Introduction
It is possible that the plasmid purified in a miniprep may not contain the desired DNA insert due to contamination or mislabeling. It is therefore important to verify the identity of the insert before continuing with additional research involving the cloned fragment. The simplest way to do this is to digest the recombinant plasmid with a restriction enzyme that will produce a characteristic fragmentation pattern, then separate those fragments using agarose electrophoresis to observe a banding pattern appropriate for those fragments.
In order to select an appropriate restriction enzyme, one must examine a restriction map for the DNA sequence to be verified. For genes or genomes that have not been sequenced, restriction maps must be developed from logical analysis of experimental data, as we did in the exercise last week. If the DNA has been sequenced, maps can be generated from the sequence data using computer software or they may be available online in the case of commercially available products (such as plasmid vectors). One usually looks for a restriction enzyme that will cut several times to produce fragments large enough to be observed on a gel. It has been determined that the restriction enzyme Ava II will produce a different fragmentation pattern for each of the three unknown DNA fragments.
Necessary maps
Because our recombinant plasmids are a combination of selected genes and our choice of vector (pUC19) we will not find complete maps of them prepared for us anywhere. We must infer the maps from separate maps of the vector and the genes.
Fig. 1 Restriction map of the pUC19 cloning vector. Total length: 2686 bp. Image from New England Biological technical information
Figure 1 shows the locations of recognition sites on the pUC19 plasmid for some commercially available restriction endonucleases. Those restriction enzymes that cut the plasmid only one time are shown in bold type and those that cut twice are shown in normal type. (Obviously those in the MCS cut only once.) The numbers represent the distance in basepairs from an arbitrary point on the plasmid.
Fig. 2 Restriction map of Id2 PCR product used as a source for an unknown fragment
Fig. 3 Restriction map of P15 PCR product used as a source for an unknown fragment
Fig. 4 Restriction map of MAX PCR product used as a source for an unknown fragment
Figures 2-4 show the approximate locations of recognition sites for several restriction endonucleases on the sequences of the genes used as the source of the three unknown DNA fragments. The site on the end of each fragment labeled "BamH I or Xba I" is either one or the other, depending on which one was used in the double digest before the ligation of the fragment into the MCS of pUC19. The numbers represent the distance in basepairs from the end of the sequence nearest the part of the gene coding for the amino terminal end of the protein. You will use these four maps to predict the fragments that will be cut in the diagnostic Ava II digest.
