Visualization of diagnostic heteroduplex DNAs from cystic fibrosis deletion heterozygotes provides an estimate of the kinking of DNA by bulged bases.
Previous studies (Hsieh, C.-H., and Griffith, J. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4833-4837) of DNAs containing extra or bulged bases on one of the two strands of a duplex showed that they exhibit slower electrophoretic mobility than non-bulged DNAs, indicating that bulges create stiff kinks in the DNA. Here we paired a 97-base single-stranded DNA from the intact cystic fibrosis (CF) gene with a complementary 94-base strand containing a central 3-base deletion (delta F508), common to many CF patients. This produced a 94-base pair DNA with a central 3-base bulge. Visualization of these DNAs by electron microscopy showed that twice as many bulge-containing DNAs had a central kink as compared with the non-bulged controls. Examination of the distribution of kinking angles showed that the bulged population contained 5-7-fold more molecules with a central kink of 80 +/- 10 degrees than did the control molecules. When the 3-base bulge was replaced by a 3-base gap, the resulting duplex DNA showed central kinks with a somewhat lower frequency but greater range of kinking angles.
Wang
Y H
YH
University of North Carolina Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599-7295.
Barker
P
P
Griffith
J
J
D001483:Base Sequence; D002872:Chromosome Deletion; D003550:Cystic Fibrosis / Q000235:genetics*; D004247:DNA / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D006579:Heterozygote; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D016133:Polymerase Chain Reaction
D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D004247:DNA