Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: DNA-PKcs is critical for telomere capping
Figure Lengend Snippet: DNA-PKcs-deficient MEFs exhibit similar telomere lengths and telomerase activities when compared with wild-type MEFs. ( A ) Southern analysis of MEFs. Early passage MEFs from independently isolated littermates were prepared from wild-type (lanes 7 and 8), DNA-PKcs+/− (lanes 1, 2, 5, and 6), and DNA-PKcs−/− (lanes 3 and 4). Gel plugs containing genomic DNAs were digested with Rsa I and Hin fI (odd number lanes) or undigested (even number lanes), fractionated by pulse-field gel electrophoresis, and hybridized with the telomeric specific [TTAGGG] 3 probe. The approximate sizes of the products (kb) are indicated based on molecular weight markers. The Southern hybridization signal observed with the [TTAGGG] 3 probe under these conditions was sensitive to BAL-31 exonuclease digestion, suggesting that this is telomeric DNA (data not shown). ( B ) PCR analysis for genotyping. Using the specific primer pairs (see Materials and Methods ), wild-type and targeted alleles were amplified as products of 450 bp and 360 bp, respectively. Lanes 2 and 4 show the DNA-PKcs+/− pattern, lane 3 shows the DNA-PKcs−/− pattern, and lane 5 shows the wild-type pattern. Lane 1 contains a size marker. ( C ) Telomerase activity in DNA-PKcs-deficient MEFs. TRAP assay was performed after 30 PCR cycles on cell extracts (10, 10 2 , and 10 3 cells) prepared from DNA-PKcs−/− (lanes 1–3), DNA-PKcs+/− (lanes 4–6), and wild-type (lanes 7–9) MEFs. In lanes 10–12, a serial dilution of HeLa cell lysate was run as a positive control for quantitating relative telomerase activity levels. Lane 13 contains a negative control without cell lysate. IC denotes a standard internal control for PCR efficiency.
Article Snippet: DNA was digested with Hin fI and Rsa I (New England Biolabs) and electrophoresed on a 1% TBE agarose gel at 14°C, using a CHEF DR-II pulsed-field apparatus (Bio-Rad).
Techniques: Isolation, Nucleic Acid Electrophoresis, Molecular Weight, Hybridization, Polymerase Chain Reaction, Amplification, Marker, Activity Assay, TRAP Assay, Serial Dilution, Positive Control, Negative Control