Page 9 - JMR-Vol.03-No.08

Journal of Mosquito Research, 2013, Vol.3, No.8, 58-64

ISSN 1927-646X

http://jmr.sophiapublisher.com

63

gene and 10 ng of genomic DNA. Reactions were

performed in a (BIORAD PCR System iCycler)

thermal cycler. The PCR condition consisted of initial

denaturation step for 4 min at 94

?

followed by 35

cycles of 1 min at 94

?

, 1

min at 40

?

,

and 2 min at

72

?

.

A final extension step was performed at 72

?

for 10 min. A negative control (PCR mix without

DNA) was also prepared alongwith the other samples.

4.4.

Purification of PCR-product

The

0.6

volume of PEG-NaCl was added into the

PCR-product. After a brief vortexing the samples were

incubated at 37

?

for 30~40 minutes followed by

centrifugation at 10000 rpm for 30 minutes. The pellet

was washed twice by adding 100 µL of 70% chilled

ethanol followed by centrifugation at 10 000 rpm for

30

minutes. Finally, the pellet was allowed to air dry

and re-suspended in 10 µL of triple distilled water.

4.5.

Restriction digestion of PCR product

4.5.1.

Single digestion

The purified PCR product of four species were

digested using 10 µL of nuclease free water, 2 µL of

10

X fast digest buffer, 2 µL of DNA, and 1 µL of fast

digest enzymes

Dra

I

,

EcoR

I

,

Taq

I

,

Mbo

I

and

Bgl

II at

37

?

for 5 minutes. A volume of 15 µL of control was

also prepared with all the above components.

Followed by incubation at 65

?

was carried out for 5

minutes to inactivate the enzyme.

4.5.2.

Double digestion

Double digestion was performed in a reaction volume

of 18 µL consisting 10 µL of nuclease free water, 2 µL

of 10 X Fast Digest Buffer, 2 µL of DNA sample and

2

µL of each enzyme was added. A combination of

Mbo

I

+ Dra

I

and

Mbo

I

+ Taq

I was taken. Double

digestion was performed by the procedure similar to

that of single digestion.

4.6.

Statistical analysis

Polymorphism in above mosquito species was

analyzed by scoring the polymorphic and

monomorphic bands. If same band of DNA is present

in all indivisuals or the sample populations under

study it is said to be monomorphism whereas in

polymorphism is defined as discontinuous variation in

a single population. The genetic distance and the

polymorphism among the population were interpreted

by using POPGENE version 1.31 software. A suitable

dendogram was generated with the help of above

software.

Authors' Contributions

Sharma A.K.: The main author of the paper and have made

contribution to conception and design and also the analysis of

data; Tyagi V: This author is involved in the acquisition of

data by using various methods; Chandel K: Helped in the

general supervision of the research group; Mendki MJ:

Involved in drafting of Manuscript; Tikar SN: Revising the MS

critically for important intellectual contents; Sukumaran D:

Have given final approval of the version to be published.

Acknowledgement

The authors are thankful to Prof. (Dr.) M. P. Kaushik, Director,

Defence Research & Development Establishment, Gwalior,

Madhya Pradesh, India for providing all necessary facility to

conduct this research work. Sincere thanks also to the scientists

and supportive staff of Vector Management Division for their

kind cooperation for carrying out the above work.

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