SCIENCE AND AGRO STUDIES

Objective:- To perform blotting technique.  Principle:- All the three blotting methods are fairly simple and usually consist of four separate steps: i. Electrophoretic separation of protein or of nucleic acid fragments in the sample, ii. Transfer to and immobilization on paper support, iii. Binding of analytical probe to target molecule on paper, and iv. Visualization of bound probe. > Molecules in a sample are first separated by electrophoresis and then transferred on to an easily handled support medium or membrane. This immobilizes the protein or DNA fragments, provides a faithful replica of the original separation, and facilitates subsequent biochemical analysis. > After being transferred to the support medium the immobilized protein or nucleic acid fragment is localized by the use of probes, such as antibodies or DNA, that specifically bind to the molecule of interest. > Finally, the position of the probe that is bound to the immobilized target molecule is visualized ...

PCR For 16S rRNA Sequence Analysis:- Principle:- > Polymerase chain reaction (PCR) is molecular technique used to amplify specific regions of DNA for applications such as sequencing and genetic analysis.  >Typically, there is a limited amount of DNA in the sample to study and amplification is required.  > PCR is carried out in a test tube with the DNA template, primers specific for the region that is desired, DNA polymerase, and reagents that stabilize the reaction.  > Once the reaction is put together, it will go into a thermocycler (PCR machine) that will create the conditions for DNA replication to occur.  > Each round of PCR requires three steps, denaturation, annealing, and elongation, each of which doubles the amount of DNA template present in the reaction.  > By repeating this process multiple times, usually 30, this will amplify the DNA exponentially. PCR bead method:- Materials:- i. 27F primer (20uM stock) ii. 1492R primer (20uM stock)...

Objective:- To separate DNA fragments by Agarose Gel Electrophoresis. Principle:- Nucleic acids are separated by applying an electric field, so these negatively charged molecules will move through an agarose matrix towards the anode, and the biomolecules are separated by size in the agarose gel matrix, where the distance travelled by a DNA molecule is inversely correlated with its size. Materials:- i. Agarose powder ii. 1X TBE buffer (89 mM Tris-base, 89 mM boric acid and 2 mM EDTA) prepared from 10X TBE iii. Ethidium Bromide (5 mg/ml) iv. Gel loading dye (Glycerol and orange dye) v. 1 kb and 100 bp DNA ladder vi. Horizontal electrophoresis apparatus vii. Power supply. Procedure:- > Measure the desired grams of agarose to make 1% agarose gel. > Heat the solution to boiling in the microwave to dissolve the agarose to produce a homogeneous mixture. > Add 4 µl of ethidium bromide CARFULLY to the dissolved agarose and mix. > Get a gel plate and a comb. Put the two dams into th...

Okazaki fragments:- These are short segments of newly synthesized DNA that are produced on the lagging strand during DNA replication. Why are they formed?:- > DNA polymerase can only synthesize DNA in the 5′ → 3′ direction. Leading strand:- Synthesized continuously toward the replication fork. Lagging strand:- Synthesized discontinuously away from the replication fork as short pieces called Okazaki fragments. Size:- Prokaryotes:- Approximately 1,000–2,000 nucleotides long. Eukaryotes:- Approximately 100–200 nucleotides long. Attenuation:- Attenuation is a regulatory mechanism used in bacterial operons to ensure proper transcription and translation. In bacteria, transcription and translation are capable of proceeding simultaneously. The need to prevent unregulated and unnecessary gene expression can be prevented by attenuation, which is characterized as a regulatory mechanism. i. Transcriptional-attenuation:- It is characterized by the presence of an attenuator within the DNA s...

Dinucleotide Repeats in Z-DNA:- Dinucleotide repeats are short DNA sequences in which a pair of nucleotides is repeated many times. In Z-DNA, the most common repeat is: (CG)n = CGCGCGCG... where n represents the number of repeated CG units. Key Features:- > Z-DNA is a left-handed DNA double helix. > It forms most easily in regions containing alternating purine and pyrimidine bases, especially CG repeats. > Common sequences that form Z-DNA:     i. (CG)n (most stable)     ii. (CA/TG)n (under certain conditions) Why Dinucleotide Repeats Favor Z-DNA:- > Alternating purine (G) and pyrimidine (C) bases allow the DNA backbone to adopt the characteristic zigzag shape of Z-DNA. > Negative supercoiling, high salt concentration, or cytosine methylation further stabilize these repeats in the Z-DNA conformation. Biological Importance:- > Helps regulate gene expression. > Involved in DNA recombination. > Plays a role in genome stability and DNA repair. ...