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Genetics

Microbial Genetics

Plant Genetics

Microbial Genetics

Staphylococcus Aureus

Nocardis Otitidis

Pseudomonas Aeruginosa

Serratia Marcescens

Enterobacter Aerogenes

Klebsiella Pneumoniae

Microbial Identification
(16S rDNA)

Nocardis Otitidis ATCC 14630


PROJECT REPORT

NAME OF SCIENTIST:
Mr. Mahendra Kumar Trivedi

SERVICE:
DNA FINGERPRINTING SERVICE (BY RAPD)

SERVICE NO.:
DF 58

Project Title	DNA Fingerprinting by RAPD Analysis
Cat#:	CSER 48
Name of Scientist:	Mr. Mahendra Kumar Trivedi
Institute:	Life Energies ltd, Mumbai.
Sample name:	Nocardia otitidis ATCC 14630

Objective:

The objective of this project is to Genotype five samples (CONTROL Versus TREATED- A, A1, B, B1) of the same line with five primers on Nocardia otitidis ATCC 14630.

Job Requirement:

Isolation of genomic DNA from Nocardia otitidis samples.
Generating RAPD-Fingerprinting profiles showing Polymorphic bands with five RAPD primers

Gram-positive material and primers

Three series of Inoculums (Control, Series A and Series B) were prepared for Nocardia otitidis. The Control series was procured from Bangalore Genei while the Series "A" and series 'B' were handed over to Mr. Mahendra Kumar Trivedi in sealed tubes on 23rd January 2008 during his visit to our R&D facility at Bangalore. On the same day, we provided one isolated room to Mr. Trivedi where he subjected the Series 'A' and Series 'B' to his treatment modality.

Whilst handing over these cultures to Mr. M. K. Trivedi for treatment purposes, optimum precautions were taken to avoid the contamination.

After the presumed treatment, Mr. Trivedi returned the samples to Ms. Bindu Damodaran (Designation - Executive Manager - Microbiology) in the same condition as handed over to him in an hour's time on the same day.

We did not know the treatment modality as it was kept confidential throughout the project.

MATERIALS & METHODS:

Sub culturing of treated samples.

1% Inoculum from two treated tubes (series A and series B) was taken and inoculated to fresh 5 ml medium and incubated at 37 C with 160 rpm for 18 hrs and labelled as series A1 and B1 respectively. The cultures were spun down and genomic DNA was isolated.

Genomic DNA was isolated using Ultrapure Genomic DNA prep Kit (Cat# KT 83).

Steps Involved:

Genomic DNA was isolated from Nocardia otitidis samples (Control, Treated A, Treated B provided by Mr. M. K. Trivedi and sub cultured samples Treated A1, Treated B1 provided by Bangalore Genei) using Ultrapure Genomic DNA prep Kit (Cat# KT 83).
Performed RAPD reactions using five RAPD primers (RBA Series used separately) to find polymorphism between control and treated samples under PCR conditions as mentioned:

Cycle condition:

94°C	94°C	35°C	72°C	94°C	38°C	72°C	72°C
7 min	60 sec	60 sec	120 sec	60 sec	60 sec	90 sec	7 min
	8 cycles			35 cycles

12 of PCR products were loaded on to 1.5 % agarose gel and resolved by electrophoresis. The gel was subsequently stained with ethidium bromide and viewed under UV-light. Photographs were documented subsequently (attached).
Polymorphic DNA bands are marked by arrows on the copy of the report.

Fig. 1. RAPD Profile of Nocardia otitidis samples Generated using Genei
RAPD primer RBA 8A, RBA 13A & RBA 20A.

Lane Description:

1: Control
2: Treated A
3: Treated A.1
4: Treated B
5: Treated B.1
M:100 bp Ladder (GeneiTM) Cat # RMBD19S;

Fig. 2. RAPD Profile of Nocardia otitidis samples Generated using Genei
RAPD primer RBA 10A & RBA 15A.

Lane Description:

1: Control
2: Treated A
3: Treated A.1
4: Treated B
5: Treated B.1
M:100 bp Ladder (GeneiTM) Cat # RMBD19S;

Calculation:
	A/ Bx100 = Percent polymorphism

Where,
	A= Polymorphic bands in treated plant B= No of bands in control

Sl. No	Bands scored	Description	Primer
1	19	No band is Common to Control and Treated samples; One band is unique to Control sample; One band is unique to Treated sample A; Five bands are unique to Treated sample A1; One band is unique to Treated sample B; No band is unique to Treated sample B1.	RBA 8A
2	21	Two bands are Common to Control and Treated samples; Three bands are unique to Control sample; Two bands are unique to Treated sample A; No band is unique to Treated sample A1; Four bands are unique to Treated sample B; No band is unique to Treated sample B1.	RBA 13A
3	15	Two bands are Common to Control and Treated samples; Three bands are unique to Control sample; One band is unique to Treated sample A; No band is unique to Treated sample A1; Four bands are unique to Treated sample B; No band is unique to Treated sample B1.	RBA 20A
4	19	No bands are Common to Control and Treated samples; Two bands are unique to Control sample; Three bands are unique to Treated sample A; One band is unique to Treated sample A1; Three bands are unique to Treated sample B; No band is unique to Treated sample B1.	RBA 10A
5	25	Two bands are Common to Control and Treated samples; Two bands are unique to Control sample; Three bands are unique to Treated sample A; No band is unique to Treated sample A1; One band is unique to Treated sample B; No band is unique to Treated sample B1.	RBA 15A

RESULTS & DISCUSSION:

Control & Treated sample A

1. The level of true polymorphism between Control & Treated sample A was evaluated at 15 % with RBA 8A primer.

2. The level of true polymorphism between Control & Treated sample A was evaluated at 53 % with RBA 13A primer.

3. The level of true polymorphism between Control & Treated sample A was evaluated at 50 % with RBA 20A primer.

4. The level of true polymorphism between Control & Treated sample A was evaluated at 54 % with RBA 10A primer.

5. The level of true polymorphism between Control & Treated sample A was evaluated at 56 % with RBA 15A primer.

Control & Treated sample A1

6. The level of true polymorphism between Control & Treated sample A1 was evaluated at 53 % with RBA 8A primer.

7. The level of true polymorphism between Control & Treated sample A1 was evaluated at 60 % with RBA 13A primer.

8. The level of true polymorphism between Control & Treated sample A1 was evaluated at 35 % with RBA 20A primer.

9. The level of true polymorphism between Control & Treated sample A1 was evaluated at 90 % with RBA 10A primer.

10. The level of true polymorphism between Control & Treated sample A1 was evaluated at 31 % with RBA 15A primer.

Control & Treated sample B

11. The level of true polymorphism between Control & Treated sample B was evaluated at 15 % with RBA 8A primer.

12. The level of true polymorphism between Control & Treated sample B was evaluated at 100 % with RBA 13A primer.

13. The level of true polymorphism between Control & Treated sample B was evaluated at 57 % with RBA 20A primer.

14. The level of true polymorphism between Control & Treated sample B was evaluated at 36 % with RBA 10A primer.

15. The level of true polymorphism between Control & Treated sample B was evaluated at 25 % with RBA 15A primer.

Control & Treated sample B1

16. The level of true polymorphism between Control & Treated sample B1 was evaluated at 7 % with RBA 8A primer.

17. The level of true polymorphism between Control & Treated sample B1 was evaluated at 60 % with RBA 13A primer.

18. The level of true polymorphism between Control & Treated sample B1 was evaluated at 35 % with RBA 20A primer.

19. The level of true polymorphism between Control & Treated sample B1 was evaluated at 54 % with RBA 10A primer.

20. The level of true polymorphism between Control & Treated sample B1 was evaluated at 18 % with RBA 15A primer.

Treated sample A & Treated sample A1

21. The level of true polymorphism between Treated sample A & Treated sample A1 was evaluated at 53 % with RBA 8A primer.

22. The level of true polymorphism between Treated sample A & Treated sample A1 was evaluated at 71 % with RBA 13A primer.

23. The level of true polymorphism between Treated sample A & Treated sample A1 was evaluated at 22 % with RBA 20A primer.

24. The level of true polymorphism between Treated sample A & Treated sample A1 was evaluated at 91 % with RBA 10A primer.

25. The level of true polymorphism between Treated sample A & Treated sample A1 was evaluated at 52 % with RBA 15A primer.

Treated sample B & Treated sample B1

26. The level of true polymorphism between Treated sample B & Treated sample B1 was evaluated at 7 % with RBA 8A primer.

27. The level of true polymorphism between Treated sample B & Treated sample B1 was evaluated at 68 % with RBA 13A primer.

28. The level of true polymorphism between Treated sample B & Treated sample B1 was evaluated at 33 % with RBA 20A primer.

29. The level of true polymorphism between Treated sample B & Treated sample B1 was evaluated at 100 % with RBA 10A primer.

30. The level of true polymorphism between Treated sample B & Treated sample B1 was evaluated at 62 % with RBA 15A primer.

Treated sample A & Treated sample B

31. The level of true polymorphism between Treated sample A & Treated sample B was evaluated at 0 % with RBA 8A primer.

32. The level of true polymorphism between Treated sample A & Treated sample B was evaluated at 47 % with RBA 13A primer.

33. The level of true polymorphism between Treated sample A & Treated sample B was evaluated at 7 % with RBA 20A primer.

34. The level of true polymorphism between Treated sample A & Treated sample B was evaluated at 18 % with RBA 10A primer.

35. The level of true polymorphism between Treated sample A & Treated sample B was evaluated at 31 % with RBA 15A primer.

Treated sample A1 & Treated sample B1

36. The level of true polymorphism between Treated sample A1 & Treated sample B1 was evaluated at 46 % with RBA 8A primer.

37. The level of true polymorphism between Treated sample A1 & Treated sample B1 was evaluated at 0 % with RBA 13A primer.

38. The level of true polymorphism between Treated sample A1 & Treated sample B1 was evaluated at 0 % with RBA 20A primer.

39. The level of true polymorphism between Treated sample A1 & Treated sample B1 was evaluated at 36 % with RBA 10A primer.

40. The level of true polymorphism between Treated sample A1 & Treated sample B1 was evaluated at 13 % with RBA 15A primer.

CONCLUSION:

Polymorphism was detected between Control & Treated sample A. The percentage of true polymorphism observed between Control & Treated samples of Nocardia otitidis sample is on an average 45%.
Polymorphism was detected between Control & Treated sample A1. The percentage of true polymorphism observed between Control & Treated samples of Nocardia otitidis sample is on an average 53%.
Polymorphism was detected between Control & Treated sample B. The percentage of true polymorphism observed between Control & Treated samples of Nocardia otitidis sample is on an average 46%.
Polymorphism was detected between Control & Treated sample B1. The percentage of true polymorphism observed between Control & Treated samples of Nocardia otitidis sample is on an average 34%.
Polymorphism was detected between Treated sample A & Treated sample A1. The percentage of true polymorphism observed between Control & Treated samples of Nocardia otitidis sample is on an average 57%.
Polymorphism was detected between Treated sample B & Treated sample B1. The percentage of true polymorphism observed between Control & Treated samples of Nocardia otitidis sample is on an average 52%.
Polymorphism was detected between Treated sample A & Treated sample B. The percentage of true polymorphism observed between Control & Treated samples of Nocardia otitidis sample is on an average 20%.
Polymorphism was detected between Treated sample A1 & Treated sample B1. The percentage of true polymorphism observed between Control & Treated samples of Nocardia otitidis sample is on an average 19%.
The treatment modality of Mr. Mahendra Kumar Trivedi is not known to us, the company is only concerned about detecting DNA polymorphism in Control & Treated lines of Nocardia otitidis samples.

Approved by: Dr. Avijit Roy

Manager R & D
Bangalore Genei.

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