| First Author | Duff K | Year | 1993 |
| Journal | Mouse Genome | Volume | 91 |
| Issue | 1 | Pages | 118-20 |
| Mgi Jnum | J:4222 | Mgi Id | MGI:52718 |
| Citation | Duff K, et al. (1993) Novel polymorphic AC repeat within the amyloid procursor protein gene on chromosome 16. Mouse Genome 91(1):118-20 |
| abstractText | Full text of Mouse Genome contribution: NOVEL POLYMORPHIC AC REPEAT WITHIN THE AMYLOID PRECURSOR PROTEIN GENE ON CHROMOSOME 16. K. Duff, Korenblat@, M. Wragg@, J. Brown and A. Goate@ Dept. of Biochemistry and Molecular Genetics, St. Mary's Hospital Medical School, London W2 1PG *Present address and address for correspondence: Alzheimer's Disease Research Lab, Psychiatry Center, University of South Florida, Tampa, Florida 33613; Fax. (813) 974 2952 @present address: Dept. of Psychiatry, Washington University Medical School, St Louis, Missouri 63110; Fax. (314) 362 8649 Introduction Sequencing of the mouse homolog of the amyloid precursor protein gene (APP) mapped to 16C3-ter (1) identified a 20 unit AC repeat in the intronic sequence between exons 17 and 18. The repeat was initially identified in strain C129. Amplification of the AC repeat using PCR revealed that the repeat was polymorphic in a number of inbred strains and between the inbred strains and Mus spretus. Materials and methods DNA amplification Two oligomers which flank the AC repeat were synthesized and used to amplify a region of DNA 247 bp long in C129. Primer sequences are as follows: PCR-F GAT TGA AGT AGG GGG AA PCR-R GCC TAC CTA CAT CAA CCT CT PCR mix components are given in table 1. Table 1 Nucleotides (dCTP, dGTP & TTP); Stock conc.: 5mM; Vol for 10 samples (ul): 5. Cold dATP; Stock conc.: 0.625mM; Vol for 10 samples (ul) 2.5. Forward primer; Stock conc.: 20pm/ul; Vol for 10 samples (ul): 5. Reverse primer; Stock conc.: 20pm/ul; Vol for 10 samples (ul): 5. S35SalphadATP; Stock conc.: 600Ci/mM; Vol for 10 samples (ul): 2.5. 1.5mM Mg2+ buffer (eg. Cambio); Stock conc.: 10X; Vol for 10 samples (ul): 12.5(1X). Taq Polymerase (eg. Cambio); Stock conc.: (5U/ul); Vol for 10 samples (ul): 1. Water; Vol for 10 samples (ul): 41.5. 5 ul of genomic mouse DNA (2ng/ul) was added to 7.5 ul of the PCR mix. The sample was overlaid with approximately 50 ul of mineral oil and spun in a microcentrifuge briefly. The PCR conditions are given in table 2. Table 2 Number of cycles: 1; Temperature (degrees C): 94; Time (mins): 3. Number of cycles: 30; Temperature (degrees C): 94; Time (mins): 1; Temperature (degrees C): 51; Time (mins): 1; Temperature (degrees C): 72; Time (mins): 1. Number of cycles: 1; Temperature (degrees C): 72; Time (mins): 10. After completion of the PCR program, 3 ul of formamide containing dye mix (eg. USB stop/loading dye for sequencing reactions) was added to the PCR reactions. The samples could be stored at Ð20 degrees C for 1-2 weeks. Gel electrophoresis Samples were denatured by incubation in a 100 degree C dryblock for 3 minutes. 4ul of each PCR product was then loaded onto a standard 6 % denaturing, polyacrylamide (sequencing) gel. Size marker lanes (G, A, T, C termination reaction products of M13 sequenced from the -40 primer) were loaded alongside the DNA samples. The gel was run in 1X TBE at 70W for 2 hours at 50 degrees C. The gel was dried down onto Whatman paper and exposed to autoradiography film at room temperature overnight. Results PCR was performed at three different annealing temperatures, 50 degrees, 51 degrees and 55 degrees C. Non specific annealing was seen at 50 degrees C whereas at 55 degrees C the reaction was specific but amplification of the product band was very poor. An annealing temperature of 51 degrees C gave a good yield of a single amplified band (Fig. 1). Table 3 summarizes the approximate length of the PCR product amplified from different mouse strains. Table 3 Strain: BALB/c; PCR Product length (bp): 245. Strain: C57BL/6; PCR Product length (bp): 245. Strain: C129; PCR Product length (bp): 247. Strain: CBA; PCR Product length (bp): 245. Strain: M. spretus; Product length (bp): 251. A PCR product length of 247 bp corresponds to a repeat length of 20 AC repeat units in C129. It is expected that the PCR product length polymorphism seen in some of the strains reflects their varying numbers of AC units, although this has not been confirmed by sequencing. The PCR primers used for the mouse failed to anneal either to hamster or human DNA, presumably due to intronic sequence divergence. Figure 1. (Legend). Autoradiograph showing the resolution of AC repeats by Polyacrylamide gel electrophoresis. The repeat size was determined by comparison to M13 DNA sequenced from the -40 universal primer. 0nly the ddATP and ddTTP termination products are shown. Lanes 1 to 5 contain DNA amplified from the following mouse strains. Lane 1: strain BALB/c, lane 2: strain C57BL/6, lane 3: strain C129, lane 4: strain CBA, lane 5: Mus spretus. The polymorphic repeat unit appears to be highly informative and may prove useful in genomic mapping studies. Acknowledgements The authors wish to thank Elsie Damien for donating the genomic DNAs. References 1. Lovett, M., Goldgaber, D., Ashley, P., Cox, D.R., Gajdusek, D.C., and Epstein, C.J. (1987) Biochem. Biophys. Res. Commun. 144: 1069-1075. |
