Quantitation of specific fragments in DNA restriction digests: application to the cohesive fragments in lambda DNA digests.
A procedure for the quantitation of reactions between specific members of a set of DNA restriction fragments is presented. Quantitation of the cohesive fragments in NruI nuclease digests of lambda DNA is used as an example. Restriction fragments are resolved on agarose gels and their amounts are estimated from densitometer scans of photographic negatives of ethidium bromide-stained gels. A linear relationship is found between the peak height of given fragment on the scan and the logarithm of the molecular weight of the fragment, arising in part from the stoichiometry of the digest; this relationship allows simple interpolation between the peak heights of the nonreacting fragments in each gel lane to determine the theoretical maximal amount of each reactive fragment in that gel lane. Similar procedures should be applicable to enzymatic ligation or to site-specific cleavage of specific restriction fragments or to autoradiographic detection of the fragments. Since each lane of the gel is analyzed independently, the method is largely self-correcting for variations in amounts applied to the gel.
Zimmerman
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Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Trach
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D010582:Bacteriophage lambda / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004279:DNA, Viral / Q000032:analysis* / Q000378:metabolism; D003720:Densitometry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D005821:Genetic Techniques; D008970:Molecular Weight
D004279:DNA, Viral; D004262:DNA Restriction Enzymes; C055654:endodeoxyribonuclease NruI; D015252:Deoxyribonucleases, Type II Site-Specific