Tools & techniques (Molecular & Biochemical to Study Physiological Processes & to Screen & Asses Stress Response in Plants)

1. Title :- Tools & techniques (Molecular & Biochemical to
Study Physiological Processes & to Screen & Asses
Stress Response in Plants)
Guided by:-
S. S. Shinde
Assist. Professor
Department of Agril. Botany
Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani
College of Agriculture, Parbhani
Department:- Agricultural Botany
Presented by:-
Akshay Deshmukh
Ph.D Scholar
2019 A/05P

2. Tools & techniques
(Molecular & Biochemical to Study Physiological
Processes & to Screen & Asses Stress Response in Plants)
INDEX…..
 DNA & RNA Extraction
 Semi-quantitative & quantitative RT-PCR
 cDNA Synthesis & Library Construction
 Northern Blotting
 Immunoassays

3. DNA Extraction
 What is DNA extraction
“Extracting DNA from the cell” this is the simplest definition for DNA extraction.
or
“Isolation of DNA by breaking the cell membrane and nuclear membrane with the help
of chemicals, enzyme or physical disruptions is defined as a DNA extraction”.
 Purpose of DNA extraction
Isolation of specific DNA in plant or animal cell for diagnostic purpose or gene
cloning.
History of DNA extraction
The first DNA extraction attempt had performed by Friedrich
Miescher in 1869. He had isolated the cell material and
named it as the “nuclei”
in 1958 Meselson and Stahl developed a full-function
protocol for DNA extraction.

4. Different types of DNA extraction methods

5. The method is also called as a phenol-chloroform and isoamyl alcohol, PCI method of
DNA extraction.
This method is one of the best methods of DNA extraction. The yield and quality of DNA
obtained by the PCI method is very good.
Different Chemicals Role of chemicals
Tris DNA is pH sensitive, Tris buffer maintains the pH of the solution
EDTA EDTA is a chelating agent and used to block DNase activity
Sodium dodecyl sulphate helps to break cell membrane and nuclear envelope
NaCl creates the ionic bond, It will help to protect DNA from denaturation
MgCl2 protects DNA by mixing with other cell organelles.
Phenol, chloroform and
isoamyl alcohol
Helps in the removal of protein.
Phenol-chloroform method of DNA extraction

6. Use of chemicals in Steps of DNA Extraction
DNA extraction step Chemical
Lysis of cell wall/ cell membrane and Lysis of
nuclear membrane
Tris, MgCl2, EDTA, NaCl, SDS, CTAB,
Triton X100
Digestion of protein CTAB, SDS, phenol, chloroform, Nonidet
P40, different chaotropic, urea, guanidium
isothiocyanate, guanidium thiocyanate, N-
Lauroyl sarcosine
Precipitation of DNA Isopropanol, ethanol, methanol, NaCl,
sodium acetate
Washing of DNA alcohol
Dissolving DNA TE buffer, distil water

8. Remaining Methods Principal
Proteinase K DNA extraction
Method
 Called as an enzymatic method of DNA extraction
 Use in BLOOD samples
Silica column-based DNA
extraction method
 A positively charged silica particles bind with the negatively
charged DNA and hold it during centrifugation.
 rapid and gives “PCR ready DNA” for the downstream of the
applications.
DNA extraction by magnetic
beads
 The DNA is separated under the magnetic field.
 Positively charged magnetic beads attract the negatively charged
DNA.
CsCl density gradient method of
DNA extraction
 the DNA is separated based on the density of it with the
centrifugation.

9. RNA Extraction
 What is RNA extraction
“Extracting RNA from the cell” this is the simplest definition for RNA extraction.
or
“Isolation of RNA by breaking the cell membrane with the help of chemicals, enzyme or
physical disruptions is defined as a RNA extraction”.
 Purpose of RNA extraction
cDNA library construction
Isolation of specific RNA in plant cell for diagnostic purpose.
Microarray analysis
Northern analysis
RT-PCR

10. Methods of RNA Extraction
Method Typical kit
Guanidinium-acid-phenol Extraction TRIzol and TRI reagent
Silica technology, glass fiber filters RNeasy and its variations
Density gradient centrifugation using
cesium chloride or cesium
trifluoroacetate
Magnetic bead technology Dynabeads mRNA DIRECT Micro
Lithium chloride and urea isolation
Oligo(dt)-cellulose column
chromatography
Non-column poly (A)+
purification/isolation

11. Acid-Guanidinium-Thiocyanate-phenol Chloroform
Extraction
 .

13. Semi-quantitative & quantitative RT-PCR
 Reverse transcription polymerase chain reaction (RT-PCR) is an
laboratory technique combining reverse transcription of RNA into
DNA
 Reverse transcription polymerase chain reaction (RT-PCR) also
called as Semi-quantitative.
 (called complementary DNA or cDNA) and amplification of specific
DNA targets using polymerase chain reaction (PCR).

14. PCR RT-PCR qPCR
PCR is a laboratory technique used
to make multiple copies of a
segment of DNA.
RT-PCR is an laboratory
technique combining reverse
transcription of RNA into DNA
and amplification of specific DNA
targets using PCR.
Quantitative PCR (qPCR) is
used to detect, characterize
and quantify nucleic acids for
numerous applications.
DNA is use as the template to
amplify.
RNA is use as the template to
amplify.
It is the remain half part of
RT-PCR
DNA is transcribed into
complementary ssDNA, using
DNA polymerase.
RNA is reverse transcribed into
complementary DNA (cDNA),
using reverse transcriptase.
After the formation of cDNA
qPCR is subsequently carried
out
It includes three steps denaturation,
annealing, and elongation (one
cycle of amplification).
The first step of RT-PCR is the
synthesis of a DNA/RNA hybrid.
Then ssDNA molecule is
completed by the DNA-dependent
DNA polymerase activity of the
reverse transcriptase into cDNA.
fluorescent labeling

16. Types of RT-PCR
One Step (RT-PCR)
All reaction components are
mixed in one tube prior to
imitation of the reaction.
Two Step (RT-PCR)
RNA is first reverse transcribed
into cDNA
Then cDNA is use as template for
subsequent PCR amplification
using primer specific for one or
more gene

17. Application of RT-PCR
 Use in research:- This technique is commonly use in molecular biology to
detect RNA expression
Helps in development of cDNA library
 Diagnostic use:- Helps in genetic study of Cancer
 Microbiological use:- use by microbiologist in filed of food safety
 Detection GMO crop

18. cDNA Synthesis & Library Construction
 All eukaryotes have an mRNA. Each specialized cell have a specific mRNA that
encodes information for a specific Protein
 This information can be transformed back into DNA or in other words a DNA
copy of mRNA (using reverse transcriptase enzymes)
 This copy of transcribed DNA from mRNA is termed as cDNA which can be
stored in plasmids or phages.
 cDNA contains only the expressed genetic information which allows to study the
amino acid sequence directly from the DNA

19. Construction of cDNA Library
 The first step in creating a cDNA library is to isolate
mRNA from the cells.
 All mRNA have a POLY-A tail which distinguishes the
mRNA from the remaining RNA i.e. tRNA and rRNA
 By using this tail as the primer site we isolate only mRNA
 Once the mRNA is isolated it is treated with an enzymes
reverse transcriptase . This enzymes will crates a (ss) cDNA
intermediates from the mRNA
 By hybridizing the POLY-A of the mRNA with oligo-dT a
primer is created. Reverse transcriptase recognizes this
template and will add bases to 3’ end.

20. Packaging the cDNA
The final step is to ligate the sticky ends of the
cDNA with the λ-Phage arms that have
complementary sticks ends, thereby inserting the
Double strand cDNA into the Vectors

21. Conclusion
mRNA is isolated from the
RNA
mRNA is treated with
Reverse Transcriptase
enzyme.
cDNA thus obtained is fused
with any Vectors.

22. Applications of cDNA
 To express eukaryotic genes in prokaryotic
 cDNA does not have intron and can be expressed in prokaryotic cells.
 Most useful in reverse genetics where the additional genomics information is of
less use.
 It is useful for subsequently isolating the gene that codes for that mRNA

23. Blotting
The process of detection macromolecules that we deals with it
Southern Blotting: if we detect DNA
Northern Blotting: if we detect RNA
Western Blotting: if we detect Protein
Northern Blotting
The northern blot, or RNA blot, is a technique used in molecular biology research to
study gene expression by detection of RNA (or isolated mRNA) in a sample.
The northern blot technique was developed in 1977 by James Alwine, David Kemp, and
George Stark at Stanford University

24. The steps involved in Northern Blotting analysis
RNA isolation (total or poly(A) RNA)
Probe generation.
Denaturing agarose gel electrophoresis.
Transfer to solid support and immobilization.
Prehybridization and hybridization with probe.
Washing.
Detection.
Stripping and reprobing (optional)

27. Advantage and Disadvantages of Northern
Blotting
Advantages
Simplest Method
Highly Specific
Quality and quantity of gene can be measured
Disadvantages
Detect small number of gene at a time
Detection with multiple probes is difficult
Application
Study RNA degradation
Study RNA Splicing
Study gene expression

28. Immunoassays
 “Immuno” refers to an immune response that causes the body to generate
antibody
 “Assay” refers to a test
 An immunoassay is a biochemical test that measures the presence or
concentration of a macromolecule in a solution through the use of an antibody
(usually) or an antigen (sometimes).
 Immunoassays is the test that uses immune complexing when antibody and
antigens are brought together.
 Immunoassays may measures either the antibody or antigen.
 An antibody is a protein produced in a body to a foreign substance.
 An antigen is the substance that the body is trying to eliminate by mounting an
immune response.
 An analyte is anything measured by a laboratory test.

29. Principle of Immunoassays
 Immunoassays rely on the ability of an antibody to recognize and bind a specific
macromolecule in what might be a complex mixture of macromolecules.
 In immunology the particular macromolecule bound by an antibody is referred to
as an antigen and the area on an antigen to which the antibody binds is called
an epitope.
 In some cases, an immunoassay may use an antigen to detect for the presence of
antibodies, which recognize that antigen, in a solution.
History
Rosalyn Sussman Yalow and Solomon Berson are
credited with the development of the first immunoassays
in the 1950s.
Yalow awarded the Nobel Prize for her work in
immunoassays in 1977.

30. Illustration of the basic components of an
immunoassay
An Analyate =
An antibody =
A detectable lable =

31. Labels
Lables Test
Enzymes Use in Enzyme-linked immunosorbent
(ELISAs), or sometimes enzyme
immunoassays (EIAs).
Radioactive isotopes Radioimmunoassay.
DNA reporters Use in real-time immunoquantitative PCR
(iqPCR)
Fluorogenic reporters Use in Protein microarrays.
Electrochemiluminescent tags Electrochemiluminescence
Label-free immunoassays
Immunoassays employ a variety of different labels to allow for detection of
antibodies and antigens.

32. Classifications of immunoassay
 Competitive, homogeneous immunoassays
 Unlabeled analyte in a sample competes with labelled analyte to bind an antibody.
 The amount of labelled, unbound analyte is then measured.
 The amount of labelled, unbound analyte is proportional to the amount of analyte in the sample.
 Competitive, heterogeneous immunoassays
 Unlabeled analyte in a sample competes with labelled analyte to bind an antibody.
 The labelled, unbound analyte is separated or washed away, and the remaining labelled, bound analyte is
measured.
 One-site, noncompetitive immunoassays
 The unknown analyte in the sample binds with labelled antibodies.
 The unbound, labelled antibodies are washed away, and the bound, labelled antibodies are measured.
 The intensity of the signal is directly proportional to the amount of unknown analyte.

33. Two-site, noncompetitive immunoassays
 The analyte in the unknown sample is bound to the antibody site, then the labelled
antibody is bound to the analyte.
 The amount of labelled antibody on the site is then measured.
 It will be directly proportional to the concentration of the analyte because the labelled
antibody will not bind if the analyte is not present in the unknown sample.
 This type of immunoassay is also known as a sandwich assay as the analyte is
"sandwiched" between two antibodies.

34. THANK YOU…………