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Quantification of DNA is an essential step after its extraction in conjunction with the confirmation of the quality. NanoDrop spectrophometer is a powerful instrument in determining both purity and concentration of nucleic acids on the basis of absorbance values obtained by adding small amount of sample (1-2 μl) on the measurement surface.

Principle: The instrument works on the basic principle of spectrophotometry, which takes highly sensitive fluorescent measurements of the microvolumes. It takes use of microvolume sample retention technology based on surface tension property of the liquid and fibre optic technology. Together, these features make it possible to detect sample even in microlitres, which is much more than the traditional method that need cuvette-sized sample volume. The concentration of nucleic acid sample is normally determined by measuring its absorbance at 260 nm.

To Estimate the DNA Using Diphenylamine

Introduction

DNA estimation is helpful to know about the total amount of DNA of prokaryotic as well as eukaryotic system. One method used for DNA estimation is Diphenylamine assay, is that of Burton who modified the original procedure of Dische. This procedure is based on treatment of nucleic acid with hot perchloric acid to hydrolyze DNA nucleotides. Diphenylamine reacts specifically with deoxyribose in DNA to form a blue-colored complex. Since diphenylamine does not react with ribose, one can assay for DNA in the presence of contaminating RNA.

Principle

The DPA method is based on the colorimetry measurement of products formed by the reaction of ù- hydroxylevulinaldehyde (from the deoxyribose released after depurination of DNA) with diphenylamine in 2.5 N perchloric acid.

When DNA is treated with diphenylamine under acidic condition (PCA) a blue complex is formed with a sharp absorption maximum at 595 nm (600 nm). In acid solution, the straight chain form of the deoxypentose is converted to highly reactive ω – hydroxylevulinaldehyde that reacts with diphenylamine to give blue complex. The reaction yields two mixture of product which together have absorbance maximum at 595 nm (600 nm). Interferences include proteins, lipids, saccharides and RNA. In DNA the only deoxyribose of the purine nucleotides reacts, so that the value obtained represents half of the total deoxyribose present.

Introduction

Both beneficiary and deleterious microorganisms are present in food. Beneficiary microbes are used for the preparation of a number of dairy and meat products and are called Probiotics, whereas deleterious microbes can cause food poisoning. The quantitative analysis of food products is necessary to find out the presence of types of microorganisms.

Requirements

1. Food sample
2. Mechanical blender
3. Sterilised distilled water
4. Steilised distilled water blanks (9 ml)
5. Petri plates
6. Sterile Pipettes (1 ml)
7. Eiosin Methylene Blue agar medium
8. Incubator
9. Colony counter
10. Marker

Procedure

1. Weigh 1 g of well blended

RAPD stands for Random Amplification of Polymorphic DNA. It is a type of PCR reaction, but the segments of DNA that are amplified are random. The scientist performing RAPD creates several arbitrary, short primers (8-12 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify. By resolving the resulting patterns, a semi-unique profile can be gleaned from a RAPD reaction.

No knowledge of the DNA sequence for the targeted gene is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where. This makes the method popular for comparing the DNA of biological systems that have not had the attention of the scientific community, or in a system in which relatively few DNA sequences are compared (it is not suitable for forming a DNA databank). Because it relies on a large, intact DNA template sequence, it has some limitations in the use of degraded DNA samples. Its resolving power is much lower than targeted, species specific DNA comparison methods, such as short tandem repeats. In recent years, RAPD is used to characterize, and trace, the phylogeny of diverse plant and animal species.

RAPD stands for Random Amplification of Polymorphic DNA. It is a type of PCR reaction, but the segments of DNA that are amplified are random. The scientist performing RAPD creates several arbitrary, short primers (8-12 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify. By resolving the resulting patterns, a semi-unique profile can be gleaned from a RAPD reaction.

No knowledge of the DNA sequence for the targeted gene is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where. This makes the method popular for comparing the DNA of biological systems that have not had the attention of the scientific community, or in a system in which relatively few DNA sequences are compared (it is not suitable for forming a DNA databank). Because it relies on a large, intact DNA template sequence, it has some limitations in the use of degraded DNA samples. Its resolving power is much lower than targeted, species specific DNA comparison methods, such as short tandem repeats. In recent years, RAPD is used to characterize, and trace, the phylogeny of diverse plant and animal species.

Objective: To amplify DNA using RAPD primers and study on agarose gel.

Background information:

RAPD markers are decamer (10 nucleotide length) DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence and which are able to differentiate between genetically distinct individuals. RAPD is applied to analyze the genetic diversity by random primers. Unlike traditional PCR analysis, RAPD does not require any prior DNA sequence information of the target organism: the identical 10-mer primers will or will not amplify a segment of DNA, depending on positions complementary with primers' sequence. For example, no fragment is produced if primers annealed too far apart or 3' ends of the primers are not facing each other. Therefore, if a mutation has occurred in the templ that was earlier complementary with primer, a PCR product will not be produced, resulting in a different pattern of amplified DNA segments on the gel. RAPD is a dominant marker means it cannot distinguish between heterozygous; single copy and homozygous; two copies. Rarely RAPD markers may be Co-dominant where different fragment length of DNA is amplified from the single locus. PCR is a molecular technique used for amplification of DNA, a complete enzyme based chemical reaction, therefore, the concentration and quality of template DNA, concentrations of PCR components, and the PCR cycling conditions may greatly influence the outcome. Thus, the RAPD technique is notoriously laboratory dependent and needs carefully developed laboratory protocols to be reproducible. Mismatches between the primer and the template may result in the total absence of PCR product or decreased amplified product. Thus, the RAPD results can be difficult to interpret.

Rapeseed
Rapeseed (Brassica napus L.), a member of the Brassicaceae family, is one of the primary oilseed crops cultivated for biodiesel production, particularly in Europe and Canada (Hoekman et al., 2012; Demirbas, 2009). Its seeds contain 40-45% oil, making it one of the most efficient oil crops for biofuel production (Ramos et al., 2009). The extracted oil is known as canola oil, which is preferred for both food and fuel applications due to its low erucic acid and glucosinolate content (Atabani et al., 2012). Rapeseed (Brassica napus L.), is one of the biofuel species that has achieved global attention, primarily due to its high yield, climatic versatility as well as its low environmental impact than fossil fuels (Singh et al., 2010). The crop is being grown all over the temperate region because its ability to resist cold temperatures and pests is a major advantage for the practice of sustainable agriculture. Moreover, the rapeseed has been positively affected by the development of biotechnology and the utilization of hybrid breeding that has been significantly increasing the oil yield and disease resistance of rapeseed, thus, facilitating the biodiesel production from this plant (Zanetti et al., 2013). Biodiesel from rapeseed has ecological advantages, significantly reducing greenhouse gas emissions by up to 50% compared to regular diesel (Hill et al., 2006). It also supports rural economic development through the diversification of farmer income and diminishing reliance on imported petroleum (FAO, 2021). The increasing price of the biofuel, chiefly supported by sustainable energy policies and reducing carbon footprint policies, is one of the driving factors for increasing demand of rapeseed as global demand for renewable energy sources rises (Euopean Commission 2021).
Sunflower
Sunflower (Helianthus annuus L.) is an important oilseed crop used for biodiesel production, especially in temperate regions like Europe and North America (Demirbas, 2009). The oil content of sunflower seeds ranges from 25-50%, making it a suitable feedstock for biofuel (Atabani et al., 2012). The characteristics of sunflower oil, such as the high content of unsaturated fatty acids, lead to better cold flow properties of its biodiesel (Ramos et al., 2009). In addition, the creation of high-oleic sunflower varieties allowed considerable improvement in oxidative stability and led to the fact that sunflower biodiesel is more suitable for storage and combustion (Dimitrijevic et al., 2017). A second

The chemistry of humic substances is the least comprehended in the field of Soil Science. The many important functions of humus will remain obscure if the structural chemistry of the humic and fulvic acids is not known. A variety of functional groups have been reported in humic substances. However, considerable disagreement still exists in respect of the exact amounts present and, in some cases, proof of existence is lacking.

Some important structural groups of organic molecules are given below:

The polymerase chain reaction (PCR) stands out as a highly potent technology within the realm of molecular biology. With PCR, particular sequences present within a DNA or cDNA template can undergo replication, termed “amplification,” increasing their quantity manifold, ranging from thousands to millions. This process entails the utilization of sequence-specific oligonucleotides, a DNA polymerase capable of withstanding high temperatures, and thermal cycling.

Real Time PCR, a variant of the standard PCR technique, serves as a prevalent method for quantifying DNA or RNA within a sample. Through the use of sequence-specific primers, the quantity of copies of a specific DNA or RNA sequence can be assessed. This is achieved by monitoring the amount of amplified product at each stage throughout the PCR cycle. Earlier cycles exhibit amplification if a particular sequence (DNA or RNA) is abundant in the sample, while amplification in later cycles indicates scarcity of the sequence.

Introduction 
A broad definition of bioprocessing is the process of creating a valuable product from a live source. The system’s most important element is that the source organism is living and sensitive to its surroundings. Accordingly, the framework states that it will respond to relatively small changes in its physico-chemical environment by modifying its physiology to maximise efficiency. The use of entire cells and enzymes to catalyse bioprocesses in industrial settings is becoming more popular these days than conventional chemical synthetic methods. Among the many benefits of biotransformations in this context are their extensive substrate portfolio (including liquid, solid, and gas waste), one-pot reactions in mild circumstances and environmental friendliness. However, a number of drawbacks, including the unstable nature of biocatalysts and subpar performance in specific reaction conditions, the low solubility of some substrates in the reaction medium, substantial production expenses brought on by intricate downstream processing and product isolation and the lack of expertise in microbiology or bioprocess design, frequently make it difficult to scale up bioprocesses from the lab to the manufacturing facility. There is a growing need for innovative bioprocesses that can get over the aforementioned obstacle in industrial and environmental biotechnology in order to create effective, sustainable, and affordable processes. As a result, the industrial sector requires new methods that use biocatalysts that are highly active and stable under a variety of reaction conditions (typically accomplished through metabolic engineering), boost feedstock utilisation by using inexpensive waste resources, use green chemistry solutions that enable the use of fewer toxic solvents and reagents, or develop cost-effective biorefinery concepts that can transform industrial waste and byproducts into products with added value, thereby establishing new value chains. Figure 1.1 shows the three stages of a typical bioprocess: upstream processing, bioreaction and downstream processing which includes separation and purification steps. Every stage of these phases offers multiple chances to enhance a certain bioprocess.

ABSTRACT

Plant growth promoting (PGP) bacteria play an immense role in promoting crop growth mainly by direct mechanisms such as N_2-fixation, phosphate solubilization and phytohormone production and indirectly may help to suppress pathogens or contribute to enhance absorption of nutrients leading to increase yields. The N₂fixers are grouped based on their niche adaptation as endophytic nodular symbionts (Rhizobium spp.), free living (Azotobacter spp.), associative symbionts (Azospirillum spp.) and endophytic non-nodular symbionts (Azoarcus and Gluconacetobacter diazotrophicus). In the recent past, phosphate solubilizing and phytohormone synthesizing bacterial communities also gained momentum.

Abstract

The structure and function of proteins involved in plant-microbe interactions is investigated through large-scale proteomics technology in a complex biological sample. Since the whole genome sequence are now available for several plant species and microbes, proteomics study has became easier, accurate and huge amount of data can be generated during plant-microbe interactions. Proteomics approaches are highly important and relevant in many studies and showed that only genomics approaches is not sufficient as much significant information are lost as the proteins and not the genes coding them are final product which is responsible for the observed phenotype.

Restriction enzymes cut only at specific sequences of 4 to 8 bases

A long restriction site is rare. Cutting rare sites will produce a few large pieces of DNA.

Restriction enzymes with long sites → small number of large fragments

Restriction enzymes with short sites → large number of small fragments

Abstract

Production technology has been worked out for crops and several inputs have been recommended which are to be applied in proper quantity at most suitable time to get the maximum return. Nutrition in the form of fertilizers is a must. However, injudicious use of agro-chemicals has resulted in diseases called ‘iatrogenic plant diseases’. Under the circumstances it is important to know the relationship between soil nutrients and plant pathogens which are causing the diseases of economic importance. Author has given adequate emphasis to micronutrients as at a very minor quantity they reduce the disease to an acceptable level. He has also discussed in detail about all the major and minor nutrients required by the plants and how their deficiency or over doses affect the plant pathogens and in turn the diseases.

Keywords:  Soil nutrients, plant pathogens, diseases, relationship

Climate change produces long-term alterations in temperature, precipitation, wind and atmospheric carbon dioxide. Because insects are ectothermic organisms, these environmental changes strongly influence their physiology, ecology and behaviour. Rising temperatures can modify development, movement, feeding, mating, reproduction, diapause, migration and survival. Altered rainfall and extreme weather may affect habitat suitability, host availability and interactions with natural enemies. Climate-related behavioural changes can influence pest abundance, crop damage, geographical distribution and the timing of infestations. This chapter examines how insects respond behaviourally to changing environmental conditions and explains why understanding these responses is essential for forecasting pest outbreaks and developing effective climate-resilient crop-protection strategies.

Introduction

Restriction enzymes, recognizes specific base sequences in double-helical DNA and cleaves at specific places, both strands of a duplex containing the recognized sequences. To biochemists, these exquisitely precise scalpels are marvelous gifts of nature. They are indispensable for analyzing chromosome structure, sequencing very long DNA molecules, isolating genes, and creating new DNA molecules that can be cloned. Werner Arber and Hamilton Smith discovered restriction enzymes, and Daniel Nathans pioneered their use in the late 1960s.

Restriction Enzymes

Restriction enzymes are enzymes isolated from bacteria that recognize specific sequences in DNA and then cut the DNA to produce fragments, called restriction fragments. Restriction enzymes play a very important role in the construction of recombinant DNA molecules, as is done in gene cloning experiments. Another application of restriction enzymes is to map the locations of restriction sites in DNA.

Restriction endonucleases, often called restriction enzymes, cut DNA within the molecule by hydrolyzing the phosphodiester bonds between the nucleotides. Restriction enzymes were discovered in bacteria and there are now more than 1200 known restriction enzyme types. These enzymes are named using a simple system. EcoRI, for example, was isolated from E. coli and was the first enzyme isolated from a particular strain, hence the designation of I. HaeIII was isolated from Haemophilus aegyptius. HindIII was isolated from Haemophilus influenzae, and was the third enzyme discovered in a particular strain.

Usually, restriction enzymes only cut the DNA at or near a very specific nucleotide sequence known as a recognition site. This “restrictive” nature of these enzymes allows molecular biologists to pin-point exactly where the DNA is to be cut. In many cases, the recognition site of a restriction enzyme consists of a palindromic sequence where the order of the bases is read the same on each side of the helix. The two sides of the helix are antiparallel and the recognition sequence reads from 5’ to 3’ on either side of the helix. For example the recognition site for EcoRI is as follows;

Introduction

The primary tools used by the molecular biologists in manipulating DNA are restriction enzymes and other DNA/RNA modifying enzymes. Over the years, the number and uses of these enzymes have increased as new molecules have been discovered. Restriction endonucleases are bacterial enzymes that cleave double-stranded DNA. They are present in bacteria presumably to destroy DNA from foreign sources. By cleaving the foreign DNA at specific sites. The host bacterial DNA os protected from cleavage because specific recognition sites are modified, usually by methylation form methylase at one of the bases in the site, making the site no longer a substrate for RE cleavage. Host bacteria used to propagate cloned DNA in the laboratory arte usually mutant in the host restriction genes, thus their intracellular enzyme activities will not destroy the foreign recombinant sequences. The endonuclaase with its accompanying methylase is called a restriction modification (R-M) system.

At least four different kinds of R-M systems exist characterized on the basis of the subunit composition, cofactor requirements and type of DNA cleavage. Most characterized enzymes belong to type II class, together with type IIS class. They comprise the commercially available restriction enzymes used for DNA analysis and manipulation. Type I and Type III enzymes are relatively uncommon and a few additional enzymes fit none of the classes.

Among more than 3000 different enzymes isolated from bacterial strains many share common specificities. Restriction enzymes that recognize identical sequences have been called as isoschizomers.