Authors: Sushanta Kumar Barik, Rupenangshu Kumar Hazra, Manas Ranjan Prusty, Animesha Rath & Santanu Kumar Kar
We represent a simplified and rapid version of DNA extraction protocol from different mosquito species which is suitable for polymerase chain reaction(PCR) and other molecular biology works. The protocol involves three steps like lysis, phenol : chloroform (1:1) extraction and two fold isopropanol precipitation at -20 degree celcius using 1X STE buffer (50mM NaCl, 50mM Tris-Hcl,100mM EDTA,PH 8.0).The proposed extraction protocol has an advantage of DNA extraction from mosquitoes using 1X STE buffer at 37ºcelcius which prevent DNA degradation at higher temperature and kept DNA stability in long term storage . The protocol proved by three different mosquito species and removing for potential contamination showed that the protocol yields good quantity and quality DNA, typically better than commercial kits .The protocol was evaluated by polymerase chain reaction (PCR). The protocol of DNA extraction is a time saving as well as economies with available laboratory chemicals, consumables and basic equipments. The protocol is adoptable for laboratories in external and internal funded research projects with large numbers of mosquito samples in a small period .The protocol minimize the time for other DNA extraction protocols in two to three days and provides a workflow for mapping of malaria vectors ,their vectorial transmission in the area of translational research.
Insect research including population genetics ,taxonomic and evolutionary field always invite molecular techniques .Molecular techniques followed good quality protocols like DNA extraction from a large number of samples in terms of time , money and equipment access. Several methods of DNA extraction have been published using Chelex-100, Sarcosil with Proteinase K, CTAB, Polyvinyal pyrolidone and mercaptoethanol ,Only CTAB ,CTAB with mercaptoethanol ,CTAB with SDS, DIGSOL buffer etc.1,2,3. A non destructive DNA extraction procedure have great potential on insect specimen rather than a destructive and time consuming protocol 4 .A universal and rapid salt of high quality genomic DNA extraction method was used to perform hundreds of pcr based reactions 5.
DNA extraction at room temperature prevent DNA degradation and is suitable for polymerase chain reaction using EDTA , Proteinase K and Guanidinium thiocyanate 6 . Most commercial insect DNA extraction kits (Merk , prep G, DNeasy kit, Qiagen, Germany EM, Charge Switch and Aquapur DNA extraction kits ) are not cost effective and involve use of expensive laboratory equipments. We developed a simple and very efficient DNA isolation protocol from mosquito species for pcr related works in our laboratory.
The reagents which were used to make the STE(Sodium Chloride ,Tris –HCl, EDTA, Ph. 8.0) buffer acting as a lysis buffer in this protocol.
This protocol consists of three phases like( 1)Incubation phase (2)Washing /Extraction phase (3) Precipitation phase.
The end of DNA isolation is a 70% ethanol washing step contribute to removal of proteins , Then dissolved in nuclease free water or TE buffer (Ph 8.0). The total DNA isolation protocol completed within 4 hours 30 minutes for a single mosquito but two to three days in case of other published protocols
The protocol described here offers a rapid and efficient DNA extraction suitable for polymerase chain reaction. The protocol is used for identification of mosquito species in a multiplex polymerase chain reaction.
In this protocol, the quality and quantity of DNA is generally good. The purity value of DNA samples ranges from >1.8 and is suitable for polymerase chain reaction . The amount of DNA ranges from 50-150ng from a single mosquito.The protocol was proved for mosquito species like Anopheles, Culex and Aedes using a pair of D3 primers(D3 forward 5’-GACCCGTCTTGAAACACGGA-3’and D3 reverse 5’ TCGGAAGGAACCAGCTACTA-3’) in the 28s rDNA region 7 . The PCR conditions were 1X PCR buffer ,2mM MgCl2,500mM dNTP ,1mM of each primer(20pico mole) and 1 unit of Taq DNA polymerase per 10 µl reaction volume. (MP biomedicals).The DNA template 1 µl was used for this pcr reaction mix. The reaction conditions were 94º celcius for 5 minutes before 35 cycles of amplification,94ºcelcius 30 sec,50º celcius for 45 sec and 72 º celcius for 1 minute, followed by a final extension 8 minutes. The 10 microliter PCR amplification product was subjected to electrophoresis in a 1% agarose(Bangalore genei) stained with ethidium bromide in 1X TBE buffer
Thanks to Indian Council Of Medical Research providing financial assistants in the malaria translational research.
Manuscript: A simple, rapid and very efficient protocol for DNA isolation from mosquito species
Flow chart diagram of DNA extraction from mosquitoes: A simple,rapid and very efficient protocol from mosquito species
The protocol description is attached in a manuscript form
Sushanta Kumar Barik, Rupenangshu Kumar Hazra, Hazra"s Lab group, Regional Medical Research Centre Indian Council Of Medical Research Chandrasekharpur Bhubaneswar India
Manas Ranjan Prusty, Animesha Rath & Santanu Kumar Kar, Unaffiliated
Correspondence to: Rupenangshu Kumar Hazra ([email protected])
Source: Protocol Exchange (2013) doi:10.1038/protex.2013.007. Originally published online 15 January 2013.