Wednesday, March 28, 2018

Quality Control, Risk and Quality Risk Management

Quality Control, Risk and Quality Risk Management

Quality: Concepts

What is quality?
It is a multifaceted question, difficult to answer in the abstract. Four fitness that explains the evolution process of quality is
1.   Fitness to standard
2.   Fitness to use
3.   Fitness to cost
4.   Fitness to the latent requirement
ISO 9000:2000 and international standard on quality vocabulary define as:
The degree to which a set of inherent characteristics fulfills the requirement.
The Americans who brought the messages of quality to Japan and the rest of the world. Prominent figures lying on the group are W. Edwards Deming, Joseph M Juran, Philip Crosby
Japanese who developed new concepts to the American messages. Prominent figures lying on this group are: Kaoru Ishikawa, Sigeo Shingo, Yoshio Kondo
Quality Control and Quality Assurance
Quality control is a subset of Quality Assurance

Quality Assurance

“Quality assurance” is a wide-ranging concept covering all matters that individually or collectively influence the quality of a product. It is the totality of the arrangements made with the object of ensuring that pharmaceutical products are of the quality required for their intended use.
Quality Assurance is all those planned and systemic actions necessary to provide confidence that an entity will fulfill requirements for quality. It is a process of creating standards thorough planning. Assurance comes through the knowledge of what will be, is being, of has been done, rather than doing it. The means to provide the confidence, assurance need to build into the process, such as documenting plans, documenting specifications, creating records, reporting reviews etc. Such documents and activities also serve to control quality as well as assure it. ISO 9001 is a quality assurance standard, designed for use in assuring customers that suppliers have a capacity of meeting their requirements. Quality assurance also incorporates Good manufacturing practices (GMP) and other factors.
Quality control
Quality control is operational techniques and activities which are used to fulfill requirements of quality. It is a process of maintaining standards. It prevents the undesirables changes in the quality of product or service being supplied. If you do not have control quality products are produced by chance not design.

What is risk?

It is commonly understood that risk is defined as the combination of the probability of occurrence of harm and the severity of that harm.
In relation to pharmaceuticals, although there are a variety of stakeholders, including patients and medical practitioners as well as government and industry, the protection of the patient by managing the risk to quality should be considered of prime importance.
It is important to understand that product quality should be maintained throughout the product lifecycle such that the attributes that are important to the quality of the drug product remain consistent with those used in the clinical studies. An effective quality risk management approach can further ensure the high quality of the drug product to the patient by providing a proactive means to identify and control potential quality issues during development and manufacturing.
Product Lifecycle: 1. Introduction, 2. growth, 3. maturity, 3. Decline

What is quality risk management?

Quality risk management (QRM) can improve the decision-making if a quality problem arises. Effective QRM can facilitate better and more informed decisions, can provide regulators with greater assurance of a company’s ability to deal with potential risks, and can beneficially affect the extent and level of direct regulatory oversight.
Quality risk management specifically provides guidance on the principles and some of the tools of quality risk management that can enable more effective and consistent risk-based decisions, by both regulators and industry, regarding the quality of drug substances and drug products across the product lifecycle.
Tools for quality risk management that can be applied to different aspects of pharmaceutical quality. These aspects include development, manufacturing, distribution, inspection, and submission/review processes throughout the lifecycle of drug substances, drug products, biological and biotechnological products (including the use of raw materials, solvents, excipients, packaging and labeling materials in drug products, biological and biotechnological products).
  

Primary Principles of Risk Management

· The evaluation of the risk to quality should be based on scientific knowledge & ultimately link to the protection of the patient and
· The level of effort, formality & documentation of the quality risk management process should be commensurate with the level of risk.
Principles and common practices
Core principles of quality risk management according to the ICH Q9 guideline include the following: International Conference on Harmonization (ICH) provides an excellent high-level framework for the use of risk management in pharmaceutical product development and manufacturing quality decision-making applications
1.    Compliance with applicable laws: Risk assessment should be used to assess how to ensure compliance and to determine the resulting prioritization for action—not for a decision regarding the need to fulfill applicable regulations or legal requirements.
2.    Risk can only be effectively managed when it is identified, assessed, considered for further mitigation, and communicated. This principle embodies the four stages of an effective quality risk-management process as defined by ICH Q9: risk assessment (i.e., risk identification, analysis, and evaluation); risk control (i.e., risk reduction and acceptance); risk communication; and risk review.
3.    All quality risk evaluations must be based on scientific and process-specific knowledge and ultimately linked primarily to the protection of the patient. Risk assessment is based on the strong understanding of the underlying science, applicable regulations, and related processes involved with the risk under analysis. Collectively, these components should be assessed first and foremost with regard to the potential impact on the patient.
4.   Effective risk management requires a sufficient understanding of the business, the potential impact of the risk, and ownership of the results of any risk-management assessment.
5.  The risk assessment must take into account the probability of a negative event in combination with the severity of that event. This principle also serves as a useful working definition for risk (i.e., risk represents the combination of the probability and severity of any given event).
6.   It is not necessary or appropriate to always use a formal risk-management process (e.g., standardized tools). Rather, the use of an informal risk-management process (e.g., empirical assessment) is acceptable for areas that are less complex and that have a lower potential risk. Risk decisions are made by industry every day. The complexity of the events surrounding each decision and the potential risk involved are important inputs in determining the appropriate risk-assessment methodology and a corresponding level of analysis required. For less complex, less risky decisions, a qualitative analysis (e.g., decision tree) of the options may be all that is required. In general, as the complexity and/or risk increases, so should the sophistication of the risk-assessment tool used. In the same regard, the level of documentation of the risk-management process to render an appropriate. 
The Components of the Quality Risk Management Process (ICH Q9)
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SALMONELLA - Morphology, Laboratory diagnosis, Cultural characteristics

SALMONELLA - Morphology, Laboratory diagnosis, Cultural characteristics

SALMONELLA
TU Note: SALMONELLA - Morphology, Laboratory diagnosis, Cultural characteristics

The Salmonella are facultative anaerobic Gram-negative bacilli, motile, non‐capsulated, non-sporing organism. Salmonella currently comprises of about 2,500 serotypes or species. All of them are potentially pathogenic Salmonella produce 3 main types of diseases in human.  These are enteric fever, food poisoning (Gastroenteritis) and Septicaemia. Most of the other Salmonella are chiefly pathogenic in animals like poultry, pig, rodent, cattle, parrot and other.

Morphology 

Salmonella is gram-negative bacilli arranged mainly singly or in pairs. It is non‐capsulated, non‐sporing. Most of the strains are motile due to the presence of peritrichous flagella except S. Gallinarum and S. Pullorum, which are non‐motile. Measuring 1‐3 μm long 0.6 μm wide.

Taxonomy of the Salmonellae

The problems involved in the taxonomy and nomenclature of this group of bacteria g p can only be understood in the historical perspective. At first, the genus Salmonella appeared to comprise species that differed only in their antigen structures. Species names were therefore used for what turned out to be serovars. More recent molecular studies have demonstrated that the genus Salmonella contains only two species that can be subdivided into seven subspecies. All of the important human pathogen salmonellae belong to the subspecies enterica. The (false) species names for the serovars had, however, already become normal usage. In view of the fact that the causative pathogens in typhoid salmonelloses, a clinical picture clearly differentiated from Salmonella gastroenteritis, are only serovars of the same species/ subspecies, the official committee has, however, not adopted the new nomenclature as yet.

Current nomenclature system

All antigenic formulae of recognized Salmonella serovars are listed in the White‐Kauffmann‐Le Minor scheme, and newly recognized serovars are reported every year in the journal Research in Microbiology. The nomenclature system used at the United States Centers for Disease Control and Prevention (CDC) is based on recommendations from the WHO Collaborating Centre, which is responsible for regular updates of the scheme.
In order to avoid unnecessary confusion between serovars and species, the serovar name is not italicized and starts with a capital letter. As not all serovars have a name, for those designated by their antigenic formulae the subspecies name is written in Roman letters, followed by their antigenic formulae – including O (somatic) and both phase 1 and phase 2 H (flagellar) antigens.
Approximately 60% of all Salmonella serotypes belong to Salmonella enterica subspecies enterica. Within this subspecies, the most commonO‐antigen serogroups are A, B, C1, C2, D and E. It must be noted that strains in these serogroups cause approximately 99% of Salmonella infections in humans and warm‐blooded animals.
The nomenclature of the genus Salmonella has evolved from the initial one serotype‐one species concept proposed by Kauffmann on the basis of the serologic identification of 'O' (somatic) and 'H' (flagellar) antigens.
  • Each serotype was considered a separate species (for example Salmonella paratyphi A, Salmonella newport and Salmonellaenteritidis); this concept, if used today, would result in 2463 species of Salmonella.
  • The defining development in Salmonella taxonomy occurred in 1973 when Crosa et al., demonstrated by DNA‐DNA hybridization that virtually all Salmonella belonged in a single species name Salmonella enterica which is separated into 7 distinct subspecies. Most of the serotypes that cause human diseases are in subgroup I.The single exception is Salmonella bongori which was made that there be only 2 species of Salmonella, Salmonella enterica (include the 2462 Serovars previous species) and Salmonella bongori
Antigenic structure Salmonella possesses the following antigens based on which they are classified and identified :
  1. Flagellar (H) antigen
  2. Somatic (O) antigen
  3. Capsular (K) Antigen
The serovars are determined by O and H antigens. The Kauffman–White scheme is used to arrange them (see Table). This taxonomic arrangement classifies the serovars in groups characterized by certain O antigens (semibold).
This results in a clinically and epidemiologically useful grouping since certain serovars are responsible for typhoid salmonelloses and others for enteric salmonelloses. The serovars are determined by means of antisera in the slide agglutination test. Phase Variations of the H Antigens H antigens occur with two different antigen structures. The primary structure of flagellin is determined by two genes on the chromosome, only one of which is read off. Whether a gene is read off or not is determined by spontaneous inversion of a DNA sequence before the H2 gene, which inversion occurs with a frequency of approximately 10–4 per cell division.
O antigen
Characteristics of somatic (O) antigen:
  • The somatic O antigen is a phospholipid protein-polysaccharide complex, which forms a part of the cell wall.
  • It can be extracted from the bacterial cell by treatment with phenol or alcohol.
  • The O antigen is unaffected by boiling, alcohol or weak acids.
  • It is present both in motile and non‐motile Salmonella.
  • For serological test (Widal test) "O" suspension is prepared either from non‐motile strains or by heat or alcohol treatment of motile strains, which destroy H antigen.
  • When mixed with antiserum "O" antigen suspensions produce compact, chalky granular.
  • The optimum temperature for "O" agglutination is at 50‐55°C.
  • The "O" antigen is less immunogenic than H antigen.

H antigen

Characteristics of flagellar (H) antigen:
  •  This antigen is present on the flagella of the bacterium.
  • It is hat labile protein.
  • It is destroyed by boiling or by treatment with alcohol but not formaldehyde.
  • For serological test (Widal test) H suspension is prepared by addition of formalin to young motile broth culture.
  • When mixed with anti‐serum, H suspension agglutinate rapidly and producing large loose floccules.
  • The H antigen is strongly immunogenic and induces antibody formation rapidly in high titre following infection.
  • The optimum temperature for H agglutination is at 37°C.

VI antigen

Characteristics of (Vi) antigen:
  • Vi‐antigen is external to the cell wall.
  • Vi‐antigen is a virulent (Vi for virulence) for mice.
  • Vi‐antigen interferes with agglutination of the freshly isolated organism by "O" antigen.
  • It is a polysaccharide in nature and heat-labile.
  • It destroyed by boiling for one hour.
  • It is not affected by 2% formalin and alcohol.
  • It is lost on serial subcultures.
  • It produces a low titre antibody

    Virulence factors

    • Type III secretion systems (bacterial proteins): Two separate type III secretion systems of bacterial proteins mediate the initial invasion of Salmonella into the mucosa of small intestine Other factors
    • Invasins: Invasins are proteins that mediate adherence to, and penetration of, intestinal epithelial cells. Synthesis of these fimbrial proteins by the bacterial cells is under the control of inv genes.
    • Factors involved in resistance to acid pH: Acid tolerance response (ATR) gene of chromosome
    • Vi (virulence) antigen: The surface antigen (Vi antigen) has antiphagocytic properties.

    Clinical manifestations

    The signs and symptoms of enteric fever are
    1. Gradually increasing fever
    2. Mental clouding of consciousness (typhus means cloud)
    3. Malaise
    4. Headache
    5. Constipation
    6. Coated tongue
    7. Anorexia
    8. Splenohepatomegaly 
    9. Bradycardia (Slow heart rate) 
    10. Rose spots may appear on chest and abdomen in 2nd and 3rd week of infection.

    Laboratory diagnosis

     Specimen collection
    • Blood, urine, stool, bone marrow, and aspirated duodenal fluid are suitable specimens for diagnosis of typhoid fever.

    Pathogenesis

    • Salmonella is strict parasites of animals or human beings. It causes enteric fever, septicemia, and gastroenteritis.
    • Enteric fever is most usually caused by Salmonella Typhi or Salmonella Paratyphi A, B or C but can be caused by any Salmonella serotypes.
    • Enteric fever is a potentially life-threatening systemic illness characterized by high fever and abdominal complaints. The term enteric fever encompasses both typhoid and paratyphoid fever.
    • Enteric fever is a generalized acute infection characterized by cyclic course, definitive temperature curve, general intoxication, bacteremia and affection of the lymphatic apparatus of the small intestine through which the infection implants itself in the host upon entrance of the causative agent into the gastrointestinal tract.

    Virulence factors

    • Type III secretion systems (bacterial proteins): Two separate type III secretion systems of bacterial proteins mediate the initial invasion of Salmonella into the mucosa of small intestine Other factors
    • Invasins: Invasins are proteins that mediate adherence to, and penetration of, intestinal epithelial cells. Synthesis of these fimbrial proteins by the bacterial cells is under the control of inv genes.
    • Factors involved in resistance to acid pH: Acid tolerance response (ATR) gene of chromosome
    • Vi (virulence) antigen: The surface antigen (Vi antigen) has antiphagocytic properties.

    Clinical manifestations

    The signs and symptoms of enteric fever are:
    1. Gradually increasing fever
    2. Mental clouding of consciousness (typhus means cloud)
    3. Malaise
    4. Headache
    5. Constipation
    6. Coated tongue
    7. Anorexia
    8. Splenohepatomegaly
    9. Bradycardia (Slow heart rate)
    10. Rose spots may appear on chest and abdomen in 2nd and 3rd week of infection.

    Microscopic examination

    Microscopic examination of Gram staining smear shows Gram-negative bacilli arranged mainly in single.

    Cultural characteristics

    It is aerobic and facultatively anaerobic. The range of temperature for growth is 15‐41°C and the optimum pH for growth is 6.8. Grow on a general purpose or basal medium.

    MacConkey agar

    Specimens are inoculated into the MacConkey agar; incubate at 37°C for 24 hours. The colonies are large 2‐3 mm in diameter, circular, low convex, smooth, translucent, colorless due to non‐lactose fermentation.

    Broth medium

    It produces uniform turbidity, no pellicle formation.

    Blood agar

    Colonies are large 2‐3 mm in diameter, colorless, circular, low convex, translucent, smooth and non-hemolytic.

    Deoxycholate citrates agar (DCA)

    Salmonellae produce non‐lactose fermenting pale colored colonies, which have the black center on DCA due to H2S production.
    ENTEROCOCCUS: Morphology, Lab Diagnosis and Control

    ENTEROCOCCUS: Morphology, Lab Diagnosis and Control

    Enterococcus/Group D Streptococcus:

    Group D Streptococci

    • Former Lancefield Group D is classified in 1980 into two groups.

    Enterococci

    • Fecal Streptococci has been reclassified as separate Genus called Enterococcus- containing different species, such as Enterococcus faecalis, Enterococcus faecium.
    Non-Enterococcal Group D
    Streptococcus bovis
    Enterococci
    • Enterococci contain cell wall polysaccharide that reacts with group D antisera. Therefore, in the past, they were considered group D streptococci. Today, DNA analysis and other properties have placed them in their own genus, Enterococcus.
    • The clinically most important species are E.faecalis and E. faecium. Enterococci can be Alpha, Beta, or nonhemolytic. As a rule, enterococci are not very virulent, but they have become prominent as a cause of nosocomial infections due to their multiple antibiotic resistance.

    Enterococcus faecalis

    • Enterococcus faecalis is the main pathogen in the genus Enterococcus, causing about 95% of enterococcal infection.

    Morphology

    • They are Gram‐positive cocci arranged in pairs or short chains. They are non-capsulated.

    Pathogenesis

    • Enterococci are a commensal organism and do not have potent toxin and well-defined virulence factors.
    • Their greatest significance is their resistance to many commonly used antibiotics.

    Disease:

    It produces:
    1. Urinary tract infection
    2. Biliary tract infection
    3. Ulcers (e.g. bed sores)
    4. Wound infection (particularly abdominal)
    5. Occasionally endocarditis and meningitis

    Laboratory diagnosis

    Specimen collection: Possible pathological specimens are
    1. Urine
    2. Pus swab
    3. Blood
    4. CSF.
    Microscopic examination:
    • Microscopic examinations of Gram‐staining smear shows Gram‐positive cocci in pairs or in short chains.
    Culture characters: 
    • Enterococci are aerobic; organisms are capable of growing a wide range of temperature (10‐45°C). They can withstand heat at 60°C for 30 minutes. Grow at pH 9.6, and 6.5 % NaCl broth. Grow at 40 % Bile.
    Blood agar: 
    • Enterococci are generally non-hemolytic but some strains show alpha or beta hemolysis.
    Fig: Enterococcus facalis in blood agar
    MacConkey agar: 
    • E. faecalis ferments lactose, produce small, dark‐red colonies.
    Bile Esculin Agar:
    • Produces black colored colonies due to hydrolysis of esculin to esculetin which forms the black precipitate.
    Cysteine lactose electrolytedeficient agar (CLED): 
    • It produces small yellow colonies on CLED agar.
    TU Notes: ENTEROCOCCUS: Morphology, Lab Diagnosis and Control

    Biochemical reactions

    • Enterococci are distinguished from the non‐Group D streptococci by their ability to survive in the presence of bile and to hydrolyze the polysaccharide esculin.
    • Unlike non-enterococcal group D streptococci, enterococci grow in 6.5 percent NaCl and yield a positive PYR test.
    • E. faecalis can be distinguished from E. faecium by their fermentation patterns, which are commonly evaluated in clinical laboratories.

    Treatment

    • Most Enterococci are sensitive to ampicillin and resistant to cephalosporin.

    Antibiotic Resistance

    • A major problem with the enterococci is that they can be very resistant to antibiotics. E faecium is usually much more antibiotic‐resistant than E. faecalis.
    • Enterococci are naturally resistant to BetaIactam antibiotics and aminoglycosides but are sensitive to the synergistic action of a combination of these classes. In the past, the initial regimens of choice were penicillin + streptomycin, or ampicillin + gentamicin.

    Vancomycin Resistance

    • The glycopeptide vancomycin is the primary alternative drug to a penicillin (plus an aminoglycoside) for treating enterococcal infections. These enterococci are not synergistically susceptible to vancomycin plus an aminoglycoside. Vancomycin resistance has been most common in E faecium, but vancomycin‐resistant strains of E. faecalis also occur.
    • The gene encoding for the enterococcal Beta‐lactamase is the same gene as found in Staphylococcus aureus. The gene is constitutively expressed in enterococci and inducible in staphylococci. Because enterococci may produce small amounts of the enzyme, they may appear to be susceptible to penicillin and ampicillin by routine susceptibility tests

     NON-ENTEROCOCCAL GROUP D STREPTOCOCCI

    • Streptococcus bovis is the most clinically important of the non-enterococcus group D streptococci. Part of normal fecal flora, they are either alpha‐ or nonhemolytic. S. bovis occasionally causes urinary tract infections and subacute bacterial endocarditis, especially in association with bowel malignancy. 
    • The organism is bile‐ and esculin positive, but is PYR‐negative, and does not grow in 6.5 percent salt (unlike the enterococci). It tends to be sensitive to penicillin and other antibiotics.
    Agarose and Polyacrylamide Gel Electrophoresis Matrices

    Agarose and Polyacrylamide Gel Electrophoresis Matrices

    Introduction

    TU Note: Agarose and Polyacrylamide Gel Electrophoresis Matrices
    Fig: Polyacrylamide Gel Electrophoresis (PAGE)
    During the last years molecular biology techniques, such as polymerase chain reaction (PCR), have become widely used for medical and forensic applications, as well as research, and detection and characterization of infectious organisms. In the virology field, it has been demonstrated that the employment of PCR technique offers the advantages of high sensitivity and reproducibility in viral genomic detection and strains characterization. However, the sensitivity in the detection of DNA fragments is also linked to the sensitivity of the electrophoresis matrix applied for PCR product development.
    Electrophoresis through agarose or polyacrylamide gels is a standard method used to separate, identify and purify nucleic acids since both these gels are porous in nature. In this blog post, the evaluation of the sensitivity of agarose and polyacrylamide gel electrophoresis matrices in the detection of PCR products is analyzed. For this purpose, rotavirus PCR amplicons were used as a model. Human rotaviruses have been recognized as the most common cause of dehydrating diarrhea in infants and young children on a worldwide scale. These viruses are characterized by the presence of 11 segments of double-stranded RNA surrounded by three separate shells, the core, inner capsid and outer capsid. Currently, rotaviruses are dual classified into G and P genotypes according to the differences of VP7 and VP4 neutralization antigens which form the outer capsid of the virion. Two rotavirus vaccines have been licensed in the year 2006 in many countries. Although large-scale safety and efficacy studies of both rotavirus vaccines have shown excellent efficacy against severe rotavirus gastroenteritis (Ruiz-Palacios et al., 2006; Matson, 2006), the lack of clear data about the protection against genotypes not included in the vaccine formulations underlines the importance of virological surveillance, rotavirus strain characterization and the evaluation of the impact of these vaccines in diminishing the diarrhea illness in our region (Gentsch et al., 2005; Perez-Schael et al., 1990; Velazquez et al., 1996). In addition, the presence of multiple G and/or P genotypes in individual specimens may offer a unique environment for mixed infection acquisition and thereby for the reassortment of rotavirus genes. This could affect both, rotavirus evolution and efficacy performance of current and future vaccines. In this context, knowledge of both the rotavirus genotypes circulating in a community and the incidence of rotavirus mixed infections is essential for acquiring an in-depth understanding of the ecology and distribution of rotavirus strains and anticipating antigenic changes that could affect vaccine effectiveness. For this purpose, rotavirus G and P genotypes are determined by extraction of the viral RNA from fecal specimens followed by analysis by semi-nested reverse-transcriptase PCR (RT-PCR) with primers specific for regions of the genes encoding the VP7 or VP4. The genotype-specific PCR products are then analyzed on an agarose or polyacrylamide gel followed by ethidium bromide staining or silver staining, respectively. The matrix used for electrophoresis should have adjustable but regular pore sizes and be chemically inert, and the choice of which gel matrix to use depends primarily on the sizes of the fragments being separated (Guilliatt, 2002). As commented before, although the importance of specificity and sensitivity of PCR is well known, the mechanism by which the results are measured is equally important (Wildt et al., 2008).

    General characteristics of agarose and polyacrylamide matrices Agarose gel electrophoresis (AGE)

    Agarose is a natural linear polymer extracted from seaweed that forms a gel matrix by hydrogen-bonding when heated in a buffer and allowed to cool. For most applications, only a single-component agarose is needed and no polymerization catalysts are required. Therefore, agarose gels are simple and rapid to prepare (Chawla, 2004). They are the most popular medium for the separation of moderate and large-sized nucleic acids and have a wide range of separation but a relatively low resolving power, since the bands formed in the gels tend to be fuzzy and spread apart. This is a result of pore size and cannot be largely controlled. These and other advantages and disadvantages of using agarose gels for DNA electrophoresis are summarized here

    Advantages

    • Nontoxic gel medium
    • Gels are quick and easy to cast
    • Good for separating large DNA molecules
    • Can recover samples by melting the gel, digesting with enzyme agarose or treating with chaotropic salts

    Disadvantages

    • High cost of agarose
    • Fuzzy bands
    • Poor separation of low molecular weight samples

    Polyacrylamide gel electrophoresis (PAGE)

    Polyacrylamide gels are chemically cross-linked gels formed by the polymerization of acrylamide with a crosslinking agent, usually N,N’-methylenebisacrylamide. The reaction is a free radical polymerization, usually carried out with ammonium persulfate as the initiator and N,N,N’,N’-tetramethylethylendiamine (TEMED) as the catalyst. Although the gels are generally more difficult to prepare and handle, involving a longer time for preparation than agarose gels, they have major advantages over agarose gels. They have a greater resolving power, can accommodate larger quantities of DNA without significant loss in resolution and the DNA recovered from polyacrylamide gels is extremely pure (Guilliatt, 2002). Moreover, the pore size of the polyacrylamide gels can be altered in an easy and controllable fashion by changing the concentrations of the two monomers. Anyway, it should be noted that polyacrylamide is a neurotoxin (when unpolymerized), but with proper laboratory care, it is no more dangerous than various commonly used chemicals (Budowle & Allen, 1991). Some advantages and disadvantages of using polyacrylamide gels for DNA electrophoresis.

    Advantages

    • Stable chemically cross-linked gel
    • Sharp bands
    • Good for separation of low molecular weight fragments
    • Stable chemically cross-linked gel

    Disadvantages

    • Toxic monomers
    • Gels are tedious to prepare and often leak
    • Need new gel for each experiment

    Gel concentration

    Agarose gel concentration

    The percentage of agarose used depends on the size of fragments to be resolved. The concentration of agarose is referred to as a percentage of agarose to the volume of buffer (w/v), and agarose gels are normally in the range of 0.2% to 3% (Smith, 1993). The lower the concentration of agarose, the faster the DNA fragments migrate. In general, if the aim is to separate large DNA fragments, a low concentration of agarose should be used, and if the aim is to separate small DNA fragments, a high concentration of agarose is recommended

    Concentration of agarose (%) DNA size range (bp)

    Concentration of agarose (%)DNA size range (bp)
    0.2 0.4 0.6 0.8 1 1.5 2 35000-40000 5000-30000 3000-10000 1000-7000 500-5000 300-3000 200-1500 100-1000



    The choice of acrylamide concentration is critical for optimal separation of the molecules (Hames, 1998). Choosing an appropriate concentration of acrylamide and the crosslinking agent, methylenebisacrylamide, the pore sized in the gel can be controlled. With increasing the total percentage concentration (T) of monomer (acrylamide plus cross-linker) in the gel, the pore size decreases in a nearly linear relationship. Higher percentage gels (higher T), with smaller pores, are used to separate smaller molecules. The relationship of the percentage of the total monomer represented by the crosslinker (C) is more complex. Researchers have settled on C values of 5% (19:1 acrylamide/bisacrylamide) for most forms of denaturing DNA and RNA electrophoresis, and 3.3% (29:1) for most proteins, native DNA and RNA gels. For optimization, 5% to 10% polyacrylamide gels with variable cross-linking from 1% to 5% can be used. Low cross-linking (below 3% C) yields “long fiber gels” with an increased pore size (Glavač & Dean, 1996). Moreover, it should be pointed out that at low acrylamide/bisacrylamide concentrations the handling of the gels is difficult because they are slimy and thin. Table 4 gives recommended acrylamide/bisacrylamide ratios and gel percentages for different molecular size ranges 
    Polyacrylamide gel concentration

    Acrylamide/Bis Ratio
    Gel %
    Native DNA/RNA (bp)
    Denatured DNA/RNA (bp)
    19:1
    4
    6
    8
    10
    12
    100-1500
    60-600
    40-500
    30-300
    20-150
    70-500
    40-400
    20-200
    15-150
    10-100
    29:1
    5
    6
    8
    10
    12
    20
    200-2000
    80-800
    60-400
    50-300
    40-200
    <40
    70-800
    50-500
    30-300
    20-200
    15-150
    <40

    Polyacrylamide gel concentration for resolving DNA/RNA molecules. 

    Note: Recommended applications for each formulation are shown in bold.

    Electrophoretic buffer systems

    Effective separation of nucleic acids by agarose or polyacrylamide gel electrophoresis depends upon the effective maintenance of pH within the matrix. Therefore, buffers are an integral part of any electrophoresis technique. Moreover, the electrophoretic mobility of DNA is affected by the composition and ionic strength (salt content) of the electrophoresis buffer (Somma & Querci, 2006). Without salt, electrical conductance is minimal and DNA barely moves. In a buffer of high ionic strength, electrical conductance is very efficient and a significant amount of heat is generated. Different categories of buffer systems are available for electrophoresis: dissociating and non-dissociating, continuous and discontinuous.

    Dissociating and non-dissociating buffer systems

    The electrophoretic analysis of single stranded nucleic acids is complicated by the secondary structures assumed by these molecules. Separation on the basis of molecular weight requires the inclusion of denaturing agents, which unfold the DNA or RNA strands and remove the influence of shape on their mobility. Nucleic acids form structures stabilized by hydrogen bonds between bases. Denaturing requires disrupting these hydrogen bonds. The most commonly dissociating buffer systems used include urea and formamide as DNA denaturants. Denatured DNA migrates through these gels at a rate that is almost completely dependent on its base composition and sequence. Denaturing or dissociating buffer systems for proteins include the use of sodium dodecyl sulfate (SDS). In the SDS-PAGE system, developed by Laemmli (1970), proteins are heated with SDS before electrophoresis so that the charge-density of all proteins is made roughly equal. Heating in SDS, an anionic detergent, denatures proteins in the samples and binds tightly to the uncoiled molecule (with net negative charge). Consequently, when these samples are electrophoresed, proteins separate according to mass alone, with very little effect from compositional differences. DNA molecules are negatively charged; therefore the addition of SDS in the gel preparations is only with the aim of enhancing the resolution power of the bands (Day & Humphries, 1994). In the absence of denaturants, double stranded DNA (dsDNA), like a PCR product, retains its double helical structure, which gives it a rodlike form as it migrates through a gel. During the electrophoresis of native molecules in a non-dissociating buffer system, separation takes place at a rate approximately inversely proportional to the log10 of their size.

    Continuous and discontinuous buffer systems

    In the continuous buffer systems, the identity and concentration of the buffer components are the same in both the gel and the tank. Although continuous buffer systems are easy to prepare and give adequate resolution for some applications, bands tend to be broader and resolution consequently poorer in these gels. These buffer systems are used for most forms of DNA agarose gel electrophoresis, which commonly contain EDTA (pH 8.0) and Trisacetate (TAE) or Tris-borate (TBE) at a concentration of approximately 50mM (pH 7.5-7.8). TAE is less expensive but not as stable as TBE. In addition, TAE gives better resolution of DNA bands in short electrophoretic separations and is often used when subsequent DNA isolation is desired. TBE is used for polyacrylamide gel electrophoresis of smaller molecular weight DNA (MW<2000) and agarose gel electrophoresis of longer DNA where high resolution is not essential. Discontinuous (multiphasic) systems employ different buffers for tank and gel, and often two different buffers within the gel. Discontinuous systems concentrate or “stack” the samples into a very narrow zone prior to separation, which results in improved band sharpness and resolution. The gel is divided into an upper “stacking” gel of low percentage of acrylamide and low pH (6.8) and a separating gel with a pH of 8.8 and much smaller pores (higher percentage of acrylamide). The stacking gel prevents any high-molecular-weight DNA present in the sample from clogging the pores at the top of the running gel before low molecular weight DNA has entered. Both, the stacking and the separating gels, contain the only chloride as the mobile anion, while the tank buffer contains glycine as its anion, at a pH of 8.8. The major advantage of the discontinuous buffer system over continuous buffer system is that this gel system can tolerate larger sample volumes (Rubin, 1975).

    Loading buffer

    This is the buffer to be added to the DNA fragment that will be electrophoresed. This buffer contains glycerol or sucrose to increase the density of the DNA solutions; otherwise, the samples would dissolve in running buffer tank and not sink into the gel pocket. The gel loading buffer also contains dyes that facilitate observation of the sample during gel loading and electrophoresis, such as bromophenol blue or xylene cyanol. Because these molecules are small, they migrate quickly through the gel during electrophoresis, thus indicating the progress of electrophoresis (Chawla, 2004). The components and concentrations of the 6X loading dye usually used are: 0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol; or 0.25% bromophenol blue, 50 mM EDTA, 0.4% sucrose.

    Voltage/current applied

    The higher the voltage/current, the faster the DNA migrates. If the voltage is too high, band streaking, especially for DNA≥12-15kb, may result. Moreover, high voltage causes a tremendous increase in buffer temperature and current in very short time. The high amount of the heat and current built up in the process leads to the melting of the gel, DNA bands smiling, decrease of DNA bands resolution and fuse blowout. Therefore, it is highly recommended not exceed 5-8 V/cm and 75 mA for standard size gels or 100 mA for minigels. On the other side, when the voltage is too low, the mobility of small (≤1kb) DNA is reduced and band broadening will occur due to dispersion and diffusion.

    Visualizing the DNA

    After the electrophoresis has been completed there are different methods that may be used to make the separated DNA species in the gel visible to the human eye. 7.1 Ethidium bromide staining (EBS) The localization of DNA within the agarose gel can be determined directly by staining with low concentrations of intercalating fluorescent ethidium bromide dye under ultraviolet light. The dye can be included in both, the running buffer tank and the gel, the gel alone, or the gel can be stained after DNA separation. For a permanent record, mostly instant photos are taken from the gels in a dark room. It is important to note that ethidium bromide is a potent mutagen and moderately toxic after an acute exposure. Therefore, it is highly recommended to handle it with considerable caution.

    Silver staining (SS)

    Silver staining is a highly sensitive method for the visualization of nucleic acid and protein bands after electrophoretic separation on polyacrylamide gels. Nucleic acids and proteins bind silver ions, which can be reduced to insoluble silver metal granules. Sufficient silver deposition is visible as a dark brown band on the gel. All silver staining protocols are made of the same basic steps, which are: i) fixation to get rid of interfering compounds, ii) silver impregnation with either a silver nitrate solution or a silver-ammonia complex solution, iii) rinses and development to build up the silver metal image, and iv) stop and rinse to end development prior to excessive background formation and to remove excess silver ion (Chevallet et al., 2006)