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Abstract Telomere length dynamics are an established biomarker of health and ageing in animals. The study of telomeres in numerous species has been facilitated by methods to measure telomere length by real‐time quantitative PCR (qPCR). In this method, telomere length is determined by quantifying the amount of telomeric DNA repeats in a sample and normalizing this to the total amount of genomic DNA. This normalization requires the development of genomic reference primers suitable for qPCR, which remains challenging in nonmodel organism with genomes that have not been sequenced. Here we report reference primers that can be used in qPCR to measure telomere lengths in any vertebrate species. We designed primer pairs to amplify genetic elements that are highly conserved between evolutionarily distant taxa and tested them in species that span the vertebrate tree of life. We report five primer pairs that meet the specificity and reproducibility standards of qPCR. In addition, we demonstrate an approach to choose the best primers for a given species by testing the primers on multiple individuals within a species and then applying an established computational tool. These reference primers can facilitate qPCR‐based telomere length measurements in any vertebrate species of ecological or economic interest.
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Determining Trap Compliances, Microsphere Size Variations, and Response Linearities in Single DNA Molecule Elasticity Measurements with Optical Tweezers
We previously introduced the use of DNA molecules for calibration of biophysical force and displacement measurements with optical tweezers. Force and length scale factors can be determined from measurements of DNA stretching. Trap compliance can be determined by fitting the data to a nonlinear DNA elasticity model, however, noise/drift/offsets in the measurement can affect the reliability of this determination. Here we demonstrate a more robust method that uses a linear approximation for DNA elasticity applied to high force range (25–45 pN) data. We show that this method can be used to assess how small variations in microsphere sizes affect DNA length measurements and demonstrate methods for correcting for these errors. We further show that these measurements can be used to check assumed linearities of system responses. Finally, we demonstrate methods combining microsphere imaging and DNA stretching to check the compliance and positioning of individual traps.
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- Award ID(s):
- 1716219
- PAR ID:
- 10286835
- Date Published:
- Journal Name:
- Frontiers in Molecular Biosciences
- Volume:
- 8
- ISSN:
- 2296-889X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fmolb.2021.605102
