Retrieval and amplification of single-copy genomic DNA from a nanometer region of chromosomes: a new and potential application of atomic force microscopy in genomic research.
Using a modified tip of the atomic force microscope (AFM), we harvested several strands of genomic DNA from a nanometer region of mouse chromosomes. We have also co-developed a random PCR method to amplify the recovered genomic DNA, in which a single DNA molecule of several kilobasepairs could be amplified efficiently. A subsequent fluorescence in situ hybridization (FISH) indicated that the amplified DNA originally came from the tip-manipulated regions of mouse chromosomes. Several fragments containing unique sequences were identified using Southern hybridization after subcloning the PCR products into pUC18 plasmid. The present results showed a potential application of AFM to genomic analysis.
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Laboratory of Biodynamics, Tokyo Institute of Technology, Yokohama, Japan.
Ikai
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D016475:3T3 Cells; D000818:Animals; D015139:Blotting, Southern / Q000379:methods; D002874:Chromosome Mapping / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers; D005784:Gene Amplification; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D051379:Mice; D018625:Microscopy, Atomic Force / Q000379:methods*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004247:DNA