Restriction digests of the clone give the following sizes (kb): EcoRI--9; HindIII--9; PstI--3.2, 2.1, 1.05, 0.76. 0.32; BamHI--2.6, 2.1, 1.7, 1.15, 0.41. The vector is pcD minus the SV40 early region promoter. The BamHI fragment (2355/3466) contains exons 1 - 8 and has been used to detect the TaqI polymorphism. A 1.7 kb HindIII(2337)/BglII(4903) fragment has been used as a single copy probe. The insert, which is a full-length copy of the cDNA, is the same as that described for pLDLR-2. The Alu repeat sequences in the cDNA occur in a 1.7 kb BamHI fragment (5874-7600). A SmaI(5191)/XbaI(2349) or SmaI/SalI(2343) or SmaI/HindIII(2337) double digest should generate a single-copy probe. Recorded as not polymorphic by Collaborative Research, meaning either not polymorphic in 0.8% agarose gels, or difficult to work with. The AvaII polymorphic site is in exon 3. The non-polymorphic XbaI site is in intron 12 and is used to yield more separable bands. The PvuII site polymorphism is part of an Alu repeat in intron 15. The sequence of one allele is CGGCTG, the other is CAGCTG (PvuII site). ApaLI digest shows 3 alleles: 26.4, 22.4, 19.6 kb. ApaLI/BamHI digest shows 4 alleles. (E) alleles detected by 3' exon 18 probe from pLDLR3 map to 3' flanking region. D and E alleles are in linkage disequilibrium. D2 in not detected in the absence of Enzyme(s) not detecting polymorphism: BamHI, BglII, EcoRI, EcoRV, HindIII, KpnI, SacI, SstI, SstII, SphI, XbaI, XhoI. |
