PCR amplification of CAG repeats located in exon 1 of HTT in HD cell lines used forward (5′-ATGAAGGCCTTCGAGTCCCTCAAGT CCTTC-3′) and reverse (5′-CTGAGGCAGCAGCGG CTGTGCCTGCG-3′) primers. PCRs were performed in a volume of 25 μl containing 100 ng genomic DNA, 1.6 mM of each dNTP, 4 pmol of each primer, and 0.5 U Q5 DNA polymerase (New England Biolabs). After an initial denaturation of 4 min at 98 °C, 40 cycles of 45 s at 98 °C, 1 min at 68 °C, and 3 min at 72 °C were carried out, followed by a final extension of 10 min. PCR products were resolved in a 7.5% (wt/vol) denaturing polyacrylamide gel, followed by Southern blot analysis using a 32P-labeled (CTG)5 oligonucleotide probe. PCR products were then visualized by an Amersham Typhoon phosphor imager as described (49 (link)).
To analyze the expanded CTG repeats derived from Polθ-catalyzed primer extension, DNA bands (32P-labeled) were excised and eluted from gels, then PCR-reamplified for 35 cycles by REDTaq DNA polymerase (Sigma-Aldrich) using primers 5′-ACGTTGTAAAACGACGGCCA-3′ (forward) and 5′-CATGATTACGAATTC-3′ (reverse), essentially as described above. PCR products were cloned into a pGEM-T vector (Promega) and transfected into E. coli DH5-Alpha (Thermo Fisher). Plasmid DNAs were isolated and subjected to Sanger DNA sequencing (Source BioScience).
To analyze the expanded CTG repeats derived from Polθ-catalyzed primer extension, DNA bands (32P-labeled) were excised and eluted from gels, then PCR-reamplified for 35 cycles by REDTaq DNA polymerase (Sigma-Aldrich) using primers 5′-ACGTTGTAAAACGACGGCCA-3′ (forward) and 5′-CATGATTACGAATTC-3′ (reverse), essentially as described above. PCR products were cloned into a pGEM-T vector (Promega) and transfected into E. coli DH5-Alpha (Thermo Fisher). Plasmid DNAs were isolated and subjected to Sanger DNA sequencing (Source BioScience).
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