title: A method for generating selective DNA probes for the analysis of C-negative regions in human chromosomes.
abstract: Linker-adapter polymerase chain reaction (LA-PCR) is among the most efficient techniques for whole genome DNA amplification. The key stage in LA-PCR is the hydrolysis of a DNA sample with restriction endonucleases, and the choice of a restriction endonuclease (or several endonucleases) determines the composition of DNA probes generated in LA-PCR. Computer analysis of the localization of the restriction sites in human genome has allowed us to propose an efficient technique for generating DNA probes by LA-PCR using the restriction endonucleases HaeIII and RsaI. In silico hydrolysis of human genomic DNA with endonucleases HaeIII and RsaI demonstrate that 100- to 1,000-bp DNA fragments are more abundant in the gene-rich regions. Applying in situ hybridization to metaphase chromosomes, we demonstrated that the produced DNA probes predominantly hybridized to the C-negative chromosomal regions, whereas the FISH signal was almost absent in the C-positive regions. The described protocol for generating DNA probes may be successfully used in subsequent cytogenetic analysis of the C-negative chromosomal regions.
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Division, Russian Academy of Sciences, Novosibirsk. morozkin @ niboch.nsc.ru
authors.forename: E S
authors.initials: ES
authors.lastname: Morozkin
authors.forename: E M
authors.initials: EM
authors.lastname: Loseva
authors.forename: T V
authors.initials: TV
authors.lastname: Karamysheva
authors.forename: V N
authors.initials: VN
authors.lastname: Babenko
authors.forename: P P
authors.initials: PP
authors.lastname: Laktionov
authors.forename: V V
authors.initials: VV
authors.lastname: Vlassov
authors.forename: N B
authors.initials: NB
authors.lastname: Rubtsov
mesh_terms: D001483:Base Sequence; D002877:Chromosomes, Human / Q000737:chemistry / Q000235:genetics; D003582:Cytogenetics / Q000379:methods*; D003596:Cytosine / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D015894:Genome, Human; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008677:Metaphase; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D012339:RNA, Ribosomal, 28S / Q000737:chemistry / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D012339:RNA, Ribosomal, 28S; D003596:Cytosine; D004247:DNA; D004262:DNA Restriction Enzymes