for Medical Science, Shionogi & Co., Ltd., Osaka, Japan.
Kawai
J
J
Taga
C
C
Ozawa
N
N
Watanabe
S
S
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping; D013687:Telencephalon / Q000737:chemistry
D004247:DNA