Detection and location of single-base mutations in large DNA fragments by immunomicroscopy.
A technique whereby single-base mutations can be detected by immunomicroscopy of DNA heteroduplexes is described. Four constructs of the filamentous phage M13 were prepared so as to differ by a single base at the same site. Heteroduplexes were prepared and reacted with a water-soluble carbodiimide, with polyclonal antibodies specific for the carbodiimide, and then with a second antibody linked to an electrondense marker. Electron microscopy of the heteroduplexes indicated that the label was located at 4.9 to 5.1 kb in the 7.2-kb phage. The known site of the mismatch was 4.96 kb. Also, plasmids containing inserts of a fragment from the 5' end of hemoglobin A or hemoglobin S were prepared. The median location of the label in heteroduplex molecules was 2.9 kb. The known site of the mismatch was 2.65 kb in the 4.9-kb plasmid. The procedure requires about 10 days to analyze two samples of plasmid or phage DNA.
Ganguly
A
A
Department of Biochemistry and Molecular Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Rooney
J E
JE
Hosomi
S
S
Zeiger
A R
AR
Prockop
D J
DJ
D000755:Anemia, Sickle Cell / Q000235:genetics; D000906:Antibodies; D001483:Base Sequence; D003028:CME-Carbodiimide / Q000031:analogs & derivatives*; D002234:Carbodiimides; D003090:Coliphages / Q000235:genetics; D004247:DNA / Q000235:genetics* / Q000648:ultrastructure; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000235:genetics / Q000648:ultrastructure; D006451:Hemoglobin, Sickle / Q000235:genetics; D006801:Humans; D008854:Microscopy, Electron / Q000379:methods*; D009693:Nucleic Acid Hybridization
D000906:Antibodies; D002234:Carbodiimides; D004279:DNA, Viral; D006451:Hemoglobin, Sickle; C060331:1-cyclohexyl-3-(2-(4-morpholinyl)ethyl)carbodiimide; D003028:CME-Carbodiimide; D004247:DNA