Effectiveness of the postponed isolation (post-frozen isolation) method for PCR-quality Sarcoptes mite gDNA.
The aim of the present study was to assess whether individual Sarcoptes mites collected from frozen skin ('postponed isolation' method) are suitable sources of PCR-quality genomic DNA, and to test the effectiveness of this method in comparison with the 'direct isolation' method, often used through force of habit. Hundreds of single Sarcoptes scabiei samples, resulting from direct (live) or postponed (post-frozen) isolation, were tested using a approximately 450 bp product (ITS-2) and multi-locus 10x genotyping with microsatellite markers. No statistical difference in yield of soluble DNA was found between the two isolation methods. Nevertheless, 19% of the reactions were classified as failed preparations in the direct isolation method, whereas the rate of unsuccessful reactions was 34% in the postponed isolation method. Consequently, post-frozen isolation is suitable and recommendable for Sarcoptes mite gDNA preparation, particularly when performing a balancing act among safety, practicability and profitability. These results have implications for mite collection for DNA extraction, and hence the needed wider leap of Sarcoptes into the genetic era.
Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Campus Las Lagunillas, s/n, 23071 Jaen, Spain. sameralasaad@hotmail.com
Samer
S
Alasaad
Dominga
D
Soglia
Sandra
S
Maione
Stefano
S
Sartore
Ramón C
RC
Soriguer
Jesús M
JM
Pérez
Roberto
R
Rasero
Luca
L
Rossi
D000818:Animals; D015925:Cryopreservation; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction; D012520:Sarcoptes scabiei / Q000235:genetics*; D017422:Sequence Analysis, DNA
D004247:DNA