Résumé
ABSTRACT Genitalia are rich source of characters in insect taxonomy. Usually, they are examined after dissection and cleaning with potassium hydroxide (KOH), procedure that may damage both genital morphological structures and intracellular molecular contents. Enzymatic procedure with Proteinase K has been used to clean muscle off the genitalia while extract DNA, but its damage to the genital structures has not been evaluated. Herein, we qualitatively compare the use of KOH and Proteinase K to prepare genital structures in minute insects (Hymenoptera: Bethylidae). We show that Proteinase K is better to preserve the genital structure and provides quality DNA.
Sujets)
Animaux , Mâle , Guêpes/anatomie et histologie , ADN/isolement et purification , Endopeptidase K/administration et posologie , Système génital/anatomie et histologie , Guêpes/classification , Guêpes/génétiqueRésumé
OBJECTIVE@#To establish optimal amplification conditions with the application of 0.2 mL test tube in single cell separation and inspection.@*METHODS@#Oral epithelium cell suspension was prepared. Five or ten cells were collected with 0.2 mL test tube. Then DNA were amplified with Identifiler Plus kit in three different conditions in which the proteinase K addition, gold enzyme concentration in PCR reaction, and PCR reaction cycles were adjusted separately. Finally the detection rate, allelic dropout rate and artificial alleles were compared among the groups.@*RESULTS@#In these 3 different conditions: addition of proteinase K, addition of 0.4 microL gold enzyme in PCR reaction, and use of 32 cycles, the detection rate was higher and allelic dropout rate was lower than the other conditions.@*CONCLUSION@#In single cell separation and inspection, the usage of 0.2 mL test tube could be an effective supplement to chip-low volume amplification.