Southern analysis of genomic DNA with unique and degenerate oligonucleotide probes: a method for reducing probe degeneracy.
We have optimized conditions for genomic Southern analysis of eukaryotic DNA with both unique and degenerate oligonucleotide probes. Using the human Factor IX gene, optimal probe concentrations and hybridization times were determined, and washing conditions with sodium chloride and tetramethylammonium chloride (TMA-Cl) were compared. With TMA-Cl, the washing temperature was independent of GC content. With these conditions, the Factor IX gene was detected in genomic DNA using a 128-fold degenerate 20-mer and a 32-fold degenerate 17-mer. This permits the detection of a gene prior to cloning and the reduction of probe degeneracy, and facilitates the isolation of that gene. We apply this method of probe degeneracy reduction using probes for the human Factor VIII gene.
Devlin
P E
PE
Biogen Research Corporation, Cambridge, MA 02142.
Ramachandran
K L
KL
Cate
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RL
D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods; D004247:DNA / Q000235:genetics*; D005164:Factor IX / Q000235:genetics; D005169:Factor VIII / Q000235:genetics; D005796:Genes; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012709:Serum Albumin / Q000235:genetics
D015345:Oligonucleotide Probes; D012709:Serum Albumin; D005169:Factor VIII; D005164:Factor IX; D004247:DNA