Methods to precisely estimate molecular weights of fragments constituting RFLP "fingerprints" and a means to standardize measurements within and across laboratories
--J.S.C. Smith, R.A. Tenborg, B. Roth, D. Grant, O.S. Smith, T. Brumback, and T. Beghtol
In order to build a database of RFLP profiles for inbred lines and hybrids using probes that are repeatedly used against membranes made from different gel runs, it is imperative to be able to report data in terms of an inherent property of the DNA fragments themselves. Relative migration in a gel (Rf) is a result not only of the size of the fragment but also of other factors including agarose concentration, buffer strength, temperature, and volt hours applied. Molecular weight is an inherent property of the DNA fragment that, once determined, properly describes that variant. Therefore, we are running gels to profile lines and hybrids with 3 lanes of molecular weight ladder. The ladder is constructed by isolating individual digested fragments of lambda with the final concentrations of each band adjusted so that the individual rungs of the marker ladder are exposed using random prime labelled lambda in approximately equal amounts. Although several commercially prepared marker ladders are available, they provide problems either in 1) having molecular weight components (below 2.0kb) that migrate into and obscure the separations coming from other combs placed lower in the gel; 2) having unequal amounts of DNA at the various rung positions, therefore, producing both over and under exposed components; and 3) having sparse representation of standards in the region of the gel from approximately 23kb to 10kb where small changes in migration equate with relatively large molecular weight differences. The rungs in the molecular weight ladder (restriction digest used to obtain the fragment in parentheses) in current use are as follows: 23.13kb (HindIII); 17.06kb (KpnI); 13.29kb (BglII); 9.42kb (HindIII); 6.56kb (HindIII); 4.36kb (HindIII); 2.32kb (HindIII); and 2.03kb (HindIII). In addition, each lane of genomic DNA is spiked with the slow moving (23.13kb) and fast moving (2.03kb) bands. In order to facilitate the preparation of the ladder, we have cloned lambda fragments below 10kb so that high yields of the smaller components of the ladder can be obtained. Thus, the time consuming testing of suitable dilutions of the ladder components need be done only infrequently. We are also seeking to replace some of the larger rungs of the ladder by using mixes of other digests of lambda. These mixes individually give multiple rungs but with no large disparities between ladder components in their ability to expose film so that adjustments in amount of fewer individual components of the ladder will be necessary. In that respect, a combination of lambda digested by EagI and XhoI provides 5 rungs between 33.5kb and approximately 12kb.
We have tested the potential ability of a BamHI/EcoRI digest of AdenovirusII (AdII)to provide a ladder that can 1) be purchased and would, therefore, obviate the need to isolate individual components of the ladder; 2) give approximately equal exposures of bands throughout the ladder; and 3) give an acceptable coverage of molecular weight standards in profiling. This digestion of AdII provides 13 rungs from 35.94kb to 1.74kb. There are four components above 10kb (35.94, 21.34, 14.33, and 10.68kb). Random prime labelled lambda did not hybridize with the AdII fragments. Individual components of the commercial product (purchased from IBI) gave approximately equal exposures after hybridization with random prime labelled AdII. This product could be used as a standard within and across laboratories to facilitate accurate and repeatable estimates of molecular weight of genomic DNAs. It could be used in one of two ways. First, as a molecular weight ladder loaded into lanes specifically designated as marker lanes. Second, as a spike in every lane of genomic sample. In this latter case, it and digest(s) of lambda would be placed in specific marker lanes also. The first hybridization of the membrane would then be made by lambda and AdII. Subsequent hybridizations would be made with lambda and genomic DNA. The lambda bands would allow an interface to be made of the genomic DNA profile and the 13 AdII BamHI/EcoRI bands that were co- electrophoresed in the genomic lanes. This latter procedure would provide the most precise means of estimating molecular weights for genomic DNA profiles. In either case, AdII is a commercially available product that could provide a good means of standardizing profile scores within and across laboratories.
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