Digital PCR

Vogelstein, Bert; Kinzler, Kenneth W.

doi:10.1073/pnas.96.16.9236

Cited by 1,516 publications

(1,102 citation statements)

References 53 publications

Supporting

Mentioning

1,065

Contrasting

Unclassified

Order By: Relevance

“…We confirmed the real‐time qPCR results using droplet digital PCR or ddPCR (Vogelstein & Kinzler, 1999), in particular for the individuals presenting the highest levels of amplification, as this second approach is more reliable in these conditions. For the ddPCR assay, 10 ng of DNA was assayed in a final volume of 20 μl containing 1× ddPCR EvaGreen ® supermix and 0.1 μM of each primer (Del97Qdir5 and Del97Qrev4, Table S1).…”

Section: Methodssupporting

confidence: 80%

Adaptive deletion in resistance gene duplications in the malaria vector Anopheles gambiae

Assogba

Alout

Koffi

et al. 2018

Evolutionary Applications

View full text Add to dashboard Cite

While gene copy‐number variations play major roles in long‐term evolution, their early dynamics remains largely unknown. However, examples of their role in short‐term adaptation are accumulating: identical repetitions of a locus (homogeneous duplications) can provide a quantitative advantage, while the association of differing alleles (heterogeneous duplications) allows carrying two functions simultaneously. Such duplications often result from rearrangements of sometimes relatively large chromosome fragments, and even when adaptive, they can be associated with deleterious side effects that should, however, be reduced by subsequent evolution. Here, we took advantage of the unique model provided by the malaria mosquito Anopheles gambiae s.l. to investigate the early evolution of several duplications, heterogeneous and homogeneous, segregating in natural populations from West Africa. These duplications encompass ~200 kb and 11 genes, including the adaptive insecticide resistance ace‐1 locus. Through the survey of several populations from three countries over 3–4 years, we showed that an internal deletion of all coamplified genes except ace‐1 is currently spreading in West Africa and introgressing from An. gambiae s.s. to An. coluzzii. Both observations provide evidences of its selection, most likely due to reducing the gene‐dosage disturbances caused by the excessive copies of the nonadaptive genes. Our study thus provides a unique example of the early adaptive trajectory of duplications and underlines the role of the environmental conditions (insecticide treatment practices and species ecology). It also emphasizes the striking diversity of adaptive responses in these mosquitoes and reveals a worrisome process of resistance/cost trade‐off evolution that could impact the control of malaria vectors in Africa.

show abstract

Section: Methodssupporting

confidence: 80%

Adaptive deletion in resistance gene duplications in the malaria vector Anopheles gambiae

Assogba

Alout

Koffi

et al. 2018

Evolutionary Applications

View full text Add to dashboard Cite

show abstract

“…The discrete nature of compartments enables digital quantification of absolute numbers of nucleic acids in a sample,3 accurate estimation of copy‐number variation,4 detection of pathogens5 and rare cancer mutations,6 as well as other applications 7…”

mentioning

confidence: 99%

DNA Nanoparticles for Improved Protein Synthesis In Vitro

et al. 2016

View full text Add to dashboard Cite

The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 104 clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription–translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro.

show abstract

“…Detailed information about the cohort and replicate counts for each age group can be found in supporting information (Appendix S1: Table S1). We then used a digital PCR‐based mutation capture assay (Vogelstein & Kinzler, 1999) to detect deletions in mtDNA samples obtained in mitochondrial preparations from cohorts of aging C. elegans (Figure 2). Each mutation capture experiment involved scanning for mtDNA deletions using a set of overlapping primer pairs.…”

Section: Resultsmentioning

confidence: 99%

Clonal expansion of mitochondrial DNA deletions is a private mechanism of aging in long‐lived animals

Lakshmanan

Yee

et al. 2018

Aging Cell

View full text Add to dashboard Cite

Disruption of mitochondrial metabolism and loss of mitochondrial DNA (mtDNA) integrity are widely considered as evolutionarily conserved (public) mechanisms of aging (López‐Otín et al., Cell, 153, 2013 and 1194). Human aging is associated with loss in skeletal muscle mass and function (Sarcopenia), contributing significantly to morbidity and mortality. Muscle aging is associated with loss of mtDNA integrity. In humans, clonally expanded mtDNA deletions colocalize with sites of fiber breakage and atrophy in skeletal muscle. mtDNA deletions may therefore play an important, possibly causal role in sarcopenia. The nematode Caenorhabditis elegans also exhibits age‐dependent decline in mitochondrial function and a form of sarcopenia. However, it is unclear if mtDNA deletions play a role in C. elegans aging. Here, we report identification of 266 novel mtDNA deletions in aging nematodes. Analysis of the mtDNA mutation spectrum and quantification of mutation burden indicates that (a) mtDNA deletions in nematode are extremely rare, (b) there is no significant age‐dependent increase in mtDNA deletions, and (c) there is little evidence for clonal expansion driving mtDNA deletion dynamics. Thus, mtDNA deletions are unlikely to drive the age‐dependent functional decline commonly observed in C. elegans. Computational modeling of mtDNA dynamics in C. elegans indicates that the lifespan of short‐lived animals such as C. elegans is likely too short to allow for significant clonal expansion of mtDNA deletions. Together, these findings suggest that clonal expansion of mtDNA deletions is likely a private mechanism of aging predominantly relevant in long‐lived animals such as humans and rhesus monkey and possibly in rodents.

show abstract

Digital PCR

Cited by 1,516 publications

References 53 publications

Adaptive deletion in resistance gene duplications in the malaria vector Anopheles gambiae

Adaptive deletion in resistance gene duplications in the malaria vector Anopheles gambiae

DNA Nanoparticles for Improved Protein Synthesis In Vitro

Clonal expansion of mitochondrial DNA deletions is a private mechanism of aging in long‐lived animals

Contact Info

Product

Resources

About