One-base excess adaptor ligation method for walking uncloned genomic DNA |
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Authors: | Yuki Tonooka Yoichi Mizukami Masahiro Fujishima |
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Institution: | (1) Department of Biology and Chemistry, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan;(2) Center for Gene Research, Yamaguchi University, Minami-kogushi 1-1-1, Ube 755-8505, Japan;(3) Department of Environmental Science and Engineering, Graduate School of Science and Engineering, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan |
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Abstract: | This report describes a novel and efficient method for walking the sequence of a genomic deoxyribonucleic acid (DNA) from
a known region to an unknown region based on an oligodeoxynucleotide (oligo) cassette-mediated polymerase chain reaction technique.
In this method, genomic DNA is digested by a restriction enzyme that generates a sticky 5′-end, followed by ligation of a
one-base excess oligo-adaptor using T4 DNA ligase. The adaptor consists of two complementary oligos that form the same sticky
end as the digested genomic DNA fragments, except that the 5′-overhang base overlaps the corresponding 3′-end base of the
restriction site. This overhanging terminal base prevents ligation between the adaptors, and the appropriate molar ratio of
adaptor to genomic DNA enables specific amplification of the target sequence. T4 DNA ligase catalyzes both the ligation of
the phosphorylated overhang base of the adaptor to genomic DNA and the excision of the corresponding 3′-terminal base of the
genomic DNA. This sequence-specific exonuclease activity of T4 DNA ligase was confirmed by ligation of an alternative adaptor
in which the 5′-terminal base was not consistent with the corresponding 3′-terminal base. Using this technique, the 3′- and
5′-flanking sequences of the catalase gene of the ciliate Paramecium bursaria were determined. |
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Keywords: | Oligo-cassette PCR Sticky end Ligase Paramecium bursaria |
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