A DNA extraction procedure that allows mite specimens to be slide mounted: phytoseiid species evaluated as a model

作者：Jeyaprakash Ayyamperumal^*; Hoy Marjorie A

来源：Experimental and Applied Acarology, 2010, 52(2): 131-140.

DOI：10.1007/s10493-010-9357-9

• Phytoseiidae
• Metaseiulus occidentalis
• DNA markers
• Guanidinium thiocyanate
• High-fidelity PCR
• High-fidelity RAPD-PCR

Four protocols for extracting DNA from mites, using phytoseiid species as exemplars, were evaluated to determine whether the DNA obtained could be used to amplify nuclear, mitochondrial or Random Amplified Polymorphic DNA (RAPD) markers from males, females and eggs. Protocol 3 was identified as the best and this allowed High-fidelity PCR (Hf-PCR) and Hf-RAPD PCR to be used successfully; it left behind the intact body of adult mites so they could be slide mounted for morphological analyses, although the eggs had to be pricked in order to yield sufficient DNA for amplifications. Protocol 3 involved soaking intact specimens in a GuSCN buffer and using a silica matrix, which binds nucleic acids, to yield DNA for amplification. The DNA isolated could be stored up to a month, indicating that the quality was good. This DNA extraction protocol will allow researchers to collect mites, store them in 95% ethanol, and subsequently extract sufficient DNA from single adults or eggs to provide diagnostic PCR products from both nuclear and mitochondrial DNA, yet leave the bodies intact for morphological analyses.

• 出版日期2010-10