Plant physiology

1. Vasantrao Naik Marathwada Agricultural
University,
College of Agriculture, Parbhani
Tools and Techniques (Molecular &Tools and Techniques (Molecular &
Biochemical) to Study Physiological ProcessesBiochemical) to Study Physiological Processes
and to Screen & Assess Stress Responses inand to Screen & Assess Stress Responses in
Plants, Such asPlants, Such as
BY
SHAIKH WASIM CHAND

2. Tools and TechniquesTools and Techniques
• DNA & RNA IsolationDNA & RNA Isolation
• cDNA Synthesis & Library ConstructioncDNA Synthesis & Library Construction
• Semiquantitative & quantitative RT-PCRSemiquantitative & quantitative RT-PCR
• Northern BlotNorthern Blot
• ImmunoassaysImmunoassays

3. DNA IsolationDNA Isolation
• Aim: To Separate the DNA that is present in the nucleus of the
cell from other cellular component.
• DNA isolation is the extraction process of DNA from various
source by using Chemical and physical methods.
• Friedrich Miescher (1869).
• Purpose: Isolation of specific DNA in plant or animal cell for
diagnostic purpose gene cloning.

4. Steps of DNA Isolation

5. DNA Isolation DiagramDNA Isolation Diagram

6. RNA ISOLATIONRNA ISOLATION by yeastby yeast
MATERIAL
• BUFFER A
• Buffer A saturated phenol (1.2 ml per sample)
• Chloroform (0.6 ml per sample)
• 3 M NaOAc (pH 5.2) (90 μl per sample)
• DEPC-treated dH2O (1.5 ml per sample)
• Absolute ethanol (2 ml per sample)
• 70 % ethanol (1 ml per sample)

7. Extraction of RNA by Yeast
• Add 500 μl of Complete Buffer A in a flask.
• Centrifuge the tubes in a microcentrifuge for 30 sec at full speed.
• Remove the layer using an RNase free blue tip. Add 600 μl of Buffer A again.
• Centrifuge the tubes in a microcentrifuge for 2-3 minutes at full speed.
• Remove aqueous layer (top layer) to a new tube.
• Add 600 μl of 1:1 phenol buffered with chloroform at room temperature.
• Mix the samples by for 20 seconds and separate the layers by centrifuging the tubes in
a microcentrifuge for 2-3 minutes at full speed.

8. • Remove the aqueous layer (top layer) to a new tube.
• Add 50 μl of 3 M NaOAc (Ph 5.2) and 1 ml of absolute ethanol.
• Resuspend the pellets in 400 μl of dH2O.
• Wash the pellets by adding 1 ml of 70% ethanol and vortexing for 20 seconds.
• Centrifuge the microcentrifuge tubes at full speed for 5 minutes.
• Remove the supernatant. Incubate the open tube at 37°C for 5 minutes to dry the
pellet.
• Dissolve the RNA in 50 μl dH2O. Vortex it and Centrifuge briefly.
• Dilute 5 μl RNA into 495 μl of dH2O. Determine the
• Dilute the RNA to 1 μg/μl

9. • Harvesting cell
• RNA extraction from yeast

10. Other methods of RNA IsolationOther methods of RNA Isolation

11. cDNA Synthesis & LibrarycDNA Synthesis & Library
ConstructionConstruction
• A cDNA library is a combination of cloned cDNA fragments inserted into
a collection of host cells which together constitute some portion of
the transcriptome of the organism and are stored as a "library".
• cDNA is produced from fully transcribed mRNA.
• In eukaryotic cells the mature mRNA is already spliced, hence the cDNA
produced lacks introns and can be readily expressed in a bacterial cell.
• Information in cDNA libraries is a powerful and useful tool.

12. cDNA Library ConstructioncDNA Library Construction
• DNA is created from a mature mRNA from a eukaryotic cell with the use
of reverse transcriptase.
• In eukaryotes, a poly-(A) tail distinguishes mRNA from tRNA and rRNA and can be
used as a primer site for reverse transcription.
mRNA extraction
• Firstly, the mRNA is obtained and purified from the rest of the RNAs.
• Several methods exist for purifying RNA such as trizol extraction and column
purification.
• Column purification is done by using oligomeric dT nucleotide.
• The rest of the RNAs are eluted out. The mRNA is eluted by using eluting buffer
and some heat to separate the mRNA strands from oligo-dT.

13. cDNA Construction
•Once mRNA is purified, oligo-dT is tagged as a complementary primer which binds to
the poly-A tail.
•The mRNA is removed by using a RNAse enzyme leaving a single stranded cDNA. This is
converted into a double stranded DNA with the help of DNA polymerase.
•Restriction endonucleases and DNA ligase are used to clone the sequences into
bacterial plasmids.
•The cloned bacteria are selected, commonly through the use of antibiotic selection.
•Once selected, stocks of the bacteria are created which can later be grown and
sequenced to compile the cDNA library.

15. Uses cDNA library ConstructionUses cDNA library Construction
• To express eukaryotic genes in prokaryotes.
• cDNA does not have introns and can be expressed in prokaryotic cells.
• Most useful in reverse genetics where the additional genomic
information is of less use.
• It is useful for subsequently isolating the gene that codes for that mRNA.

16. Semiquantitative & quantitative RT-PCRSemiquantitative & quantitative RT-PCR
• PCR was invented by Kary Mullis in the 1984.
• To make numerous copies of DNA fragments in the laboratory.
• The in vitro version of DNA Replication.
• A real-time polymerase chain reaction (RT-PCR) also known as quantitative
polymerase chain reaction (qPCR), is a laboratory technique of molecular
biology based on the polymerase chain reaction (PCR).
• It monitors the amplification of a targeted DNA molecule during the PCR.
• Real-time PCR can be used quantitatively (quantitative real-time PCR) and semi-
quantitatively, i.e. above or below a certain amount of DNA molecules (semi
quantitative real-time PCR).

17. • The PCR is a common method for amplifying DNA, for RNA-based PCR.
• The RNA sample is first reverse-transcribed to cDNA with reverse transcriptase.
• Quantitative PCR is modern methodology to study gene expression.
• Role of RT-PCR is Analysis, Detection and Quantification of the samples.
• Real-time PCR is carried out in a thermal cycler with the capacity to illuminate each
sample with a beam of light of at least one specified wavelength.
• The thermal cycler is also able to rapidly heat and chill samples.

19. Types of RT-PCRTypes of RT-PCR
One step RT-PCR
•All reaction components are mixed in one tube prior to initiation of the reaction.
•One-step RT-PCR offers simplicity and convenience and minimizes the possibility for
contamination.

20. Two step RT-PCR
•The RT reaction and PCR amplification which can be simple PCR or qPCR.
•RNA is first reverse transcribed into complementary DNA ( cDNA ) using an enzyme,
reverse transcriptase.
•The resulting cDNA is used as templates for subsequent PCR amplification using primers
specific for one or more genes.
•The resulting cDNA used for detecting multiple messages from a single RNA sample.

21. Applications
• Diagnostic uses
• Microbiological uses
• Uses in research
• Detection of phytopathogens
• Detection of genetically modified organisms
• Clinical quantification and genotyping
• Environmental remediation applications

22. Northern BlotNorthern Blot
• The northern blot is a technique used to study gene expression by detection of RNA
in a sample.
• RNAs are separated by gel electrophoresis.
• Alwine, Kemp and Stark(1977) developed a procedure for blot transfer of RNA.
• He used a chemically reactive paper (diazotization of amino benzyl oxymethyl paper
which is prepared from Whatsman 540 papers).

23. Steps involving Northern BlottingSteps involving Northern Blotting
• RNA isolation
• Separation of RNA using Gel Electrophoresis
• Blotting
• Hybridization and Washing of excess probes
• Visualization
• The Northern Blot procedure is quite similar to that of Southern blot,
except that the biomaterial used is RNA instead of DNA.

24. ProcedureProcedure

25. APPLICATIONSAPPLICATIONS
• Used for detection and quantitative.
• Estimation of hybridized mRNA.
• Study RNA degradation.
• Study RNA half life.
• Study RNA splicing.
• It is useful in the studies of gene expression.

26. ImmunoassaysImmunoassays
• “Immuno” refers to an immune response that causes the body to
generate antibodies.
• “Assay” refers to a test.
• An immunoassay is a test that uses immune complexing when
antibodies and antigens are brought together.
• Immunoassays may measure either the antigen or antibody.
• An antibody is a protein produced in the 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.

27. •Illustration of the basic
components of an
immunoassay, which
includes,
•an analyte (green),
•an antibody (black)
•a detectable label (yellow).

28. Categories of ImmunoassayCategories of Immunoassay
Competitive Assays
•Unlabeled analyte in the test sample is measured by its ability to compete with
the labeled antigen in the immunoassay.
•Less label measured in the assay means more of the unlabeled (test sample)
antigen is present.
Noncompetitive Assays
•It give the highest level of sensitivity and specificity.
•The measurement of the labeled analyte is directly proportional to the amount of
antigen present in the sample.