Short notes on X-ray Crystallography | BTF

 X-ray crystallography is the most powerful method to obtain a macromolecular structure.

X-ray crystallography is a tool used for determining the atomic and molecular structure of a crystal. The underlying principle is that the crystalline atoms cause a beam of X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a 3D picture of the density of electrons within the crystal.

X-Ray crystallography is a tool used to provide structural information about molecules. The technique was developed in 1912 by William Henry Bragg and William Lawrence Bragg (a father and son team who won the 1915 Nobel Prize in Physics for their work in the field), who built upon earlier work by Max von Laue.

X-ray crystallography remains the most robust method to determine protein structure at the atomic level. However, the bottlenecks of protein expression and purification often discourage further study. 

X-ray crystallography  is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their crystallographic disorder, and various other information.

What is X ray diffraction simple definition?

: a scattering of X-rays by the atoms of a crystal that produces an interference effect so that the diffraction pattern gives information on the structure of the crystal or the identity of a crystalline substance.

X-ray crystallography is a technique used for determining the high-resolution, three-dimensional crystal structures of atom and molecules and has been fundamental in the development of many scientific fields. In its first decades of application, it is mainly used for determining the size of atoms, the lengths and types of chemical bonds, the atomic-scale differences among various materials, as well as the crystalline integrity, grain orientation, grain size, film thickness and interface roughness of the related materials, especially minerals and alloys.

 

Through X-ray crystallography, the chemical 

structure of thousands of organic, inorganic, 

organometallic, and biological compounds 

are determined every year. 

Bragg 

Spectrometer :

X-ray was the name given to the 

highly penetrating rays which 

emanated when high energy 

electrons struck a metal target. 

What is the principle of X ray crystallography?

X-ray crystallography is a tool used for determining the atomic and molecular structure of a crystal. The underlying principle is that the crystalline atoms cause a beam of X-rays to diffract into many specific direction.  

What is X ray crystallography used for?

X-ray crystallography is a tool used for determining the atomic and molecular structure of a crystal. The underlying principle is that the crystalline atoms cause a beam of X-rays to diffract into many specific direction. 

Why is Xray crystallography important?

X-ray crystallography enables the identification of the atomic and molecular structure of a crystal. ... The X-ray technique provides direct structural information on molecules at the atomic level and is recognized as a reliable structure determination method (Gaudencio and Pereira, 2015).

Where is Xray crystallography used?

Most scientists use x-ray Crystallography to solve the structures of protein and to determine functions of residues, interactions with substrates, and interactions with other proteins or nucleic acids. Proteins can be co - crystallized with these substrates, or they may be soaked into the crystal after crystallization. 

X-ray crystallography is also a routine technique to determine how a drug interacts with its target and what modifications could improve the interaction. Prakasham et al.76 have investigated diastase enzyme immobilized on nickel-impregnated silica paramagnetic NPs and they characterized them by FTIR and X-ray crystallography.

Why do we study crystallography?

Crystallography is the study of atomic and molecular structure. Crystallographers want to know how the atoms in a material are arranged in order to understand the relationship between atomic structure and properties of these materials. Crystallography began with the study of crystals, like quartz. 

 

Applications of X-ray crystallography

X-ray crystallography is used to analyze many different molecules and has been used in many famous projects in the fields of organic and inorganic chemistry. Early structures which were resolved using the technique were simple crystals, including quartz. As well as the analysis of organic molecules (proteins, vitamins, nucleic acids) and inorganic molecules and structures, X-ray crystallography has been used to develop novel materials in both material and life sciences.

Drug identification

Investigation of bones

Characterization of textile fibers and polymers

Integrated circuits

Short notes on X-ray Crystallography | BTF

Fermentation |Biotechnify |BTF

 

Fermentation : Definition & Introduction 

The chemical breakdown of a substance by bacteria, yeasts, or other microorganisms, typically involving effervescence and the giving off of heat.

"the fermentation of organic matter by microorganisms in the gut"

Fermentation is a metabolic process that produces chemical changes in organic substrates through the action of enzymes. In biochemistry, it is narrowly defined as the extraction of energy from carbohydrates in the absence of oxygen.

The science of fermentation is known as zymology.

In microorganisms, fermentation is the primary means of producing adenosine triphosphate (ATP) by the degradation of organic nutrients anaerobically.[2] Humans have used fermentation to produce foodstuffs and beverages since the Neolithic age. For example, fermentation is used for preservation in a process that produces lactic acid found in such sour foods as pickled cucumbers, kombucha, kimchi, and yogurt, as well as for producing alcoholic beverages such as wine and beer. Fermentation also occurs within the gastrointestinal tracts of all animals, including humans.

The word "ferment" is derived from the Latin verb fervere, which means to boil.it is all about in etymology.

Fermentation, chemical process by which molecules such as glucose are broken down anaerobically. More broadly, fermentation is the foaming that occurs during the manufacture of wine and beer, a process at least 10,000 years old. The frothing results from the evolution of carbon dioxide gas, though this was not recognized until the 17th century.

How Does Fermentation Work?

Microorganisms survive using carbohydrates (sugars, such as glucose) for energy and fuel.

Organic chemicals like adenosine triphosphate (ATP) deliver that energy to every part of a cell when needed.

Microbes generate ATP using respiration. Aerobic respiration, which requires oxygen, is the most efficient way to do that. Aerobic respiration begins with glycolysis, where glucose is converted into pyruvic acid. When there’s enough oxygen present, aerobic respiration occurs.

Fermentation is similar to anaerobic respiration—the kind that takes place when there isn’t enough oxygen present. However, fermentation leads to the production of different organic molecules like lactic acid, which also leads to ATP, unlike respiration, which uses pyruvic acid.

Depending upon environmental conditions, individual cells and microbes have the ability to switch between the two different modes of energy production.

Organisms commonly obtain energy anaerobically through fermentation, but some systems use sulfate as the final electron acceptor in the electron transport chain.

Fermentation is all down to the actions of tiny natural microbes, who colonize and cultivate everything from our digestive systems, to this colorful spring in Yellowstone seen in the picture above, to the food and drink we eat. 

Microbes use carbohydrates (sugars, such as glucose) for energy to fuel their survival. To make use of that energy, organic chemicals like adenosine triphosphate (ATP) deliver it when needed to every part of a cell.

Microbes - and our own body cells - use respiration to generate ATP. The most efficient way for them to do that is through a process known as aerobic respiration, which requires oxygen.

Aerobic respiration starts with glycolysis, where glucose is converted into pyruvic acid. Then, when there's enough oxygen around, aerobic respiration takes place. 

Fermentation is similar to the kind of respiration that takes place when there isn't enough oxygen present, namely anaerobic respiration. However unlike respiration, which uses pyruvic acid, fermentation leads to the production of different organic molecules like lactic acid, which also leads to ATP.  

What are three types of fermentation? 

Lactic acid fermentation

Lactic acid fermentation is a metabolic process by which glucose and other six-carbon sugars (also, disaccharides of six-carbon sugars, e.g. sucrose or lactose) are converted into cellular energy and the metabolite lactate, which is lactic acid in solution.

Yeast strains and bacteria convert starches or sugars into lactic acid, requiring no heat in preparation. These anaerobic chemical reactions, pyruvic acid uses nicotinamide adenine dinucleotide + hydrogen (NADH) to form lactic acid and NAD+. (Lactic acid fermentation also occurs in human muscle cells. During strenuous activity, muscles can expend adenosine triphosphate (ATP) faster than oxygen can be supplied to muscle cells, resulting in lactic acid buildup and sore muscles. In this scenario, glycolysis, which breaks down a glucose molecule into two pyruvate molecules and doesn’t use oxygen, produces ATP.) Lactic acid bacteria are vital to producing and preserving inexpensive, wholesome foods, which is especially important in feeding impoverished populations. This method makes sauerkraut, pickles, kimchi, yogurt, and sourdough bread.

Ethanol fermentation/alcohol fermentation

Yeasts break pyruvate molecules—the output of the metabolism of glucose (C6H12O6) known as glycolysis—in starches or sugars down into alcohol and carbon dioxide molecules. Alcoholic fermentation produces wine and beer.

Ethanol fermentation, also called alcoholic fermentation, is a biological process which converts sugars such as glucose, fructose, and sucrose into cellular energy, producing ethanol and carbon dioxide as by-products.

Acetic acid fermentation

Starches and sugars from grains and fruit ferment into sour tasting vinegar and condiments. Examples include apple cider vinegar, wine vinegar, and kombucha

Cell Culture Techniques |Biotechnify|BTF

What is Cell Culture? 

Cell culture is the process by which cells are grown under controlled conditions, generally outside their natural environment. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions.

Essentially , cell culture involves the distribution of cells in an artificial environment (in vitro) which is composed of the necessary nutrients, ideal temperature, gases, pH and humidity to allow the cells to grow and proliferate.

• In vivo - When the study involves living biological entities within the organism.

• In vitro - When the study is conducted using biological entities (cells, tissue etc) that has been isolated from their natural biological environment. E.g. tissue or cells isolated from the liver or kidney. 

Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established.

What is Cell Culture Techniques ?

In cell culture techniques, cells (or tissues) are removed from a plant or an animal and introduced into a new, artificial environment that can support their proliferation (survival and growth). Some of the requirements of such an environment for the proliferation of the cells include: A substrate (source of nutrition).

Cell culture techniques play a key role in the development of new anticancer drugs by imposing additional constraints on those of receptor interaction alone, such as drug uptake and efflux, interaction with other cellular receptors, and cellular metabolism

Primary culture :

Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). At this stage, the cells have to be subcultured (i.e., passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth

Cell Culture Protocol :

Cell culture protocols are meant to ensure that culture procedures are carried out to the required standards. This is not only meant to prevent the contamination of the cells, but to also ensure that the researchers themselves are protected from any form of contamination.

However, the nature of the work is expected to conform to the appropriate ethical guidelines. Therefore, before anything else, it is essential to ensure that the entire procedure conforms with both medical-ethical and animal- experiment guidelines. This is because going against such legislation and guidelines can result in heavy penalties and even shutting down of the laboratory.

Before start , carry out the following procedure:

Ensure that the working are is sanitized (using 70 percent ethanol)Always use a new pair of gloves. If a pair of gloves has to be used for another cell culture procedure, they should be sanitized using 70 percent ethanol and allowed to air dry.

Any equipment that had been taken out of the cabinet should also be sanitized to prevent any contamination such equipment as pipette, glass jars and plastics to be used for the procedure should be autoclaved

Although there are a wide range of culture media for cells, it is important to keep in mind that cell cultures, and particularly primary cell cultures are easily prone to contamination in addition to the risk of containing undetected viruses. For this reason, all material should be handled as potentially infectious in order to avoid any infections.

Protocols for cell culture preparation :

Always check the information on the container to ensure that the medium is appropriate for the cell to be cultured,

Once prepared, the cell culture should be maintained under the recommended temperature range,

Monitor the culture every 30- 48 hours and check for confluency (when cells completely cover the surface of the culture) - However, this is largely dependent on the type of cells.

Once the procedure is completed and the cells have been analyzed, the culture should be appropriately discarded. Here, it is important to take a lot of caution given that by this time, cells have already proliferated and increased in numbers. Moreover, there are high chances that the specimen has been contaminated, which increase the risks of causing infections to the researcher if not handled appropriately.

What is Cell Culture used for? 

Cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the effects of drugs and toxic compounds on the cells, and mutagenesis and carcinogenesis.

What is Cell Passaging? 

Subculturing, also referred to as passaging cells, is the removal of the medium and transfer of cells from a previous culture into fresh growth medium, a procedure that enables the further propagation of the cell line or cell strain.

Applications of Cell Culture :

Cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the effects of drugs and toxic compounds on the cells, and mutagenesis and carcinogenesis. It is also used in drug screening and development, and large scale manufacturing of biological compounds (e.g., vaccines, therapeutic proteins). The major advantage of using cell culture for any of these applications is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells.

Conclusion :

In conclusion, cell culture is an indispensable tool in modern day medicine and its applications are innumerable in diagnosis of human infection. Cell culture methods are unbiased to some extent and only limited by the ability of the virus to grow in a particular cell line.

Isolation Techniques - short explanation | notes on isolation techniques

 Isolation Techniques - short explanation | notes on isolation techniques

Isolation Techniques 

1. the process of separating, or the state of being alone.

2. the physiologic separation of a part, as by tissue culture or by interposition of inert material.

3. the extraction and purification of a chemical substance of unknown structure from a natural source.

4. the separation of infected individuals from those uninfected for the period of communicability of a particular disease; see also quarantine.

5. the separation of an individual with a radioactive implant from others to prevent unnecessary exposure to radioactivity.

Definition :

Isolation refers to the precautions that are taken in the hospital to prevent the spread of an infectious agent from an infected or colonized patient to susceptible persons.

In microbiology, the term isolation refers to the separation of a strain from a natural, mixed population of living microbes, as present in the environment, for example in water or soil flora, or from living beings with skin flora, oral flora or gut flora, in order to identify the microbe(s) of interest.

History :

The laboratory techniques of isolating microbes first developed during the 19th century in the field of bacteriology and parasitology using light microscopy. Proper isolation techniques of virology did not exist prior to the 20th century. The methods of microbial isolation have drastically changed over the past 50 years, from a labor perspective with increasing mechanization, and in regard to the technologies involved, and with it speed and accuracy.

Purpose

Isolation practices are designed to minimize the transmission of infection in the hospital, using current understanding of the way infections can transmit. Isolation should be done in a user friendly, well-accepted, inexpensive way that interferes as little as possible with patient care, minimizes patient discomfort, and avoids unnecessary use.

Precautions Types

There are three types of transmission-based precautions--contact, droplet, and airborne - the type used depends on the mode of transmission of a specific disease.

Precautions :

The type of precautions used should be viewed as a flexible scale that may range from the least to the most demanding methods of prevention. These methods should always take into account that differences exist in the way that diseases are spread. Recognition and understanding of these differences will avoid use of insufficient or unnecessary interventions.

Standard precautions :

Standard Precautions define all the steps that should be taken to prevent spread of infection from person to person when there is an anticipated contact with:

Blood

Body fluids

Secretions, such as phlegm

Excretions, such as urine and feces (not including sweat) whether or not they contain visible blood

Nonintact skin, such as an open wound

Mucous membranes, such as the mouth cavity.

Standard Precautions includes the use of one or combinations of the following practices. The level of use will always depend on the anticipated contact with the patient:

Handwashing, the most important infection control method

Use of latex or other protective gloves

Masks, eye protection and/or face shield

Gowns

Proper handling of soiled patient care equipment

Proper environmental cleaning

Minimal handling of soiled linen

Proper disposal of needles and other sharp equipment such as scalpels

Placement in a private room for patients who cannot maintain appropriate cleanliness or contain body fluids.

Transmission based precautions :

Transmission Based Precautions may be needed in addition to Standard Precautions for selected patients who are known or suspected to harbor certain infections. These precautions are divided into three categories that reflect the differences in the way infections are transmitted. Some diseases may require more than one isolation category.

AIRBORNE PRECAUTIONS. Airborne Precautions prevent diseases that are transmitted by minute particles called droplet nuclei or contaminated dust particles. These particles, because of their size, can remain suspended in the air for long periods of time; even after the infected person has left the room. Some examples of diseases requiring these precautions are tuberculosis, measles, and chickenpox.

Following isolation methods are employed to isolate microbes from mixed cultures:

1. Streaking

2. Plating

3. Dilution

4. Enriched procedure, and

5. Single cell technique.

Description :

Isolation practices can include placement in a private room or with a select roommate, the use of protective barriers such as masks, gowns and gloves, a special emphasis on handwashing (which is always very important), and special handling of contaminated articles. Because of the differences among infectious diseases, more than one of these precautions may be necessary to prevent spread of some diseases but may not be necessary for others.The Centers for Disease Control and Prevention (CDC) and the Hospital Infection Control Practice Advisory Committee (HICPAC) have led the way in defining the guidelines for hospital-based infection precautions. The most current system recommended for use in hospitals consists of two levels of precautions. The first level is Standard Precautions which apply to all patients at all times because signs and symptoms of infection are not always obvious and therefore may unknowingly pose a risk for a susceptible person. The second level is known as Transmission-Based Precautions which are intended for individuals who have a known or suspected infection with certain organisms.

Frequently, patients are admitted to the hospital without a definite diagnosis, but with clues to suggest an infection. These patients should be isolated with the appropriate precautions until a definite diagnosis is made.

Notes :

It is absolutely essential that you sterilize your loop between each streaking, either by using the incinerator or by obtaining a new sterile plastic loop. This is the most common mistake students make.

Don’t leave your plate open too long or extra bacteria from the environment will fall into your plate.

Do not be disappointed if you do not get isolated colonies on your first try. This is a difficult procedure.

short notes on Bioreactors

Bioreactor :

A bioreactor refers to any manufactured device or system that supports a biologically active environment.

On the basis of mode of operation, a bioreactor may be classified as batch.

Immobilization is useful for continuously operated processes.

Why are bioreactors used?

The process can be aerobic or anaerobic. Bioreactors are used for making pharmaceutical products such as antibiotics and insulin.

Bioreactors are used for making pharmaceutical products such as antibiotics and insulin. 

What is the difference between bioreactor and fermentor?

           Bioreactors & Fermenters

Fermentation involves the breakdown of certain substances by bacteria or yeast or other microorganisms in the production of beer, wine, or liquor through the action of enzymes. Energy is extracted from carbohydrates in the absence of oxygen.

And At the end of the process, the desired products can be extracted or separated easily. Hence, these bioreactors utilize routinely in industries to produce secondary metabolites such as pharmaceuticals, vitamins and proteins.

What is a bioreactor and how does it work?

Through this mechanism, called the denitrification pathway, nitrate is removed from the tile water before it can enter surface waters.

These microorganisms "eat" the carbon from the woodchips and "breathe" the nitrate from the water. Just as humans breathe in oxygen and breathe out carbon dioxide, these microorganisms breathe in nitrate and breathe out nitrogen gas, 

Bioreactor applications?

They are used in various applications, including basic research and development, and the manufacturing of biopharmaceuticals, food and food additives.

Types :

Photoreactor :- A photobioreactor (PBR) is a bioreactor which incorporates some type of light source (that may be natural sunlight or artificial illumination).

                      Photobioreactors are used to grow small phototrophic organisms such as cyanobacteria, algae, or moss plants.

                       Bioreactors are generally used in those industries which are concerned with food, beverages and pharmaceuticals.

Bioreactors for specialized tissues :-

Many cells and tissues, especially mammalian ones, must have a surface or other structural support in order to grow, and agitated environments are often destructive to these cell types and tissues.

Currently, scaling production of these specialized bioreactors for industrial use remains challenging and is an active area of research.

 Sewage treatment :-

Conventional sewage treatment utilises bioreactors to undertake the main purification processes. In some of these systems, a chemically inert medium with very high surface area is provided as a substrate for the growth of biological film. 

                     An extremely simple version of a sewage bioreactor is a septic tank whereby the sewage is left in situ, with or without additional media to house bacteria. 

Short notes on - Laminar Air Flow

 Short notes on - Laminar Air Flow

What is laminar air flow unit?

Laminar airflow is defined as air moving at the same speed and in the same direction,  By contrast, turbulent flow creates swirls and eddies that deposit particles on surfaces randomly and unpredictably.

                   A laminar flow cabinet or tissue culture hood is a carefully enclosed bench designed to prevent contamination of semiconductor wafers, biological samples, or any particle sensitive materials. 

                    Laminar airflow is defined as air moving at the same speed and in the same direction, with no or minimal cross-over of air streams (or “lamina”). By contrast, turbulent flow creates swirls and eddies that deposit particles on surfaces randomly and unpredictably.

Who invented Laminar Air flow?

Willis Whitfield

When Willis Whitfield invented the modern-day cleanroom 50 years ago, researchers and industrialists did not believe it at first. But within a few short years, US$50bn worth of laminar-flow cleanrooms were being built worldwide and the invention is used in hospitals, laboratories and manufacturing plants today.

What is the use of laminar air flow?

The process of laminar air flow can be described as airflow where an entire body of air flows with steady,in the laboratory, Laminar Flow Cabinets are commonly used for specialised work.Laminar airflow is used to separate volumes of air, or prevent airborne contaminants from entering an area. Laminar flow hoods are used to exclude contaminants from sensitive processes in science, electronics and medicine.

                      

                       

What is the principle of laminar flow?

laminar flow towards the user. Due to the direction of air flow, Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the users.

How do you get laminar air flow?

In a vertical clean bench,laminar air is then projected vertically over the work area's..

 In these instances, HEPA-filtered air mixes with and dilutes interior airborne contaminants inside the glove box. 

What is the function of laminar air flow?

The process of laminar air flow can be described as airflow where an entire body of air flows with steady, uniform velocity. Laminar Flow Cabinets work by the use of in-flow laminar air drawn through one or more HEPA filters, designed to create a particle-free working environment and provide product protection.

What is laminar air flow in microbiology?

Laminar Hood sometimes also known as Laminar Air Flow is an enclosed bench designed to prevent contaminations .

Laminar Air Flow are equipped with a UV lamp that should be turned on about 10-20 minutes before being used to sterilize the shell or cabinet or the surface of the Laminar Air Flow to avoid any kind of contaminations.

What is HEPA filter in laminar air flow?

In a laminar flow hood the air is passed through a HEPA (High Efficiency Particulates Air) filter which removes all airborne contamination to maintain sterile conditions

Procedure of Laminar air flow.?

Turn on the switch of UV light; leave the UV on for at least 30 minutes.

Turn OFF the UV light.

Turn ON the switch of visible light.

Turn ON the switch of Air Flow.

What is laminar flow in OT?

This is re-circulated under positive pressure into the operating theatre with surgically generated contaminants being continuously removed .

True laminar flow is only achieved when approximately 100% HEPA filter coverage occurs. 

in theatre more than 300 times per hour compared to standard positive pressure theatre rates of 15-25 air changes per hour. 

How does a laminar flow work? 

In fluid dynamics, laminar flow is characterized by fluid particles following smooth paths in layers.

There are no cross-currents perpendicular to the direction of flow, nor eddies or swirls of fluids.

Laminar flow is a flow regime characterized by high momentum diffusion and low momentum convection.

Laminar flow barriers :

Laminar flow hoods are used to exclude contaminants from sensitive processes in science, electronics and medicine.

Laminar flow cabinet :

Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the user.

A laminar flow cabinet or tissue culture hoodis a carefully enclosed bench designed to prevent contamination of semiconductor wafers,

Cleaning procedure of laminar air flow:

Cleaning a Vertical Laminar Flow Hood

Begin by taking the wipe and spraying disinfectant onto the wipe. 

After Cleaning :

Some laminar airflow hoods may have UV-C Germicidal Lamps for sterilization. 

Always remember to wash your hands afterward.

When cleaning is complete, dispose of wipes, gloves, and gown in the biohazard waste.

Why is laminar flow important?

It is the smooth flow of a fluid over a surface. Though a boundary layer of air "sticks" to a wing, the air overtop should be moving quickly and smoothly to reduce friction drag. Engineers want to design aircraft with laminar flow over their wings to make them more aerodynamic and efficient.

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If you have any query about laminar air flow or doubts then ask in comment box we will help you as soon as possible. 

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DNA isolation protocol from Blood

 DNA isolation protocol from Blood

The whole explanation of how to do-Dna  isolation from blood :

Abstract: Deoxyribonucleic acid (DNA) extraction has considerably evolved since it was initially performed back in 1869.Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification.  It is the first step required for many of the available downstream applications used in the field of molecular biology. Whole blood samples are one of the main sources used to obtain DNA, and there are many different protocols available to perform nucleic acid extraction on such samples. These methods vary from very basic manual protocols to more sophisticated methods included in automated DNA extraction protocols. Based on the wide range of available options, it would be ideal to determine the ones that perform best in terms of cost-effectiveness and time efficiency. We have reviewed DNA extraction history and the most commonly used methods for DNA extraction from whole blood samples, highlighting their individual advantages and disadvantages. We also searched current scientific literature to find studies comparing different nucleic acid extraction methods, to determine the best available choice. Based on our research, we have determined that there is not enough scientific evidence to support one particular DNA extraction method from whole blood samples. Choosing a suitable method is still a process that requires consideration of many different factors, and more research is needed to validate choices made at facilities around the world.Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.

Keywords: mitochondrial, quick-prep, restriction enzyme, genomic DNA extraction, whole blood samples, solution-based DNA extraction, solid-phase DNA extraction, cost-effectiveness, time efficiency

Introduction

Recent research advances in genomic disorders have necessitated the collection of large amounts of good quality DNA that needs to be obtained from different sample sources. DNA typing is currently the most validated method for the personal identification of human bodily fluid stains found at crime scenes. Human health studies in the field of molecular biology require the use of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein samples. Successful use of available downstream applications will benefit from the use of high-quantity and high-quality DNA. Therefore, nucleic acid extraction is a key step in laboratory procedures required to perform further molecular research applications. It is essential to choose a suitable extraction method, and there are a few considerations to be made when evaluating the available options. These may include technical requirements, time efficiency, cost-effectiveness, as well as biological specimens to be used and their collection and storage requirements.

Whole blood is one of many different available sources to obtain genomic DNA (gDNA), and it has been widely used in facilities around the world. Therefore, we will focus on DNA extraction protocols using whole blood samples. Issues regarding collection, storage, and manual handling of human whole blood specimens escape the scope of this publication and will not be covered. However, they are important and they should be considered, as they could potentially impact on the performance and success of any DNA extraction technique chosen.

Initial development of DNA extraction techniques

Friedrich Miescher was the first scientist to isolate DNA while studying the chemical composition of cells. In 1869, he used leukocytes that he collected from the samples on fresh surgical bandages and conducted experiments to purify and classify proteins contained in these cells. During his experiments he identified a novel substance in the nuclei, which he called “nuclein”.² He then developed two protocols to separate cells’ nuclei from cytoplasm and to isolate this novel compound, nowadays known as DNA, which differed from proteins and other cellular substances. This scientific finding, along with the isolation protocols used, was published in 1871 in collaboration with his mentor, Felix Hoppe-Seyler.However, it was only in 1958 that Meselson and Stahl developed a routine laboratory procedure for DNA extraction. They performed DNA extraction from bacterial samples of Escherichia coli using a salt density gradient centrifugation protocol. Since then, DNA extraction techniques have been adapted to perform extractions on many different types of biological sources.

DNA extraction methods follow some common procedures aimed to achieve effective disruption of cells, denaturation of nucleoprotein complexes, inactivation of nucleases and other enzymes, removal of biological and chemical contaminants, and finally DNA precipitation. Most of them follow similar basic steps and include the use of organic and nonorganic reagents and centrifugation methods. Finally, they have developed into a variety of automated procedures and commercially available kits.

Initially, we will discuss protocols and steps aimed to achieve cell lysis, inactivation of cellular enzymes, denaturation of cellular complexes, and DNA precipitation, which require similar procedures and/or reagents during DNA extraction from whole blood samples. Key differences in steps aiming to remove biological and chemical contaminants will be highlighted when we discuss each protocol in detail.

As previously mentioned, lysis of cells is a common step in most DNA extraction protocols, and it is commonly achieved through the use of detergents and enzymes. Sodium dodecyl sulfate (SDS) and Triton™ X-100 (Sigma-Aldrich, St Louis, MO, USA) are examples of popular detergents used to solubilize cell membranes. Enzymes are also combined with detergents to target cell surface or cytosolic components. Proteinase K is a commonly used enzyme used in various protocols to cleave glycoproteins and inactivate RNases and DNases. Other denaturants such as urea, guanidinium salts, and chemical chaotropes have also been used to disrupt cells and inactivate cellular enzymes, but these can impact on quality and nucleic acid yield. 

DNA precipitation is achieved by adding high concentrations of salt to DNA-containing solutions, as cations from salts such as ammonium acetate counteract repulsion caused by the negative charge of the phosphate backbone. A mixture of DNA and salts in the presence of solvents like ethanol (final concentrations of 70%–80%) or isopropanol (final concentrations of 40%–50%) causes nucleic acids to precipitate. Some protocols include washing steps with 70% ethanol to remove excess salt from DNA. Finally, nucleic acids are resuspended in water or TE buffer (10 mM Tris, 1 mM ethylenediaminetetraacetic acid [EDTA]). TE buffer is commonly used for long-term DNA storage because it prevents it from being damaged by nucleases, inadequate pH, heavy metals, and oxidation by free radicals. Tris provides a safe pH of 7–8, and EDTA chelates divalent ions used in nuclease activity and counteracts oxidative damage from heavy metals.

Main types of DNA extraction methods from human whole blood samples

Methods as by Shows the main categories and subcategories of DNA extraction methods from whole blood samples that are generally used in research facilities worldwide.

Solution-based DNA extraction methods

As previously mentioned, solution-based protocols have two main approaches: 1) solution-based methods using organic solvents and 2) those based on a salting out technique. Further description of both methods follows.

Solution-based DNA extraction methods using organic solvents

DNA extraction protocols using organic solvents derived originally from a series of related RNA extraction methods. Some of the main steps used in these methods are: 1) cell lysis undertaken by adding a detergent/chaotropic-containing solution, including SDS or N-Lauroyl sarcosine; 2) inactivation of DNases and RNases, usually through the use of organic solvents; 3) purification of DNA and removal of RNA, lipids, and proteins; and 4) resuspension of extracted nuclei Cid. 

This method was initially developed in 1977 when an RNA extraction technique using guanidium isothyocyanate was used by Ullrich et al to isolate plasmid DNA. This technique was later modified by Chirgwin et al in 1979. It required the use of guanidium thiocyanate and long hours of ultracentrifugation through a cesium chloride cushion. In an effort to improve this method, Chomczynski and Sacchi developed in 1987 a protocol for RNA extraction using guanidium thiocyanate–phenol–chloroform and much shorter centrifugation. This last RNA extraction protocol was able to isolate RNA, DNA, and proteins, but in order to be used as a DNA extraction technique, guanidium thiocyanate–phenol–chloroform was later replaced by a mixture of phenol, chloroform, and isoamyl alcohol, as the former solvent did not completely inhibit RNase activity.Phenol is a carbolic acid that denatures proteins quickly, but it is highly corrosive, toxic, and flammable. This organic solvent is usually added to the sample and then, using centrifugal force, a biphasic emulsion is obtained. The top hydrophilic layer contains diluted DNA, and the bottom hydrophobic layer is composed of organic solvents, cellular debris, proteins, and other hydrophobic compounds. DNA is then precipitated after centrifugation by adding high concentrations of salt, such as sodium acetate, and ethanol or isopropanol in 2:1 or 1:1 ratios. Excess salt can be removed by adding 70% ethanol, and the sample is then centrifuged to collect the DNA pellet, which can be resuspended in sterile distilled water or TE buffer (10 mM Tris; 1 mM EDTA pH 8.0).

Because these techniques involve the use of toxic and corrosive organic solvents, safety is a main concern. Personal protective equipment, safety measures involving the use of a biohazard hood, and training are required. Phenol–chloroform needs to be equilibrated to an adequate pH, and protocol conditions should be optimized.In an effort to improve the safety and ease of use of these protocols, certain modifications have been introduced in order to avoid physical contact with solvents. These include incorporating a silica gel polymer or replacing solvents with other substances like benzyl alcohol.

Solution-based DNA extraction methods using salting out

Some nucleic acid extraction techniques that avoid the use of organic solvents have also been developed over the years. In 1988, Miller et al published a protocol that achieved DNA purification through protein precipitation at high salt concentration. The traditional protocol involves initial cell disruption and digestion with SDS–proteinase K, followed by the addition of high concentrations of salts, usually 6 M sodium chloride. The mixture is then centrifuged to allow proteins to precipitate to the bottom, with the supernatant containing DNA then transferred to a new vial. DNA is then precipitated using ethanol or isopropanol in the same manner as described for organic solvent methods. 

However, the use of proteinase K can be time consuming and expensive when compared with other reagents used in different solution-based approaches, so there have been a few attempts to find alternative reagents for deproteinization of DNA. In 1991, Lahiri and Nurnberger developed a DNA extraction protocol from blood samples that eliminated the use of organic solvents and prolonged incubation with proteinase K. Their protocol used Nonidet™ P-40 (NP-40; Sigma-Aldrich, St Louis, MO, USA) to lyse blood cells and high salt buffers and 10% SDS to inactivate and remove contaminants. Another protocol is the modified salting out method published in 2005 by Nasiri et al, which replaced proteinase K digestion with the use of laundry powder. This modified technique has been successfully used as a DNA extraction protocol in many facilities around the world.

Solid-phase DNA extraction methods

Purification of DNA using the liquid/solid-phase approach can be traced back to 1979, when Vogelstein and Gillespie used silica in a glass powder form in their protocol to purify DNA fragments previously separated by agarose gel electrophoresis. Solid-phase extraction methods for DNA extraction from blood samples were initially described in 1989 by McCormick who published a technique using siliceous-based insoluble particles, chemically similar to phenol, which interact with proteins to allow DNA purification. A number of different procedures using the liquid/solid DNA extraction approach have been developed since then and are used in the majority of commercially available extraction kits.

These techniques will absorb DNA under particular pH and salt content conditions through any of the following principles: 1) hydrogen binding in the presence of a chaotropic agent to a hydrophilic matrix, 2) ionic exchange using an anion exchanger under aqueous conditions, and 3) affinity and size exclusion mechanisms.^,Most of these methods follow a series of similar steps to achieve cell disruption, DNA adsorption, nucleic acid washing, and final elution. Most solid-phase techniques use a spin column to bind nucleic acid under centrifugal force. Spin columns are made of silica matrices, glass particles or powder, diatomaceous earth, or anion exchange carriers, and these compounds generally need to be conditioned using buffer solutions at a specific pH to turn them into the required chemical form. Blood cells previously degraded using particular lysis buffers are applied to the columns and centrifuged, and the DNA binds to the column aided by pH and salt concentration conditions provided by binding solutions. Some proteins and other biochemical compounds may also bind to the column, and they are later removed using washing buffers containing competitive agents during a series of washing steps. DNA is finally eluted in sterile distilled water or TE buffer.

DNA extraction methods using silica and silica matrices

Silica matrices have unique properties for DNA binding. They are positively charged and have high affinity toward the negative charge of the DNA backbone. High salt conditions and pH are achieved using sodium cations, which bind tightly to the negatively charged oxygen in the phosphate backbone of DNA. Contaminants are removed with a series of washing steps, followed by DNA elution under low ionic strength (pH ≥7) using TE buffer or sterile distilled water. Commercially available kits using a silica-based approach are manufactured by Clontech Laboratories, Inc., Mountain View, CA, USA (NucleoSpin™); MO BIO Laboratories, Inc., Carlsbad, CA, USA (UltraClean^® BloodSpin^®); QIAGEN Pty Ltd, Victoria, Australia (QIAamp^®), Promega Corporation, Fitchburg, WI, USA (Wizard^®); Epoch Life Science, Missouri City, TX, USA (EconoSpin^®); and Sigma-Aldrich, St Louis, MO, USA (GenElute™), among others. In these protocols, blood samples are incubated for a few minutes with a lysis buffer. Most protocols take about 40 minutes to 1 hour to complete, producing high yields of DNA with minimum contamination.

A substance that contains high amounts of silica (up to 94%) known as kieselguhr, diatomite, or diatomaceous earth has also been used for DNA purification. It was initially described by Boom et al in 1990. It binds DNA in the presence of chaotropic agents, followed by washing with a buffer containing alcohol, and finally DNA is eluted in a low salt buffer or sterile distilled water. Quantum Prep^® (Bio-Rad Laboratories, Hercules, CA, USA) is an example of a DNA extraction product developed using diatomaceous earth.

DNA extraction kits have also evolved, and they are incorporated into semi- and fully automated equipment able to perform protocols from sample lysis to some downstream applications like polymerase chain reaction (PCR), such as BioRobot EZ1^® Advanced (QIAGEN) and Biomek^® 4000 Laboratory Automation Workstation (Beckman Coulter, Inc., Brea, CA, USA), among others. Less risk of pipetting error, reduced number of sample transfers, and less protocol time are among the advantages of these devices. However, they should be carefully considered, given the high cost of some of the available choices of equipment. They have also been incorporated into miniaturized total chemical analysis systems, which are silicon microchips, where DNA purification separation and detection are achieved.

DNA extraction using anion exchange resins

Positively charged chemical substances able to bind to negatively charged nucleic acids or contaminants or enzymes, such as nucleases, are called anion exchange resins, and they have also been used as part of DNA extraction protocols from blood samples.

Chelex^® 100 resin (Bio-Rad Laboratories, Hercules, CA, USA) is made of styrene divinylbenzene copolymers that contain paired iminodiacetate ions. It is used in DNA extraction protocols as a chelating ion exchange resin that binds polyvalent metal ions such as nucleases commonly used in DNA extraction from forensic samples. The initial laboratory protocol, using blood as a biological source, was described by Walsh et al³⁷ in 1991. Based on this initial approach, other protocols have been developed to perform nucleic acid extraction from whole blood samples. They require small sample volumes (under 1 mL of blood) and are usually performed in a single tube reaction with different steps and reagents involved. Blood samples could be lysed using proteinase K and/or incubation at high temperature, and removal of contaminants is achieved by adding Chelex^® 100 resin, which precipitates them. Single-stranded DNA is obtained and remains suspended in the supernatant, which can be immediately used in downstream application or can be transferred to a new vial for long-term storage.

Seligson et al used anion exchange materials as part of their invention to isolate nucleic acid samples from a variety of sources, including whole blood samples. Seligson et al’s protocol uses a column containing a resin with positively charged diethylaminoethyl cellulose groups on its surface to bind negatively charged phosphates of the backbone of DNA. The strength of DNA binding to the column, as well as RNA and other impurities, can be altered through salt concentrations and pH conditions of buffers used in this nucleic acid isolation protocol. Contaminants such as protein and RNA can be washed from the DNA-containing column using medium salt buffers.

DNA extraction methods using magnetic beads

Nucleic acid extraction techniques using magnetic separation have been emerging since the early 1990s. They were originally used to extract plasmid DNA from bacterial cell lysates by Hawkins et al in 1994 and in 2006 by Saiyed et al, who developed and validated a protocol using naked magnetic nanoparticles for genomic DNA extraction from whole blood samples.Yields of DNA obtained from buccal swabs and urine are highly altered depending on the swab or the urine type, the individual being swabbed, the swabbing technique, and the number of cells captured on the swab and in the urine. 

Magnetic particles are made of one or several magnetic cores, such as magnetite (Fe3O4) or maghemite (gamma Fe2O3), coated with a matrix of polymers, silica, or hydroxyapatite with terminal functionalized groups. In the protocol developed by Saiyed et al, 30 μL of whole blood is mixed with an equal volume of 1% (weight/volume [w/v]) SDS solution. The tube is mixed by inversion two or three times and incubated at room temperature for 1 minute. Ten microliters of magnetic nanoparticles is added to this mixture, followed by the addition of 75 μL of binding buffer (1.25 M sodium chloride and 10% polyethylene glycol 6000). The solution is mixed by inversion and allowed to rest for 3 minutes at room temperature, and the magnetic pellet is immobilized using an external magnet to discard the supernatant. The magnetic pellet is washed with 70% ethanol and dried. The magnetic pellet is resuspended in 50 μL of TE buffer, and magnetic particles bound to DNA are eluted by incubation at 65°C with continuous agitation.

Choosing the appropriate protocol

The ideal extraction method should fit the following criteria: it should be sensitive, consistent, quick, and easy to use, and depending on the country in which it is used it may be important to minimize specialized equipment or biochemical knowledge. It should also pose minimum risk to users, as well as avoid possible cross-contamination of samples. Finally, and most importantly, the DNA extraction technique chosen should be able to deliver pure DNA samples ready to be used in downstream molecular 

The quality and quantity of genomic DNA extracted from blood samples is a key feature most facilities consider when choosing a protocol. Measuring ultraviolet light absorbance using spectrophotometry at different wavelengths (230 nm, 240 nm, 260 nm, and 280 nm) is an initial quick and efficient way of determining purity and concentration of nucleic acid samples. Concentration is usually calculated from DNA absorbance reading at 260 nm using Beer–Lambert law. Purity of nucleic acid samples is assessed in a 260/280 absorbance ratio, and values in the range of 1.8–2.0 are generally considered acceptable. The 260/230 absorbance ratios between 2.0 and 2.2 are also considered to be adequate as a secondary measure of purity for.. 

Lahiri et al published a study in 1991 where they compared ten solution-based extraction methods for DNA extraction using whole blood as a source. They compared a protocol previously developed by their group (method 10a and 10b which required no use of organic solvents or enzyme digestion, against nine other methods previously published and used for DNA extraction from blood. In their study, Lahiri et al extracted whole blood samples from five individuals in triplicate using the aforementioned methods. They determined DNA concentration from samples using spectrophotometry absorbance reading at 260 nm and assessed quality through 260/280 absorbance ratio and electrophoresis on agarose gel, as well as restriction of enzyme digestion and southern blot. A summary of some of the protocol features, as well as findings, is presented in All protocols tested were able to isolate DNA with relatively good purity (260/280 ratios from 1.7 to 1.94), but DNA obtained with methods 2, 5, and 6 showed different amounts of degradation evidenced in gel electrophoresis. Seven of the protocols tested, methods 3–9, required use of organic solvents and/or hazardous substances such as phenol–chloroform or chloroform. Methods 1 and 2 did not use organic compounds, but method 1 was the most time consuming. It required overnight incubation with proteinase K, a problem solved in protocol 2 by incubating samples for 30 minutes with both proteinase K and RNase A, reducing DNA extraction time to 5 hours. Method 10 (version a and b) was the quickest of all DNA isolation methods (1 hour) and removed enzyme digestion and the use of organic solvents/hazardous substances. Both versions of this protocol were able to recover similar or higher DNA yields than the other tested protocols, with about comparable 260/280 ratios. Based on their study findings, Lahiri et al were able to conclude that the DNA extraction method they developed was the quickest and safest of the solution-based methods tested, recovering DNA of comparable quality and quantity.

Abd El-Aal et al compared a combination of manual and automated extraction methods for DNA extraction from whole blood samples. Their study included six techniques: phenol–chloroform purification, DNA extraction using microwave thermal shock, DNA extraction with Wizard Genomic DNA Purification Kit (Promega Corporation), magnetic separation (LC MagNA Pure Compact Instrument; Roche Diagnostics GmbH, Manheim, Germany) both manually and partially automated using the Precision™ XS Microplate Sample processor from Biotek Instruments (Vermont, USA), and finally they modified the Wizard SV 96 Genomic DNA Purification Kit (Promega Corporation), combining it with magnetic separation using the MagNA Pure purification method. They extracted 96 blood samples and used 100 μL as the initial volume. However, they failed to mention how many samples were extracted using each method and the number of experimental replicates performed. Their results showed DNA extracted for each protocol with final concentrations ranging from 0.50 μg/μL to 0.98 μg/μL. Although phenol–chloroform, manual magnetic separation, and the combined Promega–MagNA Pure method showed relatively similar DNA concentrations (0.72–0.79 μg/μL), the magnetic separation and microwaving technique achieved the highest and lowest DNA concentrations, 0.98 μg/μL and 0.50 μg/μL, respectively. In their comparisons, they established five categories for simplicity of extraction: extremely simple, simple, less simple, more simple, and difficult, but their system can be confusing because they failed to present criteria used for each category. However, they categorized phenol–chloroform as difficult and automated MagNA Pure as extremely simple, using their previously mentioned category system. They also have five categories for cost of each protocol, with the automated MagNA Pure technique as the most expensive and microwaving as the cheapest method. Their costing categories were also not defined and there is no actual mention of specific costs for each method in their study. Based on previously mentioned findings, they concluded that magnetic separation using an automated protocol performed best in terms of simplicity of extraction, purity of extracted DNA, and speed, even though it is the one with the highest cost. They also concluded that it was essential to optimize any method chosen and they recommended the use of magnetic separation, because it required minimal starting material and it was both cost-effective and user-friendly. However, as previously stated, there is no mention of how cost-effectiveness was determined.

Lee et al extracted DNA from 22 whole blood samples using three automated extraction systems. All three protocols compared were based on solid-phase extraction techniques: QIAamp^® Blood Mini Kit (QIAGEN, Hilden, Germany) with QIAcube^®, which uses a silica membrane and resins within a spin column to bind DNA, and two other protocols that are based on magnetic-based DNA isolation techniques MagNA Pure LC Nucleic Acid Isolation Kit I with MagNA Pure LC (Roche Diagnostics GmbH, Mannheim, Germany) and Magtration-Magnazorb DNA Common Kit-200N with Magtration System 12GC (Precision System Science Co, Ltd, Tokyo, Japan). DNA concentration was measured by spectrophotometry and purity was assessed by 260/280 ratio, DNA electrophoresis on agarose gel, and PCR. Statistical analysis was performed to validate study

Results showed no statistical difference between DNA concentrations obtained among the three commercial methods, but DNA purity was slightly lower for the Magtration-Magnazorb DNA Common Kit-200N when compared with the other two methods. DNA extracted was of similar quality based on results from PCR and electrophoresis on agarose gel. Therefore, they concluded that effectiveness for all systems was equivalent and that they all produced acceptable.

Chacon-Cortes et alevaluated cost-effectiveness and time efficiency of three available DNA extraction techniques from whole blood samples: a traditional salting out method, a modified salting out method, and a commercially available kit based on a solid-phase DNA extraction method QIAamp^® DNA blood maxi kits (QIAGEN^® Pty Ltd, Clifton Hill, VIC, Australia). The modified salting out protocol replaced the sample overnight incubation step from the traditional salting out method, required for contaminant removal using proteinase K, with the use of laundry detergent to reduce time of extraction to about 1 hour. Five microliters of whole blood from six breast cancer patients was manually extracted using each protocol, and techniques were compared in terms of quality and quantity of DNA extracted, as well as cost and time required. DNA quantity was measured using spectrophotometry, and DNA quality was assessed by both 260/280 ratio and agarose gel electrophoresis of PCR product. 

Conclusion

The successful sample collection and the extraction of genomic DNA from buccal swabs, urine, and hair are noninvasive and reliable alternatives to the prickly invasive blood sampling, both for subjects and sample collectors. DNA extraction has evolved for the past 145 years and has developed into a diversity of laboratory techniques. This review highlights the currently available methods for DNA extraction from whole blood samples, and it summarizes comparison studies using different nucleic acid extraction approaches published to date. DNA extraction has evolved from solution and solid-phase manual techniques initially performed manually into incorporating these into automated methods. There is no consensus on a gold standard method for DNA extraction from whole blood samples, and they all differ in many different aspects.