eChapters

Storage

Nucleic acids are very sensitive to contaminations hence care should be taken while processing and storing DNA and RNA. The following properties of reagents and conditions need to be taken care of:

Heavy metals cause breakage of phosphodiester linkage. One can use EDTA which is an excellent heavy metal chelator.

UV rays cause extensive damage to nucleic acids. UV light at 260 nm causes breakage of thymine dimers and cross-link, including loss of biological activities.

Ethidium bromide results in photo oxidation of DNA. Oxidation products can cause phosphodiester breakage.

Nucleases are found on human skin; therefore, it is advisable to avoid direct or indirect contact between nucleic acids and fingers.

Most DNAes are not very stable; however, many RNAses are very stable and can absorb to glass or plastic and remain active.

At -20°C: extensive single and double strand breaks results. Hence for long-term storage it should be stores at -70°C. ? It is best to store DNA in high salt ( >1M) in the presence of high EDTA (>10mM) at pH 8.5, for long-term storage.

Storage of DNA in CsCl with ethidium bromide in the dark is an excellent storage medium. There is about one phosphodiester break per 200 kb of DNA per year.

Storage of ë DNA in the phage is better than storing the pure DNA.

Cell Genotypes

A large variety of bacterial strains, like E. coli, are frequently used in molecular biological experiments. Students should choose the strain or strains required for an experiment on the basis of genetic properties of the strains. It is thus pertinent that a sound knowledge of the genetic makeup of the E. coli stains is acquired.

The genetic makeup of the strains of E.coli is distinctly different from the wild type (K12 or B strains) strains from which they are derived and contains genes that can be modified. Genes are supposed to be mutated when they are not listed in a genotype. In most cases, the genes listed are mutated to the point of inactivity. In a few cases, a “-” (indicating inactive or absent or deleted), “+” (indicating active). , The genotype is a factor in choosing a bacterial strain. Chosen strains should be ascertained for their utility for the specific experiment.

Small Scale Cultures

Fresh culture from a freshly streaked plate should be taken while experimenting with E. coli cells. For preparing a small scale culture of E. coli , prepare 3-5 ml of LB medium/broth (or appropriate broth include antibiotic if the culture contains a plasmid) in two sterile 50 ml tubes. Inoculate one tube with a single colony from a fresh plate. The second tube is used as a broth control. Incubate both tubes at 37°C, shaking vigorously overnight. Inspect the tubes the next morning. The broth control should be clear and the inoculated culture should be very turbid. Make a note of any debris found in the tubes and only incubate longer if the culture is not dense. Never allow cells to overgrow. For some applications, cells can be stored at 4°C for short periods prior to use.

Abstract

The Basic Local Alignment Search Tool, or BLAST, is an online tool provided by the National Center for Biotechnology Information (NCBI) that allows users to search for matches between their query sequences and the sequences stored in NCBI databases. BLAST can be used to search for nucleotide sequences against nucleotide sequences, protein sequences against protein sequences, and protein sequences against translated nucleotide sequences in the databases. BLAST is widely used to study sequence similarity, identify target sequences, analyze the functional aspects of molecular sequences, and investigate evolutionary relationships. This chapter details on the BLAST and its applications.

Principle

Apart from PCR for generating large amounts of DNA, the second method is to use bacteria to replicate the DNA and then purify the replicated DNA from the bacteria. Plasmid DNA is usually the DNA of interest for this method. A plasmid is a double stranded circular DNA molecule that will replicate in an organism. A typical plasmid contains four important features:

The plasmid must be circular, because bacteria generally will not replicate linear ? DNA.

The plasmid must contain a sequence that functions as an origin of replication (ori).

The plasmid must contain a selection mechanism that will force the bacteria to retain the DNA; the most common type of selection mechanism used in bacteria is a gene for resistance to an antibiotic such as ampicillin or tetracycline.

The plasmid must contain a region for the insertion of the experimental DNA. A generic plasmid exhibiting these features is shown at right.

An expression plasmid that allows expression of foreign DNA should have a strong promoter segment for transcription of inserted DNA.

Must have an effective ribosome binding site that will allow efficient translation of the transcribed RNA.

Must have high copy number, i.e., the host bacterium should have many copies (usually >100)

Isolation of plasmid DNA from E. coli is a common routine in research laboratories. One can perform a widely-practiced procedure that involves alkaline lysis of cells. This protocol often referred to as a plasmid “mini-prep,” yields fairly clean DNA quickly and easily.

Procedure

Fill a microcentrifuge tube with saturated bacterial culture grown in LB broth + antibiotic. Spin tube in microcentrifuge for 1 minute, and make sure tubes are balanced in microcentrifuge. Dump supernatant and drain tube briefly on paper towel.

Repeat step 1 in the same tube, filling the tube again with more bacterial culture. The purpose of this step is to increase the starting volume of cells so that more plasmid DNA can be isolated per prep. Spin tube in microcentrifuge for 1 minute. Pour off supernatant and drain tube on paper towel.

Add 0.2 ml ice-cold Solution 1 to cell pellet and resuspend cells as much as possible using disposable transfer pipet.

Abstract

Proteins are complex molecules composed of one or more polypeptide chains made up of amino acid residues. These molecules perform a variety of essential functions in living organisms, including DNA replication, molecule transport, catalyzing metabolic processes, and providing structural support to cells. In this chapter, we explore the four levels of protein structure: primary, secondary, tertiary, and quaternary. Primary structure refers to the specific sequence of amino acids in a polypeptide chain, while secondary structure describes the folding of the chain into alpha-helices, beta-sheets, or other patterns. Tertiary structure is the three-dimensional shape of a single polypeptide chain, and quaternary structure refers to the interactions between multiple polypeptide chains to form a larger protein complex. Additionally, this chapter covers various methods used to predict protein structure, including homology modeling, ab initio prediction, and threading algorithms.

Abstract

When only sequence data is available, homology modeling has become a useful tool for predicting protein structure. Protein structure is often more important than sequence data when evaluating protein function. Homology modeling, also known as comparative modeling, is based on the principle that two sequences that exhibit a high degree of identity or similarity will also share similar structures. This chapter explores the steps involved in homology modeling, including template selection, alignment, model building, refinement, and validation. The Ramachandran plot is a valuable tool for assessing the conformation and bond angles in a protein model, and we discuss its significance in the context of homology modeling. Ultimately, homology modeling is an effective method for predicting protein structure and has numerous applications in protein engineering, drug discovery, and other fields.

Technology Scouting and Innovations in Technology Incubation
Technology scouting and innovations in technology incubation have a symbiotic relationship, fuelling each other for the benefit of budding startups and the broader technology ecosystem. Let’s dive deeper into their individual roles and how they interact:
Technology Scouting
Technology Scouting is the process of discovering, analyzing and evaluating new or existing technologies that will help them with their innovation process. In simple terms, it is a process for companies to find the necessary technology outside the company.
Hunting for promising technologies: Scouts actively search for emerging technologies, patents, research advancements, and market trends across various sectors.
Evaluating potential and fit: They assess the technical feasibility, market potential, and compatibility with the needs of incubators and their supported startups.
Connecting innovators and ventures: Scouts bridge the gap between technology creators (researchers, inventors, startups) and potential users (incubators, established companies).
Fuelling innovation pipelines: Technology scouting helps incubators access a wider range of technologies and ideas, enriching their portfolio and diversifying the innovations supported.

Solution Preparation

1. Glycerol mix (1 L)

Weigh 25 grams (liquid weight) of glycerol and

Add dH₂O to 1 Liter (Autoclave).

2. Potassium mix (1 L)

170 mL of 1M KH₂PO₄ (monobasic),

720 mL of 1M K₂HPO₄ (dibasic), and

110 mL of dH₂O (Autoclave)

Preparation of Solutions

GTE buffer

50 mM Glucose,

25 mM Tris-Cl,

10 mM EDTA,

pH 8

NaOH/SDS lysis solution

0.2 M NaOH,

1% SDS

Formation of a Rectangle

Experiments are usually laid out in square or rectangular plots for data and yield estimation, even though the field may be irregular. The plot is usually placed on the contour, parallel with a road, or in a north-and-south alignment. To mark a plot, construct a right angle and then form a rectangle or square.

Abstract

Systems biology is an interdisciplinary field that combines computer and mathematical analysis with biological research to understand the complex interactions within biological systems. This chapter explores the history and evolution of systems biology and its significant impact on modern biology. Systems biology uses a holistic approach to analyze biological systems at the molecular, cellular, and organismal levels, providing a better understanding of biological processes and the interactions between genes, proteins, and other molecules. Systems biology has immense potential in improving human health, agriculture, and the environment, and this chapter explores the ongoing efforts to translate systems biology research into practical applications.

Reagents

EDTA

Ethanol, absolute

Isopropanol

Phosphate Buffered Saline (PBS), 1X

Proteinase K

Sodium Acetate, 3 M pH 5.2 (Molecular Biology Grade)

Sodium chloride (NaCl)

Sodium Dodecyl Sulfate (SDS ), 10%

Tris EDTA (TE), pH 7.4

Tris EDTA (TE), pH 8.0

Tris HCl, pH 8.0

Reagents

Chloroform

EDTA, 0.5 M

Ethanol, absolute

Isoamyl alcohol

Phenol

Phosphate Buffered Saline (PBS), 1X

Proteinase K

RNAse A

Sodium dodecyl sulfate (SDS) solution, 10%

Reagents

Chloroform

EDTA, 0.5 M

Ethanol, absolute

Is amyl alcohol

Phenol

Proteinase K

RNase A

Sodium acetate, pH 5.2

Sodium chloride, 5 M
Sodium thiocyanate (NaSCN), 1 M

TE Buffer (Tris-EDTA), pH 7.4

Tween 20

Xylene

Principle

Restriction digestion is one of the most common reactions performed in molecular biology. Restriction endonucleases (RE) are also known as restriction enzymes, which recognize short, specific DNA sequences. There are three types of RE: Type-I, Type-II and Type-III. Type II RE, most commonly used for DNA analysis and genetic engineering, cleave double-stranded DNA (dsDNA) at specific sites within or adjacent to their recognition sequences. All Type II RE recognises a unique nucleotide sequence at which it cuts a DNA molecule and nowhere else. The recognition sequence is often a six base pair palindromic sequence (the top DNA strand from 5' to 3' is the same as the bottom DNA strand from 5' to 3'), but others recognize four or even eight base pair sequences. Many RE will not cut DNA that is methylated on one or both strands of the recognition site, although some require substrate methylation.

REs can also differ in the way they cut the DNA molecule. Some enzymes cut in the middle of the recognition sequence, resulting in a flush or “blunt end”. Other enzymes cleave in a staggered fashion, resulting in DNA products that have short single-stranded overhangs (usually two or four nucleotides) at each end. These are often called “cohesive ends”, as these single-stranded overhangs could potentially come together again through complementary base-pairing, as shown below:

Principle

Since the entire DNA sequence of the bacteriophage is known, we know exactly where a restriction enzyme will cleave the viral DNA and the size of the DNA fragments generated. This restriction enzyme digest will create a pattern of specific DNA fragments that will serves as a “DNA ladder”.

Principle of DNA Ligase Mechanism

The process of connecting two pieces of DNA together is called ligation, and is catalyzed by an enzyme called ligase. The ligase used most often in molecular biology is derived from the T4 bacteriophage, and uses ATP to supply the energy necessary for the reaction. In addition to ATP, T4 ligase requires DNA with a 5´-phosphate group and free 3´-hydroxyl groups.

Applications

Ligation of blunt or cohesive-ended DNA fragments.

Repair of nicks in double-stranded nucleic acids.

Objectives

• Better understand the law of independent assortment.

History

The law was proposed by Gregor Johann Mendel with the other laws together called as ‘Mendelian laws of inheritance’, in 1866. He used pea plant as a model organism, and studied 7 characters.

Principle

The Law of Independent Assortment, also known as “Inheritance Law”, states that alleles of different genes assort independently of one another during gamete formation. Independent assortment for two genes can be explained by assuming that the two genes are located in two different chromosomes. The two alleles of a gene will be located in the two homologues of the concerned chromosome. Independent separation of these two pairs of chromosomes at anaphase I of meiosis will lead to the independent segregation of the genes located in them. Thus any allele of one gene is equally likely to combine with any allele of the other gene and pass into the same gamete. Independent segregation of two genes produces four different types of gametes in equal proportion. A random union among these gametes gives rise to sixteen possible zygotes. These zygotes yield a 9:3:3:1 phenotypic ratio, which is known as the typical dihybrid ratio. When two pairs of independent alleles enter into F₁ combination, both of them have their independent dominant effect. These alleles segregate when gametes are formed but the assortment occurs independently at random and quite freely.

Aim

To synthesize chitosan/nanosilica coating solution for fruit coating aiming at increasing shelf life.

Introduction

Application of edible coatings is promising to improve the quality and extend shelf life of fruits and vegetables (Lin et al., 2011). Chitosan, a versatile biopolymer derived from deacetylation of chitin had been widely applied in the fresh-keep field owing to its good biocompatibility, biodegradability, antibacterial activity and capacities to form film (Lin et al., 2011).