18 .Isolation of Filarial Parasite DNA from B.malayi  microfilariae


a. Principle :


DNA and RNA exist in cell in combination with other macromolecules such as proteins and carbohydrates. Cells are first lysed with enzyme proteinase and detergents such as SDS or Sarkosyl or Triton X-100. These detergents dissolve the lipids, and thus membranes, releasing the DNA and RNA out of the cell. EDTA is added to chelate Mg++  required for nuclease activity, which otherwise destroys the nucleic acid. Proteins which complex with DNA and RNA are then removed by successive extraction with phenol. Residual phenol must be subsequently removed completely since its traces are inhibitory to most enzymes used in recombinant DNA techniques.   



B. Materials :


1)   TE buffer (pH 8)   (Appendix I)


2)   10% SDS


3)   Proteinase K   (Appendix III)


4)   5 M NaCl


5)   CTAB/ (Cetyl trimethyl ammonium bromide)/ NaCl   (Appendix III)


6)   Chloroform


7)   Isopropanol


8)   Tris saturated phenol   (Appendix III)


9)   Ethanol




c. Method :


1) Take about 2 Lakhs of B. malayi microfilariae in 0.5 ml normal saline in an eppendorf          tube. Spin for 10 minutes at 6000 rpm at room temperature and remove the supernatant.


2) To the Pellet add 567 µl of TE buffer, 30 µl 10 % SDS and 3 µl Proteinase K (20 mg / ml Double distilled Water.). The solution will be watery & sticky.


3) Incubate for 1 hour at 37° C


4) Add 100 µl of 5 M NaCl, and mix thoroughly.


5) Add 80 µl CTAB / NaCl.  mix well, incubate at 65° C for 15 minute.


6) Add 0.4 ml Chloroform and 0.4 ml Tris saturated phenol.


7) Spin for 10 minutes (12000 rpm at room temperature)


8) Take upper aqueous layer and add 0.6 volume of Iso-propanol.


9) Keep for precipitation at –20° C overnight.


10) Spin for 30 minute (10,000 rpm at 4° C).


11) Remove supernatant immediately but slowly with pipette.


12) Keep the pellet in eppendorf tube for drying till the propanol is completely evaporated.


13) Suspend the pellet in TE buffer (100µl in each) and allow it to dissolve.


14) Take out 10µl sample and check for its purity on 0.8% Agarose gel.



Agarose Gel Electrophoresis 


a. Principle:


Extracted DNA can be visualized on Agarose Gel Electrophoresis. The  DNA is simply loaded into the wells and allowed to migrate towards the anode. All molecules should have similar charge/ mass ratio, the rate of migration depends simply on the molecular weight. The bands can be visualized with the help of intercalating agent ethidium bromide under uv light. Using known standards the molecular weight of each fragment can be determined.


B. Material:


1)Agarose powder


2)Ethidium bromide (Appendix I & III)


3)TAE (Tris Acetate) buffer (Appendix I)


4)UV transilluminator.


5)Loading buffer (Appendix I)



C. Method:

1) For 1% Agarose gel weigh 1 gm of Agarose and add it into 100 ml TAE buffer.

2) Heat the slurry in a boiling water bath or in a microwave oven until the agarose dissolves.

3) Cool the solution to 37°C.  Add ethidum bromide to a final concentration of 0.5µg/ml.

4) Seal the edges of a clean dry glass plate with autoclaved tape so as to form a mold.

5) Using a Pasteur pipette, seal the edges of the mold with a small quantity of agarose solution.

6) When the seal is set, pour the rest of the agarose solution into the mold and immediately clamp the comb, the teeth of which will form the sample wells, into position near one end of the gel. Check to see that there is 0.5-1.0 mm of agarose between the bottom of the teeth and the base of the gel, so that the sample wells are completely seal.

7) After the gel is completely set (30-45 minutes at room temperature), carefully remove the comb and autoclaved tape and mount the gel in the electrophoresis tank.

8) Add just enough electrophoresis buffer to cover the gel to a depth of about 1mm.

9) Samples are mixed with loading buffer (Appendix) and are loaded into the slots of the submerged gel. Usually loading buffer is made up as a 6 fold to 10 fold concentrated solution, which is mixed with the sample and then slowly applied to the gel using a disposable micropipette, an automatic micropipetter or if you have a steady hand, a Pasteur pipette.

10)Electrophoresis is carried out at voltage up to 60-70 volts for 1-1.5 hours.

11)After the electrophoresis is over, the bands can be visualized under UV light.