Generation of digoxigenin-incorporated probes to enhance DNA detection sensitivity.
Telomere length in humans has been correlated with cancer and age-related diseases. The standard method to measure telomere length relies on Southern blot analysis with radioactively or non-radioactively labeled probes containing several telomeric DNA repeats. However, this approach requires relatively large amounts of genomic DNA, making it difficult to measure telomere length when a limited amount of sample is available. Here, we describe a non-radioactive labeling method that uses 3' fill-in combined with lambda exonuclease digestion to incorporate one or more digoxigenin (DIG) molecules into bridged nucleic acid (BNA)-containing oligonucleotides (ONTs). Using our method, we were able to generate probes to detect both C- and G-rich telomeric DNA strands. Compared with commercially available DIG-labeled telomere probes, probes generated using this new approach significantly enhance the sensitivity of telomere length measurements.
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX.
Tsung-Po
TP
Lai
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX.
Woodring E
WE
Wright
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX.
Jerry W
JW
Shay
D015139:Blotting, Southern / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D004076:Digoxigenin / Q000737:chemistry*; D015336:Molecular Probe Techniques; D015345:Oligonucleotide Probes / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D059505:Telomere Homeostasis / Q000235:genetics*
D015345:Oligonucleotide Probes; D004247:DNA; D004076:Digoxigenin
bridged nucleic acid (BNA); oligonucleotide; telomere; terminal restriction fragments (TRF)