Detection and location of single-base mutations in large DNA fragments by immunomicroscopy. Academic Article

abstract

• 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.

authors

• Prockop, Darwin

published proceedings

• Genomics

author list (cited authors)

• Ganguly, A., Rooney, J. E., Hosomi, S., Zeiger, A. R., & Prockop, D. J.

citation count

• 23

complete list of authors

• Ganguly, A||Rooney, JE||Hosomi, S||Zeiger, AR||Prockop, DJ

publication date

• January 1989

publisher

• Elsevier  Publisher

published in

• Genomics  Journal

keywords

• Anemia, Sickle Cell
• Antibodies
• Base Sequence
• CME-Carbodiimide
• Carbodiimides
• Coliphages
• DNA
• DNA Mutational Analysis
• DNA, Viral
• Hemoglobin, Sickle
• Humans
• Microscopy, Electron
• Nucleic Acid Hybridization

Digital Object Identifier (DOI)

• 10.1016/0888-7543(89)90276-0

start page

• 530

end page

• 538

volume

• 4

issue

• 4

URL

• http://dx.doi.org/10.1016/0888-7543(89)90276-0