The Significance of PCR Primer Design in Genetic Diversity Studies: Exemplified by Recent Research into the Genetic Structure of Marine Species

Delghandi, Madjid; Goddard, Stephen

doi:10.1007/978-1-0716-1799-1_1

Cited by 5 publications

(4 citation statements)

References 44 publications

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“…This failure may be linked to rapid divergence between congeneric species, leading to erroneous amplification of DNA fragments [69,70]. On the other hand, when assessing species-specific markers, no issue due to PCR artefacts affected our genotyping scoring, although multiplex amplification was performed [71,72].…”

Section: Discussionmentioning

confidence: 99%

Matching an Old Marine Paradigm: Limitless Connectivity in a Deep-Water Fish over a Large Distance

Ferrari,

Spiga,

Rodriguez

et al. 2023

Animals

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Investigations of population structuring in wild species are fundamental to complete the bigger picture defining their ecological and biological roles in the marine realm, to estimate their recovery capacity triggered by human disturbance and implement more efficient management strategies for fishery resources. The Blackspot Seabream (Pagellus bogaraveo, Brünnich 1768) is a commercially valuable deep-water fish highly exploited over past decades. Considering its exploitation status, deepening the knowledge of intraspecific variability, genetic diversity, and differentiation using high-performing molecular markers is considered an important step for a more effective stock assessment and fishery management. With one of the largest efforts conceived of and completed by countries overlooking the Atlantic and Mediterranean coasts in recent years, a total of 320 individuals were collected from different fishing grounds in the Mediterranean Sea and Atlantic Ocean and analysed using 29 microsatellite loci. We applied multiple statistical approaches to investigate the species’ connectivity and population structure across most of its described distribution area. Considering the incomplete knowledge regarding the migratory behaviour of adults, here we suggest the importance of egg and larval dispersal in sustaining the observed genetic connectivity on such a large geographical scale.

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Section: Discussionmentioning

confidence: 99%

Matching an Old Marine Paradigm: Limitless Connectivity in a Deep-Water Fish over a Large Distance

Ferrari,

Spiga,

Rodriguez

et al. 2023

Animals

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“…An important factor in DNA identification is the choice of genetic markers, as well as the choice of primer pairs. Ideal primers are responsible for the high efficiency and specificity of the amplification reaction, which will ensure good amplicon quality [54]. Universal Metazoan COI primers LCO1490 and HCO2198 [25] have been shown to have great versatility and applicability; however, they do not seem to perform well for this octopodid squid.…”

Section: Discussionmentioning

confidence: 99%

Morphological Characteristics and DNA Barcoding of the Rare Blanket Octopus Tremoctopus violaceus (Cephalopoda: Tremoctopodidae) in the Adriatic Sea

Petrić

Dragičević

Stanić³

et al. 2023

Diversity

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Tremoctopods are epipelagic argonautoid octopods characterized by their expanded dorsal webs and strong sexual size dimorphism, with dwarfed males. The scarcity of taxonomic features attributed to this genus presents a challenge, and there is growing evidence of species misidentification in Tremoctopus genus on a molecular level. In this study, we investigated four female specimens of blanket octopus Tremoctopus violaceus caught by purse seine fishing in the Central Eastern Adriatic Sea in 2019. Individuals had smooth, firm and muscular bodies, dark bluish purple on the dorsal and iridescent silvery on the ventral side, with dorsal mantle lengths of 113, 82, 80 and 78 mm. The constructed phylogenetic trees based on the 16S ribosomal RNA and cytochrome c oxidase subunit I sequences of investigated Adriatic specimens and publicly available sequences showed strong support for the T. violaceus clade, consisting of individuals collected from the Adriatic Sea, Mediterranean Sea and Atlantic Ocean, with the exclusion of Indo-Pacific clade most probably corresponding to T. gracilis. To fully understand the life-history traits of Tremoctopus species, future research should focus on DNA-based methods for correct species identification combined with morphological characters, geographic distribution and ecological information.

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“…In the overall design of the assay, we chose not to use a control reaction for the presence of fetal cfDNA. To be useful, a fetal control PCR should closely mimic the amplification efficiency of the primers for the analytical target, and this is not a trivial design problem [ 21 ]. However, a good fetal control would be desirable and for instance also help to detect sample mix up with samples from non-pregnant individuals.…”

Section: Discussionmentioning

confidence: 99%

Non-Invasive Fetal K Status Prediction: 7 Years of Experience

Rieneck

Clausen

Bergholt

et al. 2022

Transfus Med Hemother

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Introduction: In the Kell blood group system, the K and k antigens are the clinically most important ones. Maternal anti-K IgG antibodies can lead to the demise of a K-positive fetus in early pregnancy. Intervention can save the fetus. Prenatal K status prediction of the fetus in early pregnancy is desirable and gives a good basis for pregnancy risk management. We present the results from 7 years of clinical experience in predicting fetal K status as well as some theoretical considerations relevant for design of the assay and evaluation of results. Methods: Blood was collected from 43 women, all immunized against K, at a mean gestational age of 18 weeks (range 10–38). A total of 56 consecutive samples were tested. The KEL*01.01/KEL*02 single nucleotide variant that determines K status was amplified from maternal plasma DNA by PCR without allele specificity. The PCR product was sequenced by NGS technology, and the number of sequenced KEL*01.01 and KEL*02 reads were counted. Prediction of the fetal K status was based on this count and was compared with the serologically determined K status of the newborns. Results: All fetal K predictions were in accordance with postnatal serology where available (n = 34), using our current data analysis. Conclusion: We have developed an NGS-based method for the non-invasive prediction of fetal K status. This approach requires special considerations in terms of primer design, stringent preanalytical sample handling, and careful analytical procedures. We analyzed samples starting at GA 10 weeks and demonstrated the correct prediction of fetal K status. This assay enables timely clinical intervention in pregnancies at risk of hemolytic disease of the fetus and newborn caused by maternal anti-K IgG antibodies.

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The Significance of PCR Primer Design in Genetic Diversity Studies: Exemplified by Recent Research into the Genetic Structure of Marine Species

Cited by 5 publications

References 44 publications

Matching an Old Marine Paradigm: Limitless Connectivity in a Deep-Water Fish over a Large Distance

Matching an Old Marine Paradigm: Limitless Connectivity in a Deep-Water Fish over a Large Distance

Morphological Characteristics and DNA Barcoding of the Rare Blanket Octopus Tremoctopus violaceus (Cephalopoda: Tremoctopodidae) in the Adriatic Sea

Non-Invasive Fetal K Status Prediction: 7 Years of Experience

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