A differential cloning procedure of complex genomic DNA fragments.
We have developed a differential cloning procedure designed for cloning of anonymous restriction DNA fragments whose molecular sizes differ between two genomic DNA preparations from higher organisms. The procedure, which was extensively revised from the original one, consists of several steps as summarized below. (i) Digestion of two DNA preparations (target and reference DNA) with the same restriction enzyme (4 base cutter). (ii) Biotinylation of target DNA and conversion of reference DNA to nonamplifiable form by terminal dephosphorylation. (iii) Electrophoresis of the two DNA preparations through a synthetic gel with a large excess of reference DNA as a competitor. (iv) In-gel alkaline dissociation of DNA, followed by reassociation (in-gel competitive reassociation). (v) Elution of DNA from the gel and PCR after adapter ligation and adsorption of DNA onto streptavidin-coated matrix. By repeating these steps, we attained substantial enrichment (approximately 10,000-fold) of DNA fragments which were originally present at one copy or less per complex mammalian genome. The details of the procedure and its unique characteristics in cloning of altered genomic DNA fragments, particularly from mammalian genome, are discussed.
Yokota
H
H
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Amano
S
S
Yamane
T
T
Ataka
K
K
Kikuya
E
E
Oishi
M
M
D000228:Adenine Phosphoribosyltransferase / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001710:Biotin; D001921:Brain / Q000378:metabolism*; D016466:CHO Cells; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004586:Electrophoresis / Q000379:methods; D004926:Escherichia coli; D005260:Female; D007202:Indicators and Reagents; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D017208:Rats, Wistar; D015183:Restriction Mapping; D014162:Transfection
D017931:DNA Primers; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D001710:Biotin; D004247:DNA; D000228:Adenine Phosphoribosyltransferase