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81-661: Lyssavirus (from the Greek λύσσα lyssa "rage, fury, rabies" and the Latin vīrus ) is a genus of RNA viruses in the family Rhabdoviridae , order Mononegavirales . Mammals, including humans, can serve as natural hosts . The genus Lyssavirus includes the causative agent ( rabies virus ) of rabies . Lyssavirions are enveloped, with bullet shaped geometries. These virions are about 75 nm wide and 180 nm long. Lyssavirions have helical symmetry, so their infectious particles are approximately cylindrical in shape. This

162-665: A fluorescent reporter, which permits detection only after hybridization of the probe with its complementary sequence. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments ( MIQE ) guidelines propose that the abbreviation qPCR be used for quantitative real-time PCR and that RT-qPCR be used for reverse transcription–qPCR. The acronym "RT-PCR" commonly denotes reverse transcription polymerase chain reaction and not real-time PCR, but not all authors adhere to this convention. Cells in all organisms regulate gene expression by turnover of gene transcripts (single stranded RNA ): The amount of an expressed gene in

243-411: A gel or Southern blot does not allow precise quantification. For example, over the 20–40 cycles of a typical PCR, the amount of DNA product reaches a plateau that is not directly correlated with the amount of target DNA in the initial PCR. Real-time PCR can be used to quantify nucleic acids by two common methods: relative quantification and absolute quantification. Absolute quantification gives

324-543: A pitch accent . In Modern Greek, all vowels and consonants are short. Many vowels and diphthongs once pronounced distinctly are pronounced as /i/ ( iotacism ). Some of the stops and glides in diphthongs have become fricatives , and the pitch accent has changed to a stress accent . Many of the changes took place in the Koine Greek period. The writing system of Modern Greek, however, does not reflect all pronunciation changes. The examples below represent Attic Greek in

405-450: A quencher of fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease activity of the Taq polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser. An increase in

486-528: A cell can be measured by the number of copies of an RNA transcript of that gene present in a sample. In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary. The polymerase chain reaction (PCR) is a common method for amplifying DNA; for RNA-based PCR the RNA sample is first reverse-transcribed to complementary DNA (cDNA) with reverse transcriptase . In order to amplify small amounts of DNA,

567-445: A certain genetic element, one can quantify the amount of the element in the sample prior to amplification. Using taxonomic markers (ribosomal genes) and qPCR can help determine the amount of microorganisms in a sample, and can identify different families, genera, or species based on the specificity of the marker. Using functional markers (protein-coding genes) can show gene expression within a community, which may reveal information about

648-429: A dilution of 1:2 results in a (C q ) difference of 1. The cycle threshold method makes several assumptions of reaction mechanism and has a reliance on data from low signal-to-noise regions of the amplification profile that can introduce substantial variance during the data analysis. To quantify gene expression, the (C q ) for an RNA or DNA from the gene of interest is subtracted from the (C q ) of RNA/DNA from

729-451: A few seconds in each cycle, with a temperature of, for example, 80 °C, in order to reduce the signal caused by the presence of primer dimers when a non-specific dye is used. The temperatures and the timings used for each cycle depend on a wide variety of parameters, such as: the enzyme used to synthesize the DNA, the concentration of divalent ions and deoxyribonucleotide triphosphates (dNTPs) in

810-412: A housekeeping gene in the same sample to normalize for variation in the amount and quality of RNA between different samples. This normalization procedure is commonly called the ΔC t -method and permits comparison of expression of a gene of interest among different samples. However, for such comparison, expression of the normalizing reference gene needs to be very similar across all the samples. Choosing

891-477: A lack of contemporaneous evidence. Several theories exist about what Hellenic dialect groups may have existed between the divergence of early Greek-like speech from the common Proto-Indo-European language and the Classical period. They have the same general outline but differ in some of the detail. The only attested dialect from this period is Mycenaean Greek , but its relationship to the historical dialects and

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972-419: A lesser degree. Pamphylian Greek , spoken in a small area on the southwestern coast of Anatolia and little preserved in inscriptions, may be either a fifth major dialect group, or it is Mycenaean Greek overlaid by Doric, with a non-Greek native influence. Regarding the speech of the ancient Macedonians diverse theories have been put forward, but the epigraphic activity and the archaeological discoveries in

1053-885: A negative-sense, single-stranded RNA molecule that encodes five viral proteins: polymerase L, matrix protein M, phosphoprotein P, nucleoprotein N, and glycoprotein G. Genomes are linear, around 11kb in length. Based on recent phylogenetic evidence, lyssaviruses have been categorized into seven major species . In addition, five more species have recently been discovered: West Caucasian bat virus, Aravan virus, Khujand virus, Irkut virus and Shimoni bat virus. The lyssavirus genus can be divided into four phylogroups based upon DNA sequence homology. Phylogroup I includes viruses, such as Rabies virus, Duvenhage virus, European bat lyssavirus types 1 and 2, Australian bat lyssavirus, Khujand virus, Bokeloh bat lyssavirus, Irkut virus, and Aravan virus. Phylogroup II contains Lagos bat virus, Mokola virus, and Shimoni bat virus. West Caucasian bat lyssavirus

1134-539: A pair of primers to carry out the amplification, which keeps costs down; multiple target sequences can be monitored in a tube by using different types of dyes. Fluorescent reporter probes detect only the DNA containing the sequence complementary to the probe; therefore, use of the reporter probe significantly increases specificity, and enables performing the technique even in the presence of other dsDNA. Using different-coloured labels, fluorescent probes can be used in multiplex assays for monitoring several target sequences in

1215-550: A prefix /e-/, called the augment . This was probably originally a separate word, meaning something like "then", added because tenses in PIE had primarily aspectual meaning. The augment is added to the indicative of the aorist, imperfect, and pluperfect, but not to any of the other forms of the aorist (no other forms of the imperfect and pluperfect exist). The two kinds of augment in Greek are syllabic and quantitative. The syllabic augment

1296-462: A real-time PCR machine and skilled workers with experience in molecular diagnostics. In an international evaluation a single TaqMan LN34 assay could detect Lyssavirus with high sensitivity (99.90%) across the genus and high specificity (99.68%) when compared to the DFA test. It will become the primary post-mortem rabies diagnostic test where possible. Classic rabies virus is prevalent throughout most of

1377-440: A reference gene fulfilling this criterion is therefore of high importance, and often challenging, because only very few genes show equal levels of expression across a range of different conditions or tissues. Although cycle threshold analysis is integrated with many commercial software systems, there are more accurate and reliable methods of analysing amplification profile data that should be considered in cases where reproducibility

1458-608: A separate historical stage, though its earliest form closely resembles Attic Greek , and its latest form approaches Medieval Greek . There were several regional dialects of Ancient Greek; Attic Greek developed into Koine. Ancient Greek was a pluricentric language , divided into many dialects. The main dialect groups are Attic and Ionic , Aeolic , Arcadocypriot , and Doric , many of them with several subdivisions. Some dialects are found in standardized literary forms in literature , while others are attested only in inscriptions. There are also several historical forms. Homeric Greek

1539-424: A single copy. Viruses can be present in humans due to direct infection or co-infections which makes diagnosis difficult using classical techniques and can result in an incorrect prognosis and treatment. The use of qPCR allows both the quantification and genotyping (characterization of the strain, carried out using melting curves) of a virus such as the hepatitis B virus . The degree of infection, quantified as

1620-630: A standard subject of study in educational institutions of the Western world since the Renaissance . This article primarily contains information about the Epic and Classical periods of the language, which are the best-attested periods and considered most typical of Ancient Greek. From the Hellenistic period ( c.  300 BC ), Ancient Greek was followed by Koine Greek , which is regarded as

1701-510: A vowel or /n s r/ ; final stops were lost, as in γάλα "milk", compared with γάλακτος "of milk" (genitive). Ancient Greek of the classical period also differed in both the inventory and distribution of original PIE phonemes due to numerous sound changes, notably the following: The pronunciation of Ancient Greek was very different from that of Modern Greek . Ancient Greek had long and short vowels ; many diphthongs ; double and single consonants; voiced, voiceless, and aspirated stops ; and

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1782-399: Is a concern. Mechanism-based qPCR quantification methods have also been suggested, and have the advantage that they do not require a standard curve for quantification. Methods such as MAK2 have been shown to have equal or better quantitative performance to standard curve methods. These mechanism-based methods use knowledge about the polymerase amplification process to generate estimates of

1863-570: Is a literary form of Archaic Greek (derived primarily from Ionic and Aeolic) used in the epic poems , the Iliad and the Odyssey , and in later poems by other authors. Homeric Greek had significant differences in grammar and pronunciation from Classical Attic and other Classical-era dialects. The origins, early form and development of the Hellenic language family are not well understood because of

1944-418: Is added to stems beginning with consonants, and simply prefixes e (stems beginning with r , however, add er ). The quantitative augment is added to stems beginning with vowels, and involves lengthening the vowel: Some verbs augment irregularly; the most common variation is e → ei . The irregularity can be explained diachronically by the loss of s between vowels, or that of the letter w , which affected

2025-431: Is also used by microbiologists working in the fields of food safety, food spoilage and fermentation and for the microbial risk assessment of water quality (drinking and recreational waters) and in public health protection. qPCR may also be used to amplify taxonomic or functional markers of genes in DNA taken from environmental samples. Markers are represented by genetic fragments of DNA or complementary DNA. By amplifying

2106-447: Is applied to rapidly detect nucleic acids that are diagnostic of, for example, infectious diseases , cancer and genetic abnormalities. The introduction of qualitative PCR assays to the clinical microbiology laboratory has significantly improved the diagnosis of infectious diseases, and is deployed as a tool to detect newly emerging diseases, such as new strains of flu and coronavirus , in diagnostic tests . Quantitative PCR

2187-666: Is called 'East Greek'. Arcadocypriot apparently descended more closely from the Mycenaean Greek of the Bronze Age. Boeotian Greek had come under a strong Northwest Greek influence, and can in some respects be considered a transitional dialect, as exemplified in the poems of the Boeotian poet Pindar who wrote in Doric with a small Aeolic admixture. Thessalian likewise had come under Northwest Greek influence, though to

2268-435: Is commonly used for both diagnostic and basic research . Uses of the technique in industry include the quantification of microbial load in foods or on vegetable matter, the detection of GMOs ( genetically modified organisms ) and the quantification and genotyping of human viral pathogens. Quantifying gene expression by traditional DNA detection methods is unreliable. Detection of mRNA on a northern blot or PCR products on

2349-448: Is considered by some linguists to have been closely related to Greek . Among Indo-European branches with living descendants, Greek is often argued to have the closest genetic ties with Armenian (see also Graeco-Armenian ) and Indo-Iranian languages (see Graeco-Aryan ). Ancient Greek differs from Proto-Indo-European (PIE) and other Indo-European languages in certain ways. In phonotactics , ancient Greek words could end only in

2430-403: Is easier to carry out as it does not require a calibration curve as the amount of the studied gene is compared to the amount of a control reference gene. As the units used to express the results of relative quantification are unimportant the results can be compared across a number of different RTqPCR. The reason for using one or more housekeeping genes is to correct non-specific variation, such as

2511-526: Is further work to be done in this area. Although bats evolved in the Palearctic, their origins antedate that of the lyssaviruses by millions of years, which argues against their co-speciation. The evolution rate in the N gene in the Africa 2 lineage has been estimated to be 3.75×10 substitutions per site per year. This rate is similar to that of other RNA viruses. Viral replication is cytoplasmic. Entry into

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2592-584: Is often used to estimate the expression level of a gene by visualizing the abundance of its mRNA transcript in a sample. In this method, purified RNA is separated by agarose gel electrophoresis , transferred to a solid matrix (such as a nylon membrane), and probed with a specific DNA or RNA probe that is complementary to the gene of interest. Although this technique is still used to assess gene expression, it requires relatively large amounts of RNA and provides only qualitative or semi quantitative information of mRNA levels. Estimation errors arising from variations in

2673-527: Is selected for its almost constant level of expression. These genes are often selected from housekeeping genes as their functions related to basic cellular survival normally imply constitutive gene expression . This enables researchers to report a ratio for the expression of the genes of interest divided by the expression of the selected normalizer, thereby allowing comparison of the former without actually knowing its absolute level of expression. The most commonly used normalizing genes are those that code for

2754-443: Is the only virus that is a part of phylogroup III. Ikoma lyssavirus and Lleida bat lyssavirus are examples in phylogroup IV. West Caucasian bat lyssavirus was classified within its own phylogroup because it is the most divergent lyssavirus that has been discovered. Phylogenetic studies suggest that the original hosts of these viruses were bats. However, the recent discovery of lyssavirus sequences from amphibians and reptiles challenges

2835-444: Is therefore necessary to carry out an initial statistically sound methodological study in order to select the most suitable reference gene. A number of statistical algorithms have been developed that can detect which gene or genes are most suitable for use under given conditions. Those like geNORM or BestKeeper can compare pairs or geometric means for a matrix of different reference genes and tissues . Diagnostic qualitative PCR

2916-400: Is typical of plant-infecting viruses. Virions of human-infecting viruses more commonly have cubic symmetry and take shapes approximating regular polyhedra . The structure consists of a spiked outer envelope , a middle region consisting of matrix protein M, and an inner ribonucleocapsid complex region, consisting of the genome associated with other proteins. Lyssavirus genomes consist of

2997-457: Is usually evaluated at a distinct end point. The technique therefore usually provides more rapid results and/or uses fewer reactants than electrophoresis. If subsequent electrophoresis is required it is only necessary to test those samples that real time PCR has shown to be doubtful and/or to ratify the results for samples that have tested positive for a specific determinant. Unlike end point PCR (conventional PCR), real time PCR allows monitoring of

3078-708: The Greek region of Macedonia during the last decades has brought to light documents, among which the first texts written in Macedonian , such as the Pella curse tablet , as Hatzopoulos and other scholars note. Based on the conclusions drawn by several studies and findings such as Pella curse tablet , Emilio Crespo and other scholars suggest that ancient Macedonian was a Northwest Doric dialect , which shares isoglosses with its neighboring Thessalian dialects spoken in northeastern Thessaly . Some have also suggested an Aeolic Greek classification. The Lesbian dialect

3159-434: The direct fluorescent antibody (DFA) test is still the gold standard to detect lyssavirus infection. Since the new millennium reverse transcription PCR (RT-PCR) tests have been developed for rabies but only been used as a confirmatory test. Real-time PCR -based tests which have higher sensitivity and objective diagnostic thresholds and allow samples to be stored at room temperature have been promising since 2005, but require

3240-501: The present , future , and imperfect are imperfective in aspect; the aorist , present perfect , pluperfect and future perfect are perfective in aspect. Most tenses display all four moods and three voices, although there is no future subjunctive or imperative. Also, there is no imperfect subjunctive, optative or imperative. The infinitives and participles correspond to the finite combinations of tense, aspect, and voice. The indicative of past tenses adds (conceptually, at least)

3321-1031: The 5th century BC. Ancient pronunciation cannot be reconstructed with certainty, but Greek from the period is well documented, and there is little disagreement among linguists as to the general nature of the sounds that the letters represent. /oː/ raised to [uː] , probably by the 4th century BC. Greek, like all of the older Indo-European languages , is highly inflected. It is highly archaic in its preservation of Proto-Indo-European forms. In ancient Greek, nouns (including proper nouns) have five cases ( nominative , genitive , dative , accusative , and vocative ), three genders ( masculine , feminine , and neuter ), and three numbers (singular, dual , and plural ). Verbs have four moods ( indicative , imperative , subjunctive , and optative ) and three voices (active, middle, and passive ), as well as three persons (first, second, and third) and various other forms. Verbs are conjugated through seven combinations of tenses and aspect (generally simply called "tenses"):

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3402-495: The Archaic period of ancient Greek (see Homeric Greek for more details): Μῆνιν ἄειδε, θεά, Πηληϊάδεω Ἀχιλῆος οὐλομένην, ἣ μυρί' Ἀχαιοῖς ἄλγε' ἔθηκε, πολλὰς δ' ἰφθίμους ψυχὰς Ἄϊδι προΐαψεν ἡρώων, αὐτοὺς δὲ ἑλώρια τεῦχε κύνεσσιν οἰωνοῖσί τε πᾶσι· Διὸς δ' ἐτελείετο βουλή· ἐξ οὗ δὴ τὰ πρῶτα διαστήτην ἐρίσαντε Ἀτρεΐδης τε ἄναξ ἀνδρῶν καὶ δῖος Ἀχιλλεύς. The beginning of Apology by Plato exemplifies Attic Greek from

3483-691: The Classical period of ancient Greek. (The second line is the IPA , the third is transliterated into the Latin alphabet using a modern version of the Erasmian scheme .) Ὅτι [hóti Hóti μὲν men mèn ὑμεῖς, hyːmêːs hūmeîs,   Real-time PCR Two common methods for the detection of PCR products in real-time PCR are (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with

3564-530: The DNA of the pathogen and the plant is based on the amplification of ITS sequences, spacers located in ribosomal RNA gene's coding area, which are characteristic for each taxon. Field-based versions of this technique have also been developed for identifying the same pathogen. qPCR using reverse transcription (RT-qPCR) can be used to detect GMOs given its sensitivity and dynamic range in detecting DNA. Alternatives such as DNA or protein analysis are usually less sensitive. Specific primers are used that amplify not

3645-444: The DNA template; the third, at between 68 and 72 °C, facilitates the polymerization carried out by the DNA polymerase. Due to the small size of the fragments the last step is usually omitted in this type of PCR as the enzyme is able to replicate the DNA amplicon during the change between the alignment stage and the denaturing stage. In addition, in four-step PCR the fluorescence is measured during short temperature phases lasting only

3726-550: The aorist. Following Homer 's practice, the augment is sometimes not made in poetry , especially epic poetry. The augment sometimes substitutes for reduplication; see below. Almost all forms of the perfect, pluperfect, and future perfect reduplicate the initial syllable of the verb stem. (A few irregular forms of perfect do not reduplicate, whereas a handful of irregular aorists reduplicate.) The three types of reduplication are: Irregular duplication can be understood diachronically. For example, lambanō (root lab ) has

3807-419: The augment when it was word-initial. In verbs with a preposition as a prefix, the augment is placed not at the start of the word, but between the preposition and the original verb. For example, προσ(-)βάλλω (I attack) goes to προσ έ βαλoν in the aorist. However compound verbs consisting of a prefix that is not a preposition retain the augment at the start of the word: αὐτο(-)μολῶ goes to ηὐ τομόλησα in

3888-438: The center of Greek scholarship, this division of people and language is quite similar to the results of modern archaeological-linguistic investigation. One standard formulation for the dialects is: West vs. non-West Greek is the strongest-marked and earliest division, with non-West in subsets of Ionic-Attic (or Attic-Ionic) and Aeolic vs. Arcadocypriot, or Aeolic and Arcado-Cypriot vs. Ionic-Attic. Often non-West

3969-429: The copies of the viral genome per unit of the patient's tissue, is relevant in many cases; for example, the probability that the type 1 herpes simplex virus reactivates is related to the number of infected neurons in the ganglia . This quantification is carried out either with reverse transcription or without it, as occurs if the virus becomes integrated in the human genome at any point in its cycle, such as happens in

4050-419: The desired product at any point in the amplification process by measuring fluorescence (in real time frame, measurement is made of its level over a given threshold). A commonly employed method of DNA quantification by real-time PCR relies on plotting fluorescence against the number of cycles on a logarithmic scale . A threshold for detection of DNA-based fluorescence is set 3–5 times of the standard deviation of

4131-615: The dialect of Sparta ), and Northern Peloponnesus Doric (including Corinthian ). All the groups were represented by colonies beyond Greece proper as well, and these colonies generally developed local characteristics, often under the influence of settlers or neighbors speaking different Greek dialects. After the conquests of Alexander the Great in the late 4th century BC, a new international dialect known as Koine or Common Greek developed, largely based on Attic Greek , but with influence from other dialects. This dialect slowly replaced most of

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4212-399: The differences in the quantity and quality of RNA used, which can affect the efficiency of reverse transcription and therefore that of the whole PCR process. However, the most crucial aspect of the process is that the reference gene must be stable. The selection of these reference genes was traditionally carried out in molecular biology using qualitative or semi-quantitative studies such as

4293-423: The dye used is usually SYBR Green. The DNA melting temperature is specific to the amplified fragment. The results of this technique are obtained by comparing the dissociation curves of the analysed DNA samples. Unlike conventional PCR, this method avoids the previous use of electrophoresis techniques to demonstrate the results of all the samples. This is because, despite being a kinetic technique, quantitative PCR

4374-407: The efficiency of amplification is often variable among primers and templates. Therefore, the efficiency of a primer-template combination is assessed in a titration experiment with serial dilutions of DNA template to create a standard curve of the change in (C q ) with each dilution. The slope of the linear regression is then used to determine the efficiency of amplification, which is 100% if

4455-413: The environment. The agricultural industry is constantly striving to produce plant propagules or seedlings that are free of pathogens in order to prevent economic losses and safeguard health. Systems have been developed that allow detection of small amounts of the DNA of Phytophthora ramorum , an oomycete that kills oaks and other species, mixed in with the DNA of the host plant. Discrimination between

4536-577: The exact number of target DNA molecules by comparison with DNA standards using a calibration curve . It is therefore essential that the PCR of the sample and the standard have the same amplification efficiency . Relative quantification is based on internal reference genes to determine fold-differences in expression of the target gene. The quantification is expressed as the change in expression levels of mRNA interpreted as complementary DNA (cDNA, generated by reverse transcription of mRNA). Relative quantification

4617-445: The following molecules: tubulin , glyceraldehyde-3-phosphate dehydrogenase , albumin , cyclophilin , and ribosomal RNAs . Real-time PCR is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of at least one specified wavelength and detect the fluorescence emitted by the excited fluorophore . The thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of

4698-673: The forms of the Greek language used in ancient Greece and the ancient world from around 1500 BC to 300 BC. It is often roughly divided into the following periods: Mycenaean Greek ( c.  1400–1200 BC ), Dark Ages ( c.  1200–800 BC ), the Archaic or Epic period ( c.  800–500 BC ), and the Classical period ( c.  500–300 BC ). Ancient Greek was the language of Homer and of fifth-century Athenian historians, playwrights, and philosophers . It has contributed many words to English vocabulary and has been

4779-561: The historical Dorians . The invasion is known to have displaced population to the later Attic-Ionic regions, who regarded themselves as descendants of the population displaced by or contending with the Dorians. The Greeks of this period believed there were three major divisions of all Greek people – Dorians, Aeolians, and Ionians (including Athenians), each with their own defining and distinctive dialects. Allowing for their oversight of Arcadian, an obscure mountain dialect, and Cypriot, far from

4860-476: The historical circumstances of the times imply that the overall groups already existed in some form. Scholars assume that major Ancient Greek period dialect groups developed not later than 1120 BC, at the time of the Dorian invasions —and that their first appearances as precise alphabetic writing began in the 8th century BC. The invasion would not be "Dorian" unless the invaders had some cultural relationship to

4941-533: The host cell is achieved by attachment of the viral G glycoproteins to host receptors, which mediates clathrin-mediated endocytosis . Replication follows the negative stranded RNA virus replication model. Negative stranded RNA virus transcription, using polymerase stuttering , is the method of transcription. The virus exits the host cell by budding and by tubule-guided viral movement. Wild mammals, especially bats and certain carnivores, serve as natural hosts. Transmission routes are typically via bite wounds. As of 2018

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5022-401: The intended target sequence. In real-time PCR with dsDNA dyes the reaction is prepared as usual, with the addition of fluorescent dsDNA dye. Then the reaction is run in a real-time PCR instrument , and after each cycle, the intensity of fluorescence is measured with a detector; the dye only fluoresces when bound to the dsDNA (i.e., the PCR product). This method has the advantage of only needing

5103-502: The mammalian origin of lyssaviruses. The greater antigenic diversity of lyssaviruses from Africa has led to the assumption that Africa was the origin of these viruses. An examination of 153 viruses collected between 1956 and 2015 from various geographic locations has instead suggested a Palearctic origin (85% likelihood) for these viruses. Date estimates (95% likelihood) for the most recent common ancestor were very broad – between 3,995 and 166,820 years before present – which suggests there

5184-508: The older dialects, although the Doric dialect has survived in the Tsakonian language , which is spoken in the region of modern Sparta. Doric has also passed down its aorist terminations into most verbs of Demotic Greek . By about the 6th century AD, the Koine had slowly metamorphosed into Medieval Greek . Phrygian is an extinct Indo-European language of West and Central Anatolia , which

5265-433: The original sample concentration. An extension of this approach includes an accurate model of the entire PCR reaction profile, which allows for the use of high signal-to-noise data and the ability to validate data quality prior to analysis. According to research of Ruijter et al. MAK2 assumes constant amplification efficiency during the PCR reaction. However, theoretical analysis of polymerase chain reaction, from which MAK2

5346-487: The perfect stem eilēpha (not * lelēpha ) because it was originally slambanō , with perfect seslēpha , becoming eilēpha through compensatory lengthening. Reduplication is also visible in the present tense stems of certain verbs. These stems add a syllable consisting of the root's initial consonant followed by i . A nasal stop appears after the reduplication in some verbs. The earliest extant examples of ancient Greek writing ( c.  1450 BC ) are in

5427-399: The physicochemical properties of the nucleic acids and DNA polymerase . The PCR process generally consists of a series of temperature changes that are repeated 25–50 times. These cycles normally consist of three stages: the first, at around 95 °C, allows the separation of the nucleic acid's double chain; the second, at a temperature of around 50–60 °C, allows the binding of the primers with

5508-472: The presence and abundance of a particular DNA sequence in these samples. This measurement is made after each amplification cycle, and this is the reason why this method is called real time PCR (that is, immediate or simultaneous PCR). Quantitative PCR and DNA microarray are modern methodologies for studying gene expression . Older methods were used to measure mRNA abundance: differential display , RNase protection assay and northern blot . Northern blotting

5589-452: The product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter. Real-time PCR permits the identification of specific, amplified DNA fragments using analysis of their melting temperature (also called T m value, from m elting t emperature ). The method used is usually PCR with double-stranded DNA-binding dyes as reporters and

5670-421: The quantification method can be the result of DNA integrity, enzyme efficiency and many other factors. For this reason a number of standardization systems (often called normalization methods ) have been developed. Some have been developed for quantifying total gene expression, but the most common are aimed at quantifying the specific gene being studied in relation to another gene called a normalizing gene, which

5751-642: The reaction and the bonding temperature of the primers. Real-time PCR technique can be classified by the chemistry used to detect the PCR product, specific or non-specific fluorochromes. A DNA-binding dye binds to all double-stranded (ds) DNA in PCR, increasing the fluorescence quantum yield of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity measured at each cycle. However, dsDNA dyes such as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as primer dimer ). This can potentially interfere with, or prevent, accurate monitoring of

5832-448: The required wavelength allowing the generation rate to be measured for one or more specific products. This allows the rate of generation of the amplified product to be measured at each PCR cycle. The data thus generated can be analysed by computer software to calculate relative gene expression (or mRNA copy number ) in several samples. Quantitative PCR can also be applied to the detection and quantification of DNA in samples to determine

5913-410: The same methodology is used as in conventional PCR using a DNA template, at least one pair of specific primers , deoxyribonucleotide triphosphates, a suitable buffer solution and a thermo-stable DNA polymerase . A substance marked with a fluorophore is added to this mixture in a thermal cycler that contains sensors for measuring the fluorescence of the fluorophore after it has been excited at

5994-435: The same tube. The specificity of fluorescent reporter probes also prevents interference of measurements caused by primer dimers , which are undesirable potential by-products in PCR. However, fluorescent reporter probes do not prevent the inhibitory effect of the primer dimers, which may depress accumulation of the desired products in the reaction. The method relies on a DNA-based probe with a fluorescent reporter at one end and

6075-513: The signal noise above background. The number of cycles at which the fluorescence exceeds the threshold is called the threshold cycle (C t ) or, according to the MIQE guidelines, quantification cycle (C q ) . During the exponential amplification phase, the quantity of the target DNA template (amplicon) doubles every cycle. For example, a DNA sample whose C q precedes that of another sample by 3 cycles contained 2 = 8 times more template. However,

6156-517: The syllabic script Linear B . Beginning in the 8th century BC, however, the Greek alphabet became standard, albeit with some variation among dialects. Early texts are written in boustrophedon style, but left-to-right became standard during the classic period. Modern editions of ancient Greek texts are usually written with accents and breathing marks , interword spacing , modern punctuation , and sometimes mixed case , but these were all introduced later. The beginning of Homer 's Iliad exemplifies

6237-408: The transgene but the promoter , terminator or even intermediate sequences used during the process of engineering the vector. As the process of creating a transgenic plant normally leads to the insertion of more than one copy of the transgene its quantity is also commonly assessed. This is often carried out by relative quantification using a control gene from the treated species that is only present as

6318-415: The visual examination of RNA gels, northern blot densitometry or semi-quantitative PCR (PCR mimics). Now, in the genome era, it is possible to carry out a more detailed estimate for many organisms using transcriptomic technologies . However, research has shown that amplification of the majority of reference genes used in quantifying the expression of mRNA varies according to experimental conditions. It

6399-517: The world and can be carried by any warm blooded mammal. The other lyssaviruses have much less diversity in carriers. Only select hosts can carry each of these viral species . Also, these other species are particular only to a specific geographic area. Bats are known to be an animal vector for all identified lyssaviruses except the Mokola virus . Ancient Greek Ancient Greek ( Ἑλληνῐκή , Hellēnikḗ ; [hellɛːnikɛ́ː] ) includes

6480-480: Was Aeolic. For example, fragments of the works of the poet Sappho from the island of Lesbos are in Aeolian. Most of the dialect sub-groups listed above had further subdivisions, generally equivalent to a city-state and its surrounding territory, or to an island. Doric notably had several intermediate divisions as well, into Island Doric (including Cretan Doric ), Southern Peloponnesus Doric (including Laconian ,

6561-401: Was derived, has revealed that amplification efficiency is not constant throughout PCR. While MAK2 quantification provides reliable estimates of target DNA concentration in a sample under normal qPCR conditions, MAK2 does not reliably quantify target concentration for qPCR assays with competimeters. There are numerous applications for quantitative polymerase chain reaction in the laboratory . It

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