Neurological involvement in scrub typhus.
\r\n\tIn this book the authors will provide complete introduction of Polymers chemistry. The book is mainly divided into three parts. The readers will learn about the basic introduction of general polymer chemistry in the first part of the book.
\r\n\tThe second part of the book starts with a chapter which includes kinetics of polymerization. Polymer weight determination, molecular weight distribution curve and determination of glass transition temperature. The final part of the book deals polymer degradation which includes types of degradation. The chapters of the present book consist of both tutorial and highly advanced material.
Scrub typhus is also known as ‘tsutsugamushi disease’. It is a zoonotic disease. This disease was first mentioned in Japanese folklore to be associated with the jungle mite or ‘chigger’, which was named ‘dangerous bug’. Therefore, the term ‘tsutsugamushi’ is derived from two Japanese words, ‘tsutsuga’ which means something small and dangerous, and ‘mushi’ which means creature.
‘Typhus’ has been derived from the Greek word ‘typos’ which means ‘fever with stupor’. The name itself reveals a clinical aspect of the disease .
Although scrub typhus was known in China in the third century A.D., it was first described by Hakuju Hashimoto in 1810 in people living in the banks of the Shinano river and later in 1879 by Baclz and Kawakami as Japanese ‘flood fever’. It is widespread in the so-called ‘tsutsugamushi triangle’ which extends from Pakistan, India, Nepal in the West, to South Eastern Siberia, Japan, China and Korea in the North, to Indonesia, the Philippines, Northern Australia and the Pacific islands in the South. Taiwan is the centre of the tsutsugamushi triangle, and Korea has the highest reported incidence in the world . About one million new cases are identified annually.
In India, the disease had occurred among troops during World War II in the state of Assam and West Bengal. Although the disease is endemic in India, epidemics have also been reported.
Epidemiological reports confirm strong existence of scrub in hilly/rain prone areas. Outbreaks reveal an autumn-winter type and a summer type of pattern. In comparison with the summer type, the autumn-winter type is less severe.
Increasing prevalence of scrub typhus reported from some Asian countries may be related to urbanization of rural areas.
Scrub typhus is caused by an obligate intracellular gram-negative bacterium called Orientia tsutsugamushi. Ogata in 1931 isolated the organism and named it Rickettsia tsutsugamushi. Now it has been renamed as O. tsutsugamushi. The organism lacks a cell wall. There are six important serotypes of O. tsutsugamushi—Gilliam, Karp, Kato, Shimokoshi, Kawasaki and Kuroki. A new strain has been isolated from a case of scrub typhus in Australia which was quite different from the classic strains and has been named as Litchfield.
The mite has four stages: egg, larva, nymph and adult. The larval forms (chiggers) transmit the disease to humans and other vertebrates. The larvae feed on rodents particularly wild rats of subgenus Rattus, The infection in humans is acquired during outdoor recreational and agricultural activities, by the bite of the larval stage of mites. The areas are usually secondary scrub growth which grows after clearance of primary forest hence the term scrub typhus. Humans are therefore accidental hosts for the pathogen. Vertical transovarial transmission occurs in mites. One case of transplacental spread has been reported in a pregnant woman, who delivered a preterm baby with scrub typhus IgM positivity.
Following the bite, the pathogen multiplies at the site of inoculation and produces both systemic and local manifestation. Infection spreads through both haematogenous and lymphatic routes. The severity of the illness depends on both host-related and pathogen-related factors. Pathogen-related factors may be related to the different strains of O. tsutsugamushi. Host-related factors as seen in humans with G6PD deficiency who has a worse prognosis also play a role. Target cells for multiplication are the endothelial cells of the various systems. The immune response induced by the pathogen is a combination of humoral and cell-mediated immunity. There occurs a rise of cytokines during an acute infection. There also occurs a rise in macrophage colony stimulating factor, interferon gamma and granulocyte colony stimulating factor. Therefore, the macrophage and T-lymphocyte response may be the main factor in immunity against the infection. However, the parasite has also evolved to evade the immune response of the host. The pathogen can down-regulate the expression of glycoprotein 96, in infected macrophages and endothelial cells and thereby neutralize host immune response. This molecule plays a central role antigen presentation and antibody production .
Central nervous system involvement occurs in scrub typhus. It is speculated that since the pathogen is an obligatory intracellular organism, it enters the cerebrospinal fluid in a monocyte or grow through the endothelium, enter via the luminal cell membrane and release into the perivascular space . The pathogen also invades and multiplies in the vascular endothelium and cause wide spread vasculitis. Autopsy specimens have shown central nervous system pathology in scrub typhus cases in the form of diffuse or focal mononuclear cell infiltration of the leptomeninges, presence of typhus nodules (clusters of microglial cells) and haemorrhages of the brain substance .
The incubation period of O. tsutsugamushi in humans is around 10–12 days (can vary between 6 and 21 days). The clinical manifestations vary from a mild febrile illness to a severe potentially disease. The systemic features of the infection include fever, gastrointestinal disturbance, malaise, cough, myalgia and headache. A maculopapular rash starting from the trunk and spreading to the limbs is seen towards the end of the first week of the fever. Diffuse lymphadenopathy is commonly observed.
A necrotic ‘eschar’ at the bite site is almost diagnostic of scrub typhus (Figure 1).
The eschar resembles skin burn of cigarette butt. Eschar is found in 7–80% patients of scrub typhus . In the authors study, eschar was detected in 28.81% of the patients of scrub typhus and 30.77% patients of meningoencephalitis due to scrub typhus . The wide range of detection may be due to the difficulty in detecting eschars in dark-skinned individuals, difference in the eschar inducing capacity of the different strains of O. tsutsugamushi. The groin, axilla, waist and other exposed parts of the body are common sites of eschar detection. In the authors study, eschar was mostly found in the inguinal region. Different pattern of eschar distribution found in males and females due to the differences in skin folds, clothing and pressure points created by garments. The eschar not only has immense diagnostic relevance but is also important prognostically. Absence of an eschar is a risk factor for mortality .
Neurological involvement is often a prominent clinical manifestation of scrub typhus. However, they are still an unclear entity. Meningitis or meningoencephalitis can occur in upto one-fifth of affected patients. In the authors study, meningoencephalitis was found in 13.2% of scrub typhus patients . The various neurological manifestations of scrub typhus  are as given in (Table 1).
|1. Optic neuritis|
|3. Acute-disseminated encephalomyelitis|
Meningitis has features of headache, vomiting, fever, neck stiffness, along with cerebrospinal fluid (CSF) pleocytosis. Altered sensorium and fever with CSF pleocytosis are features of encephalitis. Altered sensorium with fever but normal CSF is found in encephalopathy.
Neurological manifestations in scrub typhus does not occur in isolation but are accompanied by systemic features like jaundice, breathlessness, cough, renal impairment and in some cases, with multi-organ dysfunction. In the authors study  neurological manifestations were associated with lymphadenopathy (46.15%), jaundice (53.85%), pulmonary oedema (23.08%), oliguria (15.38%), hepatomegaly (38.46%) and splenomegaly (7.69%). Multi-organ dysfunction was found in 15.38% patients of scrub typhus with neurological manifestation.
The most common symptom of scrub typhus is fever. The fever is usually mild and accompanied by myalgia. In the authors study the mean duration of fever was 5.61 days, prior to meningoencephalitis presentation.
Headache is a common symptom in scrub typhus (46–77%). A severe holocranial headache almost invariably occurs and thereby helps in identifying suspected cases. Headache occurs not only in patients of meningitis or meningoencephalitis but in other scrub typhus patients also. However, in those cases, the headache is less severe.
In scrub typhus meningitis, the severe headache is associated with neck stiffness and fever. Other signs of meningeal irritation like Kernig’s sign may also be present. These meningeal signs are detected in upto 45% patients. In the author’s study, meningeal signs were present in 76.92% patients .
Altered sensorium is present in scrub typhus patient with encephalitis and meningoencephalitis. In the author’s study, altered sensorium was found in all patients; however, other studies have reported a lower incidence.
Seizure occurs in scrub typhus with neurological involvement in 22–50% cases, though uncommon myoclonic seizure was found in one patient in the author’s series.
Cranial nerve deficits are seen in 25% patients. Most commonly sixth nerve involvement is seen, which maybe unilateral or bilateral. Facial palsy may occur in isolation or in association with Guillain Barre syndrome . Cochlear nerve involvement occurs in about 19% patients and cause sensorineural hearing loss, otalgia and tinnitus. This may be due to direct invasion by the pathogen or due to a secondary immune-mediated effect.
Other uncommon neurological manifestations of scrub typhus mentioned are infarction, cerebellitis, haemorrhages, subdural hematoma and Guillain Barre syndrome .
This is aided by serological tests in appropriate clinical setting.
Microimmunofluorescence is considered the test of choice. However, lack of fluorescent microscopes makes it difficult for most hospitals.
Latex agglutination, indirect haemagglutination, immunoperoxidase assay, ELISA and polymerase chain reaction (PCR) are also available. The nested PCR is more sensitive than the serological tests.
PCR can be used to detect rickettsial DNA in both blood and eschar samples. The PCR is targeted at the gene encoding the major 56-kDa antigen and/or 47-kDa surface antigen gene. The results are best within first week for blood samples.
ELISA (IgG and IgM) technique, particularly immunoglobulin M (IgM), capture assays are probably the most sensitive test available for rickettsial diagnosis. In cases of infection with O. tsutsugamushi, a significant IgM antibody titre is observed at the end of the first week, whereas IgG antibodies appear at the end of the second week.
Weil Felix test: the sharing of the antigen between rickettsia and proteus is the basis of this heterophile antibody test. Though this test lacks high sensitivity and specificity, it is inexpensive. The test should be carried out after 5–7 days of onset of fever.
However, due to the antigenic diversity of the pathogen, a battery of tests may be required for the diagnosis .
CSF analysis in scrub typhus meningoencephalitis reveals mild-to-moderate elevation in protein, low-to-normal glucose and mild degree of lymphocytic pleocytosis. By using nested PCR, the genotypes invading the central nervous system (CNS) may be identified. By this, it was suggested that the Karp and Boryong genotypes possibly invade the CNS more than other types . In the author’s study, tuberculous meningitis remained the close differential diagnosis of scrub typhus meningitis due to similar CSF findings . However, CSF adenosine deaminase (ADA) may be helpful, as it is elevated >10 in tuberculous meningitis, unlike scrub typhus meningitis.
Neurological involvement in scrub typhus is usually associated with a normal MRI and non-specific EEG slowing. Often MRI of brain in scrub typhus may reveal features of ischemic changes due to vasculitis or parainfectious demyelination .
Doxycycline is the drug of choice. It is bacteriostatic to O. tsutsugamushi but does not cross the blood brain barrier beyond 15–30% . Therefore, in some instances, neurological deterioration can continue despite doxycycline therapy. This may be due to resistance, immune-mediated injury, or due to drug interactions with oral antacids. Doxycycline is given in the dose of 200 mg/day, in two divided doses, for individuals above 45 kgs body weight, for a duration of 7 days. In children, doxycycline is given in the dose of 4.5 mg/kg body weight in two divided doses. Doxycycline is contraindicated in pregnant women. In complicated cases, intravenous is given followed by oral doxycycline to complete 7–15 days of therapy.
Azithromycin is another drug which can be used in a dose of 500 mg daily for 5 days and 10 mg/kg body weight in children for 5 days. It can also be given intravenously in complicated cases. Azithromycin is the drug of choice in pregnant women with scrub typhus. It is also preferred in patients of scrub typhus with renal failure, where doxycycline is not given.
In complicated cases, Chloramphenicol can also be used. It is administered intravenously at a dose of 50–100 mg/kg/day, 6 hourly doses, followed by oral therapy to complete 7–15 days of therapy.
Doxycycline and/or chloramphenicol resistant strains have been detected in South-East Asia. These strains are sensitive to Azithromycin.
Patients with meningoencephalitis due to scrub typhus can be additionally administered with dexamethasone, or mannitol, if they have altered sensorium or cranial nerve deficits.
Recovery is usually brisk with appropriate therapy.
Pre-antibiotic era mortality was more than 60%; however, recent data show a mortality of approximately 30% . In the author’s study , the mortality was 15.38%. Mortality is usually associated with multi-organ dysfunction syndrome.
Neurological complication is not uncommon in scrub typhus. They present with acute febrile illness with altered sensorium and meningeal signs. The presence of ‘eschar’ helps in early diagnosis, but they are often absent. Prompt CSF analysis is required on clinical suspicion of neurological features in scrub typhus patients. Timely initiation of therapy results in recovery and less complications.
It is said that the word “plasmid” is first proposed by the Nobel Prize winner Joshua Lederberg [1, 2]. Plasmid is an extrachromosomal small circular deoxyribonucleic acid (DNA), which duplicates independently from chromosomal DNA. Although budding yeast and fission yeast can retain plasmid, the host of the plasmid is almost bacteria. This small circular DNA is widely used as DNA vector in molecular biology, biochemistry, biotechnology, cell biology, and so on. It means that plasmid purification/isolation is very fundamental experiment in these research fields, and that this experiment is achieved in almost every laboratories on almost every day.
In biochemical aspects, to purify plasmid DNA from bacteria is to isolate only plasmid DNA from the mixture of biopolymers such as protein, ribonucleic acid (RNA), chromosomal DNA and plasmid DNA, by which bacteria cell is composed (Figure 1).
A chemical property of protein is totally different from nucleic acids; therefore, it is rather easy to separate nucleic acids and proteins. However, RNA and DNA are very similar molecules from each other. Among them, ribose in RNA is only distinguishable from deoxyribose in DNA by one hydroxyl group (−OH) at its structure. Furthermore, chromosomal DNA and plasmid DNA is both deoxyribonucleic acids that have the same chemical properties. Chromosomal DNA in almost all bacteria is circular, so is also plasmid. The distinguishable difference of them is only their size: plasmid DNA (~10 kilo base pairs) is much smaller than chromosomal DNA (4.6 million base pairs in Escherichia coli). Based on these properties, a special technique for purifying plasmid DNA among these biomolecules are required.
To purify plasmid DNA of high quantity, culture condition, or media for E. coli growth is also important .
A very simple manipulation steps enable us to recover plasmid DNA from Escherichia coli (E. coli) (Figure 2) . This experiment is called “Boiling method”. In this experiment, STET solution (100 mM sodium chloride, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 5% Triton-X or Tween 20) is added to E. coli pellet and suspended well. And then, the sample is heated to 100°C for 1 minute and centrifuged. After centrifugation, plasmid DNA is recovered in the solution, whereas insoluble heat-denatured proteins make debris as pellet fraction. After separating plasmid DNA from debris and precipitating plasmid DNA by adding alcohol, the final plasmid sample is capable for the next experiment, such as cutting plasmid by restriction enzyme and/or modifying DNA by other enzymes.
Interestingly, the pellet is rather moisty and easily removed by using toothpick and piercing it. This feature contributes to an easy handling of the experiment: we can achieve this plasmid extraction in only one tube from the start point to the end of the experiment. Therefore, boiling method has very convenient for handling many samples at a time. The modified boiling method, in which a concentrated STET solution is directly added to LB medium in which E. coli is grown, is also reported . This modified method does not require even harvesting step of the grown bacteria by centrifugation.
The major disadvantage of the boiling method is that RNA is not removed in the principle of the boiling method and that the chromosomal DNA of E. coli is not completely removed from plasmid DNA. Therefore, the purity of the finally isolated plasmid DNA is not so high. The boiling method is suitable for checking, if plasmid in E. coli transformant has an expected insert DNA (insert check), usually testing multiple samples at a time.
One substitution of the boiling method for insert check is colony polymerase chain reaction (PCR), directly adding E. coli colony to the PCR reaction mixture as a template. In colony PCR, E. coli cells are broken at the first 96°C step of PCR. Basically, a simple boiling of bacteria in the water is enough for collecting DNA from them, which has adequate quality to achieve PCR . Colony PCR is much easier experiment than boiling method. However, once we isolate plasmid DNA even by rough purity of boiling method, we can further analyze the recovered plasmid by checking restriction enzyme patterns and so on. For example, restriction map is much informative result than if the fragment is amplified by colony PCR.
To purify plasmid from E. coli, there need each step for removing unnecessary molecules, such as protein, chromosomal DNA and RNA. For this purpose, alkaline denature of E. coli is the definitive technique for removing proteins and chromosomal DNA. This method was established in 1979 , but it is so sophisticated that almost all experiment for plasmid purification today is based on this technique (Figure 3).
The principle of the alkaline lysis method is a kind of magic. After suspending the E. coli in the solvent (solution I; 25 mM Tris/HCl (pH 8.0), 10 mM EDTA), an alkaline solution (solution II; 200 mM NaOH, 1% SDS) is added to the sample. In this condition, almost all proteins are denatured. DNA double-strand structure is also denatured to single-strand. However, even in such an extreme condition, supercoiled plasmid DNA remains its structure stable and not denatured. After 5 minutes, incubation of alkaline denature, high-salt buffer (solution III; 3 M Potassium Acetate, pH 5.5) is added for the purpose of a sudden change of pH in the solution. As a result, denatured protein and chromosomal DNA do not turn back to its own structure, causing these molecules insoluble. On the other hand, plasmid DNA remains soluble, thus centrifuge step easily separates the plasmid DNA from debris of proteins and chromosomal DNA. In the alkaline lysis method, each step is very simple and easy. All we have to do is only adding solution sequentially. Moreover, the function of each solution is only changing pH and salt concentration. However, these ingeniously planned three steps enable us to recover plasmid DNA, avoiding proteins and chromosomal DNA. The most notable point of this method is that we can isolate only plasmid DNA from plasmid/chromosomal DNA mixture; both are deoxyribonucleic acids and have the same chemical properties. No other method should successfully separate chromosomal DNA and plasmid DNA by such a simple step.
In early days, original protocol of alkaline lysis method used sodium acetate as a salt in solution III . However, now potassium acetate is substituted for the major agent for solution III than sodium acetate. Potassium ion binds to dodecyl sulfate ion and forms potassium dodecyl sulfate (PDS). PDS is highly insoluble salt, which is made by adding solution III in the alkaline lysis sample. The PDS also plays a seed for insoluble debris, with which insoluble proteins and chromosomal DNA are co-precipitated. It is the great advantage that alkaline lysis method enables us to prevent protein and chromosomal DNA from plasmid at the same step.
One point we have to keep in mind is that supercoiled (closed circular) plasmid DNA is converted into nicked, relaxed (open circular) DNA by alkaline incubation. Thus, we have to keep the incubation time at solution II as in the instruction, and not to incubate the sample with solution II for a long time. Besides, RNA is not removed in a series of alkaline lysis method for plasmid purification (Figure 3). RNA is partially hydrolyzed by solution II but remains with the plasmid DNA at the final step. Therefore, a huge amount of RNA contamination is in the final plasmid DNA sample. Principally, only isolating plasmid DNA by alkaline lysis is inadequate for purifying high-quality plasmid DNA, and several schemes for further purification of the plasmid DNA, especially for removing RNA, should be needed.
Anyhow, alkaline lysis method has been the definitive way to initially purify the plasmid DNA.
Neither alkaline lysis method nor boiling method does not isolate plasmid DNA from RNA mixture. An extra step for removing RNA is needed for further purification of plasmid DNA.
Basically, to remove RNA (not to separate intact RNA) from DNA-RNA mixed solution is very easy: Only to add ribonuclease (RNase) to the solution enables us to completely digest RNA. Even when RNase is added to the solution I in the course of alkaline lysis method, RNA is completely digested in the finally corrected plasmid sample (see Figure 3). It sounds very strange that RNase in the solution I digest RNA, because the function of solution I is only to suspend the E. coli, the E. coli cell is not thought to lysed in the solution I step only. Otherwise, RNase might be still stable even in the alkaline condition of solution II, or rapidly renatured to the functional conformations in neutralized condition by solution III. RNase itself is a very stable protein, so we do not have to worry about a loss of enzyme activity at high-temperature. This character of RNase makes us very easy to handle this enzyme in the experiment, but this character often annoys us too, because a contamination of RNase to the other samples completely disturbs our RNA-handling experiment in the laboratory.
Irresponsible usage of RNase often contaminates the laboratory. Therefore, after incubating plasmid sample with RNase, the complete inactivation/removal of RNase should be needed.
Phenol or phenol/chloroform is well known as a protein denaturant. RNase is also inactivated by such as denaturant. Because RNase is very stable, repeating steps of phenol or phenol/chloroform extraction is effective for the complete removal of RNase. On the other hand, this organic reagent often inhibits enzyme activities, once contaminated with the nucleic acids samples. Besides, it is very convincing that phenol or phenol/chloroform have toxicity to cells, resulting in a decrease of transfection efficiency to cultured cells, and so on.
It is known that a certain salts selectively precipitate nucleic acids. These salts can be applied to plasmid DNA purification.
Lithium chloride (LiCl) at the final concentration of 2.5 M enables us to selectively precipitate RNA. In this condition, RNA makes a pellet by centrifugation, but not DNA. Although low-molecular-weight RNA fragment is not precipitated and remains with plasmid DNA, it is often an adequate quality for using the plasmid in the following experiments, as long as the low-molecular-weight RNA makes critical disturbance for the experiment.
In isolating plasmid DNA by boiling method, Adding LiCl to STET is a better way to do the experiment (see Figure 2). After boiling step, centrifugation makes insoluble debris, together with RNA precipitation by the function of LiCl. Therefore, one-step centrifuge is enough for removing protein and RNA. Rather low-molecular-weight RNA still remains in the solution, but normally this RNA does not disturb or inhibit the activity of restriction enzyme and so on. The insoluble pellet in the boiling method with LiCl is like a chewing gum, and is easily removed by picking with toothpick. Therefore, the combination of boiling method with LiCl is a very reasonable choice. One minor point is that LiCl is rather expensive than Calcium chloride (CaCl2).
Calcium chloride (CaCl2) is an inexpensive reagent. It is also known to precipitate RNA at the concentration of around 1 M, but DNA is not precipitated in this condition . Thus, this reagent also works in the plasmid purification process like LiCl. However, it is a luck of luckiness that when CaCl2 is added instead of LiCl in the boiling method, insoluble debris forms crumbly. This means that we cannot pick the debris up by using toothpick, and that we need another tube to transfer the supernatant after centrifugation. This disturbs a merit of the boiling method using only one tube all over the manipulations.
Polyethylene glycol (PEG) can be used to precipitate DNA . It is also reported that the size of precipitated DNA is controllable by the concentration of PEG . The principle of PEG precipitation is the same as alcohol precipitation, such us ethanol and/or isopropanol. This compound selectively precipitates DNA. Especially, low-molecular-weight RNA, such as transfer RNA, is not precipitated by PEG. There are so many products of PEG, according to their average molecular weights. Generally, PEG #3000, #4000, or #6000 has similar properties for DNA precipitation. Interestingly, to the contrast of that LiCl and CaCl2 do not precipitate low-molecular-weight RNA, the size between precipitated RNA by salts and non-precipitated RNA by PEG precipitation are complementary in the RNA length from each other.
Cesium chloride (CsCl) ultracentrifuge method  does not require RNase. It means that phenol/chloroform extraction is not needed in the experiment, so plasmid DNA purified by this method is suitable for the almost all the biochemical experiment. That is, we can apply the plasmid DNA isolated by this method to transfection of the cultured cell and so on.
An ultrapure grade of plasmid is obtained in this method, although a special expensive ultracentrifuge is required for equipment. Moreover, very long time (almost overnight) for centrifuge is needed, and ethidium bromide (EtBr) at a very high concentration (final 800 μg/mL, this is 8000 times higher concentration than agarose gel electrophoresis) is used. EtBr is widely known as a mutagen, and highly concentrated EtBr should be unwanted to handle, if possible. EtBr intercalates to the double-strand of DNA. When this compound is intercalated with DNA, the double-strand of plasmid DNA changes to the slightly unwound form. This affects the sedimentation coefficient of nucleic acids in 10% CsCl solution. Therefore, supercoiled plasmid DNA makes a single sharp band in the tube after ultracentrifugation (200,000× g,20°C, 16 hours). In this experiment, a specially customized tube should be used. The centrifuge tube is made of a soft, translucent plastic polymer, and the plasmid DNA is visualized as a band in the see-through tube. A syringe needle is inserted into the tube, and the separated plasmid band is sucked into the syringe. After transferring the sucked solution to a new tube, more extra steps are needed to get rid of CsCl and EtBr. This experiment is very sensitive to CsCl concentration. A slight change of CsCl amount causes a negative result; plasmid DNA is not separated as a single band in the tube.
CsCl ultracentrifuge method costs expensive because CsCl is an expensive reagent. Moreover, this experiment is time-consuming, hazardous, and difficult. On the contrary, once succeeded, we can obtain a large amount of ultrapure plasmid DNA. This method can even separate ccDNA from ocDNA, trusting the super high quality of plasmid DNA.
The important fact is that CsCl method is a kind of post-manipulation of alkaline lysis method. That is, alkaline lysis method is such a universal method that it works well as an initial step of CsCl method.
Two major kit for plasmid purification is available in the market (Figure 4). Both use a basic alkaline lysis method for initial steps, and also uses RNase for RNA removal. A feature of the recent plasmid isolation methods is that they do not go through phenol/chloroform extraction after RNase treatment. Organic solvents are often harmful to cultured cell and so on, so avoiding this reagent in the steps of plasmid purification is a reasonable choice.
Diethyl-aminoethyl (DEAE) group has positive charges; therefore DEAE-resin is often used to ion-exchange chromatography. DNA also has negative charges, so it binds to DEAE-resin under a certain pH or salt concentration. However, DNA purification by using DEAE was restricted to the recovery step from excised agarose gel and so on, because the binding property of nucleic acids to DEAE is rather broad and weak. Plasmid purification by anion-exchange chromatography has been reported , but it will be much better using the column as disposable to avoid contamination of samples. Therefore, DEAE was not seemed to be applicable for plasmid purification in a daily experiment. Qiagen column is famous for its ultrapure quality of purified plasmid DNA, and the principle is ion-exchange column chromatography. The precise information of Qiagen resin is confidential, but basically, it is known that the column consists of a highly condensed anion-exchange group resin . In this experiment, the agent to remove RNA is not an ion-exchange resin, but RNase. A high concentration of RNase is added in the initial step of solution I. During Qiagen chromatography steps, the solutions go through a column in a free fall, so centrifuge step is not required at each step. However, after elution of the plasmid DNA from the resin, the concentration of plasmid DNA may be low, which is inadequate for the following experiment. Therefore, on Qiagen kit, ethanol precipitation is often needed to the eluted solution for concentrating plasmid. It seems that Qiagen columns are suitable for purifying plasmid of mini or maxi scale because the column is rather expensive. Besides, it is not easy to manipulate for multiple open columns at a time.
Boom’s method is based on a paper and patent by Boom et al. , although the principle is widely known as a biochemical property of nucleic acids. The principle of Boom’s method is that glass powder or diatomaceous earth (the main ingredient is SiO2) adsorbs nucleic acids in a chaotropic condition , whereas proteins are not. In Boom’s method, guanidine hydrochloride or guanidine thiocyanate is often used for chaotropic agent. This adsorption is reversibly eluted by pure water. Hydrophobic condition keeps the adsorption of DNA to SiO2, so washing glass powder which adsorbs nucleic acids by 70% ethanol contributes to a high purity of plasmid DNA. The original Boom’s method uses grass or diatomaceous earth powder as binding agent, and each step for binding, washing, and eluting is achieved as batch technique (simply centrifuging and discarding the solution). Batch chromatography is very simple method and easy to handle, although pipetting and discarding each solutions makes the experiment rather complicating for manipulating many samples at a time. On the other hand, the commercial kit supplies a column with a silica membrane filter, which is set to 1.5 mL microtube. Once the solution is applied to column, centrifuge step forces the solution go through the column. Therefore, each steps for binding, washing, and eluting needs only several seconds (as much as 1 minute) for centrifugation. Actually, commercial kit of such a silica membrane filter is very easy and useful for handling.
The commercial kit supplies their reagents with the column, but the compositions of these reagents are always confidential . On the other hand, the principle of these kits seems almost the same, based on the DNA adsorption to silica matrix in chaotropic solution. Although it is quite natural to assume that each kit has its own special reagents, homemade solutions based on the original paper are generally available. When applied homemade reagents to commercial silica membrane column, quality and quantity of purified plasmid are almost the same as the commercial kit . It means that these solutions are available by DIY and columns are not still waste, even when reagents in the commercial kit box are expired and out of use.
A modified reagent and modified protocol are also reported to increase the recovery efficiency of the plasmid DNA by commercial column . On the other hand, not silica particles but Zirconium dioxide (ZrO2, zirconia) has also been reported as an adsorbent of DNA .
The chaotropic agent is a key factor in Boom’s method, so guanidium salt such as GuHCl should play an important role for plasmid DNA purification in Boom’s method. But it is reported that guanidium salt is not actually needed for nucleic acids to be adsorbed to silica particles. The other reagent such as high-concentration NaCl also works as chaotropic agent . More surprisingly, a high concentration of the salt in solution III of alkaline lysis method seems already adequate for making the solution to chaotropic condition . It means that adding guanidium for DNA adsorption can be skipped. To cut steps in the experiment has many advantages, especially for handling many samples at a time. Therefore, purifying plasmid samples in 96-well plate without guanidine chaotropic condition is proposed , in which method small scale and many samples at a time.
Based on the alkaline lysis method, we developed a new plasmid purification method, which ends within 1 hour and does not need RNase (Figure 5) . The principle of this method is a combination of the alkaline lysis method, CaCl2 precipitation, and PEG precipitation. Although a sequential combination of these precipitations was already reported , our new invention is that we developed a new composition of solution III. This “super solution III” contains CaCl2 to the standard solution III (Solution III: 5 M CaCl2: H2O = 2:2:1), which makes not only protein and genomic DNA debris but also a pellet of RNA in the debris. After centrifuging and recovering a supernatant, a standard PEG precipitation makes a plasmid DNA pellet and removes small RNA, which was not precipitated at the super solution III step. After all, only plasmid DNA remains in the final solution. Actually, this method needs totally 55 minutes from collecting E. coli pellet to recovering the final purified plasmid DNA. The great advantage of this method is that we are able to eliminate the use of RNase. Therefore, even RNase removal step is also eliminated. A quality and quantity are adequate for doing another experiment such as transfection into cultured cells.
In a course of doing plasmid purification for every day, I noticed several tips for the experiment.
Alkaline denaturation/renaturation steps are so sophisticated that it is the definitive method for plasmid purification. None of another method will take over the alkaline lysis method. However, RNA is not removed in alkaline lysis method, so RNA removal steps should be applied in a course of plasmid isolation by alkaline lysis method.
RNase is an easy choice to remove RNA, but should be completely removed after RNA digestion. One of the solutions is phenol/chloroform protein extraction, but phenol/chloroform may play a troublesome factor. Only a slight contamination of this reagent inhibits the activities of several enzymes and disturbs biochemical experiments. It also has toxicity to the cells, also disturbing transfection experiments. In other words, eliminating phenol/chloroform step in plasmid purification is the key point to trust its purity.
Qiagen kits and Silica-membrane kits are actually the extra steps after alkaline lysis method. These kits need RNase for RNA digest. In other words, they work as RNase remover from the solution.
RNase completely digests unwanted RNA from the plasmid sample. But this enzyme is very stable and very hard to inactivate, even disturbing RNA experiment in the laboratory. Moreover, RNase is usually isolated from animals such as bovine, which may induce allergy to the human in gene therapy .
Based on these tips, we developed a new composition of solution III on alkaline lysis method, which enables us to purify plasmid DNA without adding of RNase. This method does not need any special columns or resins, but plasmid DNA purified by this method has enough quality for applying transfection to the cultured cell, injection into the nematode, and so on. Our result indicates that plasmid DNA purification without phenol/chloroform extraction is a great advantage for the quality of purified plasmid DNA.
I thank Ms. Haruka Yano (Tokai University Graduate School) for technical suggestions.
The author has no conflicts of interest directly relevant to the content of this article.
The author has no other declarations about this article.