Restriction digests of the clone give the following sizes (kb): PstI/HindIII--7.3, 1.55; EcoRI/PvuI--9.0; EcoRI--9.0; ClaI--6.0, 2.8; BglII--9.0. Digestion with HincII gives fragments (bp) of 956, 472, 144, and 54; with Hinf1 of 797, 528, 170, 79, and 52; and with Hpa2 of 803, 747, and 38 from the insert. EcoRI with FspI or SpeI can also be used to excise the insert. EcoRI and FspI cut within the vector and SpeI cuts at nt 8739 (in ATP6). Contains human mitochondrial DNA from nucleotide 7392 to 8921. The insert contains the mutation (A to G) at nucleotide 8344 in the tRNA-Lys gene, creating a CviJI restriction site, which has been identified as the cause of MERRF. Constructed from a PCR product made with linkers to create a PstI site at the nt 7392 end and a HindIII site at the nt 8921 end. |
