chemistry of naturul products,estimation of nucleic acid,DNA and RNA

1. CLASS SEMINAR
SUB:CHEMISTRY OF NATURAL PRODUCTS
Topic –Estimation of DNA and RNA
Tuesday, 05
April 2022
1
SCHOOL OF PHARMACY,
DAVV,INDORE
PRESENTED BY
LALIT DHAKAR
M PHARM (1ST SEM)
Roll no. 2105
GUIDED BY-
Dr. MUKESH CHANDRA SHARMA
PROFESSOR,DAVV

2.  Content
 Introduction
 Method of estimation
 Spectroscopy method
 Gel electrophoresis
 Reference
Tuesday, 05
April 2022
LALIT DHAKAR,M.PHARM 1 SEM 2

3.  Introduction
 DNA and RNA are quantify to check the concentration and purity of DNA and
RNA present in solution.
 It is important to know the conc. and purity of DNA and RNA for the use in
further applications.
Tuesday, 05
April 2022
LALIT DHAKAR,M.PHARM 1 SEM 3

4.  Method of estimation
 UV absorbance
 Fluorescence dyes
 Gel electrophoresis
 Diphenylamine method
Tuesday, 05
April 2022
LALIT DHAKAR,M.PHARM 1 SEM 4

5.  Spectroscopy method :-
 Using UV absorbance is one of the most common ways to quantify DNA.
 This method involves measuring the absorbance/transmission of light through a liquid to
determine the concentration of substances in the liquid.
Tuesday, 05
April 2022
LALIT DHAKAR,M.PHARM 1 SEM 5

6.  Nano drop 2000c spectrophotometric method :-
 Can quantify nucleic acid from microvolume of 0.5 μL- 2.0 μL
 Measures DNA and RNA (A260),and protein (A280) conc. And sample purity (260/280
ratio).
 Large conc. range (2 ng/μL-15,000 ng/μL dsDNA) without dilution.
Tuesday, 05
April 2022
LALIT DHAKAR,M.PHARM 1 SEM 6

7.  Purity and quantification- nano drop
 Pure DNA sample gives a 260/280 ratio=1.8
 For pure RNA 260/280=2
 A 260?230 indicates possible contaminates.
Tuesday, 05
April 2022
LALIT DHAKAR,M.PHARM 1 SEM 7

8.  Checking contaminants
 A low 260/230 ratio indicates the contamination absorbing at 320nm or less.
 A low260/280 ratio indicates the contaminants absorbing at 280 nm or less.
 A shift in the wavelength is indicates contaminates absorbing at low
wavelengths.
 The wavelength of the sample peak should be at 260 nm if contaminants are
present the peak may shift.
Tuesday, 05
April 2022
LALIT DHAKAR,M.PHARM 1 SEM 8

9. Agarose gel electrophoresis
 Quickest way to quantify DNA, can use the agarose gel method to not only find out how
much DNA you have, but also to see whether your DNA is intact or the correct size.
 Contaminating DNA/RNA fragments can be removed using this method.
 After running the sample on gel the band of interest can be spliced out and gel extraction
can be done to purify it.
Tuesday, 05
April 2022
LALIT DHAKAR,M.PHARM 1 SEM 9

10.  Fluorescence dyes
 Another way to quantify DNA would be to use fluorescent dyes that
fluoresce when bound to DNA.
 The main distinction here is that these dyes, such as Pico Green or
SYBR Green, are specific for double strand DNA
 These methods are more sensitive than UV absorbance, especially
when you expect low concentrations in your samples and are often used
to quantify DNA for next generation sequencing.
Tuesday, 05
April 2022
LALIT DHAKAR,M.PHARM 1 SEM 10

11. Reference :-
Tuesday, 05
April 2022
LALIT DHAKAR,M.PHARM 1 SEM 11

12. Thank you
Tuesday, 05
April 2022
LALIT DHAKAR,M.PHARM 1 SEM 12