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1.
Wing Hing Wong Ywee Chieh Tay Jayanthi Puniamoorthy Michael Balke Peter S. Cranston Rudolf Meier 《Molecular ecology resources》2014,14(6):1271-1280
Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction (‘direct PCR’) and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands. 相似文献
2.
The isolation of DNA from whole blood by a modified rapid method (RM) was tested using various detergents and buffer conditions.
Extraction of DNA with either NP-40 or Triton X-100 gave a high yield of undegraded DNA in less than an hour. The concentration
of magnesium ion in the buffers was critical to obtaining intact, high molecular weight (HMW) DNA. Greater than 10 mM MgCl2 led to degradation. Addition of EDTA to the buffer inhibits this degradation. Preparation of DNA from blood stored at room
temperature or incubated at 37°C for 24 hr resulted in the same amount and quality of DNA as from samples frozen at −70°C.
DNA from blood samples that had undergone more than four freeze-thaw cycles was found to be partially degraded. The modified
RM can be applied to extract DNA from as little as 10 μl of blood (340 ng of DNA) and from dried blood samples. DNA samples
remained intact and undegraded for longer times when DNA was dissolved in higher concentrations of EDTA.
This work was supported by grants from the Indiana Department of Mental Health and PHS RO1 AG10297. 相似文献
3.
Testing and validating a modified CTAB DNA extraction method to enable molecular parentage analysis of fertilized eggs and larvae of an emerging South African aquaculture species,the dusky kob Argyrosomus japonicus 下载免费PDF全文
This study describes the successful implementation of a modified cetyltrimethyl ammonium bromide (CTAB) protocol to isolate genomic DNA and amplify 14 microsatellite markers from fertilized eggs and larvae of an emerging South African farmed marine fish species, the dusky kob Argyrosomus japonicus. To test and validate the efficiency of this method, genetic data were utilized to resolve parentage and kinship of first‐generation (F1) offspring produced in mass‐spawning events of wild broodstock fish in a commercial hatchery. 相似文献