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The ultimate goal: Sequencing DNA
Following his success in sequencing RNA, Sanger began to turn his attention to how he might sequence DNA. This he saw as his 'ultimate goal'. Before his sequencing of RNA this had seemed an impossible project. One of the major challenges with DNA was its size and the fact that there were no small DNA entities comparable to tRNA or 5S ribosomal RNA on which he could develop a sequencing method. So far the only smallest form of DNA available was from the single-stranded bacteriophage phi X174. This had been purified in 1959 by Robert Sinsheimer at the California Institute of Technology. A year later some more DNA was purified from the lambda bacteriophage by Dale Kaiser and David Hogness at Stanford University. DNA sourced from bacteriophages was thought to be made up of approximately 5,000 nucleotides. This contrasted with other more interesting DNA which were thought to contain millions of nucleotides (Sinsheimer, 1959; Kaiser, Hogness, 1960; Sanger, Dowding, 1996).
In addition to the absence of any suitable DNA to experiment on, Sanger did not have access to any suitable enzyme to cut DNA. When working on RNA he had been greatly helped by the availability of ribonuclease T1 which cut the nucleic acid at very precise points. While scientists had discovered a number of enzymes for cutting DNA, deoxyribonucleases (DNAase), none of them had so far proved a reliable tool. (Sanger, Dowding, 1996).
In 1964 Sanger welcomed into his team Ken Murray, a British postdoctoral biochemist who soon joined in his efforts to find a way to sequence DNA. Early on Murray developed a two-dimensional fractionation technique for determining the sequence of tetranucelotides, a particular block of nucleotides made up of four base pairs (adenine (A) and thymine (T), cytosine (C) and guanine (G)). He obtained the tetranucleotides from DNA that he had partially digested with various enzymes. Yet, while Murray had managed to sequence the tetranucleotides, his method had a major downside: it could not pinpoint where on the original DNA nucleotide chain the tetranucleotides were located. This posed a major hurdle in terms of sequencing whole DNA (Murray, 1970; Finch, 2008).
Ken Murray. Credit: Geoffrey Argent Studio. The son of a Yorkshire miner and school caretaker, Murray had been forced to leave school at the age of 16 because, despite having a strong academic track record, his family could not financially support his higher education. Thereafter he worked as a laboratory assistant at Boots the Chemist and then at an outstation of Glaxo. The pharmaceutical company subsequently gave him a scholarship to undertake a part-time chemistry degree at the University of Birmingham. Murray subsequently did a doctorate and then joined J Murray Luck at Stanford University purifying and characterising histones, a group of proteins closely associated with DNA. Found in the nuclei of eukaryotic cells, histones play an important role in the packaging and ordering of DNA into structural units called nucleosomes and help in the regulation of genes. Murray's source for these histones was thymus glands from calves (Brammar, Gratzer, 2014).
In addition to Murray, Sanger had the assistance of his technician Bart Barrell and of Alan Coulson, another technician who joined him in 1967. Together they began looking for ways to improve the sequencing method. So far Sanger's approach had been to break down a large molecule into smaller fragments, using enzymes, and then to break these fragments down further and piece together the sequence from the fragments' overlaps. While this degradation technique had proven effective for sequencing insulin and short strands of RNA, it did not seem practical for sequencing such a large molecule as DNA.
Bart Barrell, c.1975. Credit: Bart Barrell, Laboratory of Molecular Biology (LMB). Barrell started working as Sanger's personal assistant in 1964 when he was 18 years old, just after leaving school. He developed a passion for natural history from an early age, and became fascinated by molecular biology while still at school. Before leaving school he presented a lecture on the structure of proteins and nucleic acids to the School Scientific Society. Barrell had turned down a County Major Scholarship to attend university because he wanted to start work in science immediately. Quickly impressed with Barrell's enthusiasm and enterprise, Sanger often discussed his ideas with him for advancing his sequencing methods. Barrell soon developed an expertise in sequencing and analysis and was entrusted by Sanger to apply it on ever more challenging molecules, leaving Sanger the freedom to dedicate his time to advancing the technology.
Alan Coulson, 1987. Credit: Alan Coulson, LMB. Coulson joined Sanger in 1967 at the age of 20, shortly after completing a higher national diploma at Leicester Polytechnic. Sanger was very impressed with Coulson's skills and regarded him as his main collaborator in the laboratory, describing him as 'something of a wizard with sequences' (Sanger, 1988). They were both self-effacing and quiet so were well-matched in terms of their temperaments. Coulson carried out most of the work involved in growing bacteria in culture for multiplying bacteriohages, extracting their genetic material and producing radiolabelled DNA fragments, and was responsible for running these on the acrylamide gels to produce autoradiographs. Following Sanger's retirement, Coulson joined John Sulston and helped in the sequencing of the nematode worm C. elegans.
A possible new tool: DNA polymerase
For inspiration, Sanger looked to the work of Ray Wu and A Dale Kaiser, two molecular biologists based respectively at Cornell and Stanford Universities who had partially sequenced DNA from a lambda bacteriophage in 1968. What had helped them achieve this feat was the fact that this bacteriophage's DNA was a linear molecule with two single-stranded cohesive DNA fragments at the end. Wu and Kaiser had managed to sequence 12 nucleotides at the end of the two end DNA fragments. This was the first piece of DNA to have ever been sequenced. Wu and Kaiser's method involved a number of steps. Many of them were reminiscent of Sanger's own techniques. For example, they used the radioactive 32P isotope to label some of their nucleotides. They also broke the DNA molecule down with an enzyme. Analysis of the sequences was also carried out with the aid of ionophoresis on paper, and a two-dimensional electrophoresis method (Wu, Kaiser, 1968).
Ray Wu. Credit: Cornell University. Chinese born, Wu spent a year working with Kaiser in his laboratory at Stanford University between 1965 and 1966. He took the lead in developing the technique sequencing the lambda bacteriophage's DNA. Click here for more on Wu's work and life.
Armin Dale Kaiser. Credit: Dale Kaiser. Kaiser began working on the genetics of the lambda bacteriophages while doing a doctorate at California Institute of Technology, completed in 1955. He joined Stanford University in 1959, where he helped establish a new biochemistry department. Sanger would have become familiar with Kaiser between 1964 and 1965, because Kaiser spent a year in the LMB as a postdoctoral research fellow working alongside Hugh Huxley and Aaron Klug.
What was novel about Wu and Kaiser's method was that they made use of an enzyme known as DNA polymerase. First discovered in 1955, this enzyme is instrumental in DNA replication. In this process an enzyme, known as helicase, unwinds a double-strand of DNA into two single-strands to serve as templates. Following this, the polymerase combines the template strand with another strand of nucleotides, known as a primer, to generate a new double-strand of DNA. Both the template and primer are vital to this process, because the enzyme cannot perform synthesis independently (see figure 1).
Figure 1: Diagram showing the role of DNA polymerase in the duplication of DNA.
Wu and Kaiser had used the polymerase to generate small fragments of radiolabelled DNA (Wu, Kaiser, 1968). While intrigued by Wu and Kaiser's use of polymerase, Sanger had some reservations about adopting their method. Firstly, it entailed a lot of 'tedious' steps. Secondly, he doubted whether it could be used to sequence any DNA stretches longer than those found at the end of the lambda bacteriophage's genome (Sanger, 2001).
In addition to Wu and Kaiser's technique, Sanger began to look at one developed by Charles Weissmann and some colleagues at Zurich and Bristol University who had sequenced 175 nucleotides in RNA from the Qß bacteriophage. They did this by using a polymerase contained in the bacteriophage's own RNA. This polymerase had the advantage that it could synthesise complementary copies of the single-stranded RNA chain. With this the team were able to obtain homogeneous preparations of short, radioactive Qß RNA segments of any desired length. Weismann's group had also pioneered a short-term radioactive labelling technique (known as pulse-labelling) to produce radioactively labelled nucleotides, which they had used to deduce the RNA sequence. What was appealing about the team's work was that it offered an alternative approach to partial hydrolysis (Billeter, Dahlberg, et al, 1969; Sanger, 1980; Finch, 2008).
Soon after exploring the two methods, Sanger went to Sydney, Australia where he paid a short visit to the laboratory of Geoff Grigg, an Australian biochemist. One conversation he was to have in this laboratory was to prove invaluable to his thinking. This took place between himself, Grigg and John Griffiths, a British mathematician who had worked with Crick and was now spending a sabbatical in Grigg's laboratory. The discussion centred on a paper published by Paul Berg in 1969, which demonstrated how DNA polymerase I could be used to insert ribonucleotide triphosphates, RNA precursors, into a growing nascent DNA chain if magnesium was substituted with manganese in the polymerising reaction. This provided a means to substitute a specific deoxynucleotide with a specific ribonucleotide in the DNA chain. Such substitution opened up the possibility of cleaving a DNA chain at very specific points with an enzyme. If the method worked, Sanger would be able to obtain small enough DNA fragments with particular nucleotides on their end that would allow him to use the same sequencing methods as he had used with RNA. The key issue was how to obtain an appropriate template to kick-start the process. Griffiths suggested one solution might be to synthesise a primer from scratch. This primer would provide a tool to copy a DNA template. He hypothesised the primer could be just 6 to 10 nucleotides long (Grigg, 2001).
Once back in England, Sanger began looking for a way to implement Griffiths' idea. The first challenge was deciding which sequence to synthesise. For inspiration, he turned his attention to some work that he and his team were carrying out on a filamentous bacteriophage (f1 phage). They had already determined the amino acid sequence in its coat protein. It was made up of just two amino acids, methionine and tryptophan. These were known to correspond with a unique nucleotide sequence which was easy to figure out. It was this sequence that Sanger decided to synthesise (Sanger, 1992).
The next hurdle was to synthesise a primer to match the DNA template from the f1 phage. This was important because polymerase cannot work without the presence of both a template and primer. Nucleotide synthesis, however, was extremely difficult. Up to now the only person to have achieved this feat was Har Gobind Khorana at the Massachusetts Institute of Technology (MIT). Click here to find out more about Khorana.
Not possessing the necessary chemical expertise, Sanger invited Hans Kossel, a German molecular biologist from the University of Freiburg, whom he met by chance at a conference, to join him at the LMB to collaborate on the project. Kossel had spent some time working alongside Khorana on nucleotide synthesis and had independently been toying with the idea of making a similar sequence to the one proposed by Sanger (Sanger, 1988; Sanger, 1992).
Hans Kossel. Credit: Raina Maier. Between between 1964 and 1967 Kossel worked in the laboratory of Khorana at the University of Wisconsin on the elucidation of the genetic code. Thereafter he worked in the newly founded Institute of Biology, Freiberg, focusing on the enzymatic hydrolysis of tRNAs and on the synthesis of oligonucleotides. He was one of the first scientists to suggest the possibility of enzymatic synthesis of DNA as a DNA sequencing method. Sanger invited Kossel to work on these areas with him, which he did for a short period in 1971 and 1973.
It took Kossel and his colleagues over a year of hard work to make the primer. The primer was eight nucleotides long (octanucleotide). Sanger and his team mixed the primer with the f1 DNA template, DNA polymerase I and some radioactive nucleoside triphosphates. This they did with the help of John Donelson, an American postdoctoral biochemist researcher. Donelson had arrived in Sanger's laboratory in 1970. He was particularly suited to the task, having spent some time working with Wu at Cornell University (Donelson, Wu, 1971).
To the team's delight the octanucleotide proved an efficient primer. Much to their surprise, however, it did not replicate the DNA sequence found in the phage's coat protein. Despite this setback, the primer proved sufficiently specific for Sanger's purposes. Importantly, it provided a suitably sized piece of radioactive DNA to start testing different sequencing techniques without the need to carry out partial hydrolysis (Sanger, 1992).
In 1973 Sanger and his team published a paper that indicated that they had sequenced 50 nucleotides in the f1 DNA. The sequencing had been determined with the two-dimensional homochromatography method Sanger had devised for sequencing 5S ribosomal RNA and another two-dimensional fractionation system, known as the wandering spot method. Developed by Victor Ling, another member of Sanger's team, the wandering spot technique made it possible to read off a sequence based on the relative position of specific spots (Sanger, Dowding, 1996).
To do such sequencing, the team had partially cut up the synthesised DNA fragments with the enzyme pancreatic ribonuclease. This had cleaved the molecule at specific points where the ribonucleotide cytosine was found, providing fragments which all ended with this ribonucleotide. The sequencing was then done by piecing together the fragments' overlaps. Sanger had used the same approach for both insulin and RNA (Sanger, Donelson, Coulson, et al, 1973; Sanger, 1992; Sanger, Dowding, 1996).
The sequencing of f1 DNA was a major step forward. Not only had Sanger managed to sequence a significantly longer piece of DNA than anyone else so far, he had demonstrated the possibility of using polymerase and the ribosubstitution method for DNA sequencing. Nonetheless, determining the sequence had involved many different steps and was hard-work. The need to carry out ribosubstitution would soon be made redundant with the availability of restriction enzymes from 1970. Such enzymes recognised and cleaved DNA at specific nucleotide sequences. This meant that scientists could now easily cut up large DNA molecules into fragments with specific ends that could function as a starting point for sequencing (Hutchison, 2007).
This shows the different separation steps involved in the sequencing of f1 DNA. Credit: Sanger, Donelson, Coulson et al, 1973, figure 3.
The plus/minus technique
While Sanger and his team had managed to sequence a substantial portion of the f1 DNA, they had not completed its whole sequence. Moreover, their method was time-consuming and laborious. Eager to find a more rapid technique that would allow him to determine larger DNA sequences, Sanger continued looking for new avenues to tackle the problem.
In 1975 Sanger and his assistant Coulson published a paper (Sanger, Coulson, 1975) in which they outlined an approach radically different from the one before. What was novel about their technique was that was that it was not reliant on piecing together a sequence based on the examination of the overlaps in small DNA fragments. Furthermore, it enabled the direct visual scanning of a sequence. Sanger and Coulson had not developed the method overnight. Rather it gradually emerged as a result of some experiments, launched in April 1973, designed to test the action of different polymerases and the efficiency of different fractionation techniques (Sanger, Dowding, 1996; Garcia-Sancho, 2010).
These notes are from experiment D80. It was one of Sanger's first experiments where he began testing the possibility of copying with all four deoxytriphosphates. Sanger's DNA laboratory notebook, 1972. Credit: Wellcome Library, file SA//BIO, P/1/42.
In this note Sanger records starting 'a fairly ambitious experiment'. The aim of the experiment, labelled D93, was to find a way of extending DNA fragments, cleaved with a restriction enzyme, by the addition of all four of DNA's nucleotides, which were then to be separated with ionophoresis on gels and their sequences analysed. Results from this experiment were fairly promising. Sanger would continue to refine the method used in this experiment with the aid of Coulson and Barrell. The experiment laid the foundation for the plus and minus method. Sanger's DNA laboratory notebook, 1973-1974. Credit: Wellcome Library, file SA//BIO, P/1/43.
Eventually called the 'plus/minus' system, the technique consisted of a number of different steps. In the first instance a primer was synthesised. This was a complementary copy of a short length of the DNA sample under study. Once made, the primer was bound to a specific complementary region on a single-stranded DNA template so as to provide a starting point for DNA synthesis. The combined strands were then incubated with polymerase I (sourced from Escherichia-coli) so that it could add radioactively labelled nucleotides on to the 3' end of the primer. This process provided a random assortment of synthesised DNA fragments of varying lengths. Each was purified to remove excess nucleotides.
Following purification the samples were divided between two treatments. The first treatment, known as the 'minus' system, drew on the copying method devised by Wu and Kaiser. In this case the sample was incubated in a test tube with DNA polymerase I (sourced from Escherichia coli) together with just three out of four of DNA nucleotides (A, C, G, or T). One nucleotide was deliberately withheld from this incubation process. This took advantage of the natural biological process, whereby polymerase will carry on synthesising a DNA chain until it encounters a missing nucleotide. In the case, for example, where adenine is absent, polymerase will synthesise a string of nucleotides, coming to a halt just before this nucleotide. By leaving out a particular nucleotide, one at a time, the team were able to obtain DNA products with specific nucleotide endings.
In contrast to the 'minus' system, which relied on the removal of one specific nucleotide, the second treatment, the 'plus' system relied on the addition of one during the incubation process. Developed by Paul Englund between 1971 and 1972, the 'plus' system made use of a different polymerase, T4. This was sourced from the T4 bacteriophage. In normal circumstances this polymerase acts as a synthesising reagent. However, it will start to downgrade a DNA strand from its 3' end when it encounters an additional nucleotide. As in the case of the 'minus' system, the 'plus' system produced DNA fragments with specific nucleotide endings.
Coulson's note recording tests with T4 polymerase. Credit: Wellcome Library, Coulson papers, file: PP/COU, Notebook 'Plus and minus sequencing, T4 polymerase preparations', 1976.
Both systems relied on the preparation of four test tube reactions with the two different types of polymerase. Following treatment, the primers were separated from the template. This provided eight different DNA strands. Four of these came from the template, known as minus strands, and four came from the primer, known as plus strands. The end result was a series of overlapping fragments with each of the nucleotide endings (A, C, G, T).
Each DNA fragment was then placed alongside each other in separate lanes on acrylamide gel ready for ionophoresis. Following the application of an electrical charge the DNA fragments would migrate from the top to the bottom of the gel, with the bigger fragments moving slower than the smaller ones. In this context the gel acted like a sieve which helped sort out the fragments by their size. Once this was completed, an X-ray film was overlaid on the gel for a period of time, usually overnight, and then developed to produce an autoradiograph, which revealed the position of the radioactive tagged nucleotides in the fragments. The final image showed a series of dark bands in a ladder-line pattern which highlighted where the fragments overlapped (Hutchison, 2007; Finch, 2008).
The use of acrylamide gel for sequence analysis marked a significant change. Previously Sanger had typically carried out such work through the use of two-dimensional paper chromatography. This, however, was time-consuming because it involved lots of cutting out of bands. One of the attractions in using the acrylamide gel system was that it provided a one-dimensional reading of a sequence. The decision to use gels for fractionating DNA had initially seemed a crazy idea to Sanger. While gels were commonly used to separate intact proteins as well as DNA and DNA fragments, they had never before been used to separate individual nucleic acids (Sanger, 1992).
This shows John Donelson who played a pivotal role in the development of the acrylamide gel system for the plus and minus system. Credit: John Donelson.
When starting to develop the plus and minus method Sanger and Coulson largely carried out fractionation with homochromatography. However, they soon turned to Donelson to help with the process, which he did using an acrylamide gel-based system that he was developing for some other experiments. At first this system had appeared unpromising because initial results proved only marginally better than those obtained from homochromatography. Some progress was made through the adoption of larger gels. This produced sharper bands in autoradiographs. Yet teething problems continued. On a number of occasions the larger fragments ran faster than the smaller ones. This completely upset the sequence. Moreover, when testing the system with DNA synthesised with the ribosubstitution method for which a sequence was already known, they kept getting inverted sequences. This they attributed to a secondary structure in the DNA template. Eventually, after a lot of trial and error, the problem was resolved by the addition of 8M urea, a chemical, and the application of a high voltage, the combination of which made the gel become hot. After all these changes the team began to get much clearer results than with paper chromatography (Sanger, 1992; Sanger, Dowding, 1996).
The combination of the plus and minus method in tandem with the acrylamide gel-based fractionation system was a major step forward. Now a sequence could be read directly from an autoradiograph. This was done by scanning the picture from the bottom to the top, noting where the dark bands, the product of radioactivity, appeared. All of this could be done with the naked eye. Sanger considered this new approach one of the best ideas he ever had. The new method not only allowed researchers to directly scan a DNA sequence, but also to determine much longer stretches than ever before, 50 nucleotides at a time. It also marked a departure from the use of degradation to a copying procedure. (Sanger, 1988; Finch, 2008).
phi X 174 bacteriophage
Soon after developing the plus and minus method, Sanger's team started using it to determine the sequence of a single-stranded circular DNA from the phi X 174 bacteriophage. This was part of an ongoing project in Sanger's laboratory. The work involved sequencing larger and larger pieces of the bacteriophage's DNA. Initially this was done using Sanger's earlier sequencing methods, but by 1975 the team began largely using the plus and minus method. Much of the work was undertaken by Elizabeth Blackburn and Gillian Air. Another important person on the team was Clyde Hutchison. He had worked on phi X 174 bacteriophage since the 1960s, having studied it as part of his doctorate under the supervision of Robert Sinsheimer at Caltech. Hutchison not only had expertise in the bacteriophage's genetics, but brought with him a mutant strain of the bacteriophage which enabled the team to do some comparative sequence analysis. Later on some additional researchers joined the project: Nigel Brown, John Fildes, Pat Slocombe and Michael Smith.
By the end of 1976 the team had determined virtually the whole sequence of the bacteriophage's DNA. It was over 5,000 nucleotides long. Much of the sequencing had been carried out with the plus and minus technique. It had proven a highly effective tool for the rapid sequencing of long stretches of DNA. Within a short time the whole sequence had been completed, published in 1977. It contained 5,386 bases. The achievement marked a major breakthrough: it was the first complete genome to be sequenced (Sanger, Air, Barrell, et al 1977; Finch, 2008).
Being able to compare the DNA sequence from the mutant strain with the normal bacteriophage had also yielded some unexpected results. During this research the team had found that the 5,326 bases coded for ten genes, and that four of these overlapped. This suggested that the same string of DNA was being used to specify two different proteins. Which protein it coded for depended on the context in which it was read. Such findings challenged a hypothesis, first put forward in 1941, that genes were arranged in a linear order along the genome with one DNA sequence coding for one protein sequence. The discovery also destroyed any idea that the sequence alone contained all the information about an organism, even one as a simple as a bacteriophage. Clearly a DNA sequence was not sufficient without other extra-sequential information. One of the questions this posed was what counts as a gene and when (Sanger, Dowding, 1996; Brown, 2003).
An alternative approach: Maxam-Gilbert
Around the same time that Sanger and Coulson developed the their plus and minus method, another sequencing technique was emerging across the Atlantic. Developed at Harvard University by the American biochemist Walter Gilbert and his doctoral student Allan Maxam, this technique became available the same year as the plus and minus method. The technique shared many similarities with the one developed by Sanger and Coulson. Not only did it make use of radioactively labelled DNA fragments generated with restriction enzymes, it also deployed acrylamide gels to separate the nucleotides in the fragments and used autoradiographs to deduce the sequence. Where the two methods diverged was how they generated segments with particular nucleotide endings. In contrast to Sanger and Coulson, who achieved this by harnessing the copying mechanism of polymerase, Maxam and Gilbert did this by partial degradation which they accomplished with the help of one of four base-specific chemicals (Sanger, 1988).
By the late 1970s the Maxam-Gilbert technique had become more popular than the plus and minus method, particularly among scientists based in the United States. In part this was because it produced bands for every sequence position. The other attraction was that it could be used for sequencing double-stranded DNA. By contrast the plus and minus method was only applicable to single-stranded DNA (Hutchison, 2007; Garcia-Sancho, 2010).
Sanger would later admit that he was not 'altogether overjoyed' by the appearance of the Maxam-Gilbert technique, but considered it an 'ingenious' method. He himself had considered devising a similar chemical method, but had rejected this in favour of enzymes based on his previous work on proteins and RNA, which had shown enzymes to have greater specificity than chemical reagents. His earlier experiences had led him to assume that any rapid degradative sequencing method would need a chemical with very specific reactions. Maxam and Gilbert's method, however, showed that the specificity of chemical reactions did not matter and that strong data could be obtained without specific reactions (Sanger, 1988).
Walter Gilbert. Credit: National Library of Medicine.
Didedoxy technique
In fact Sanger was not totally satisfied himself with the plus/minus system. While much better than his previous methods, it still had some problems. One difficulty was that bands on the autoradiographs varied in strength and were sometimes totally absent. Moreover, he and Coulson had found that the bands tended to get bunched together on a relatively small area of the gel when just one incubation was carried out. To some extent this problem could be resolved by carrying out preliminary incubations, where samples were incubated with a nuclease for varying lengths of time. Such incubations provided bands of varying sizes with a wider distribution on the gel. They, however, were time-consuming so were not ideal (Sanger, Dowding, 1996).
After making little headway with other methods, Sanger and Coulson began to investigate the possibility of using dideoxynucleotides (dideoxys), a DNA nucleotide that can inhibit the action of DNA polymerase and block its attachment of nucleotides to a growing DNA chain. Sanger and Coulson's decision was inspired by some experiments conducted by Arthur Kornberg and colleagues in 1969. They had shown it was possible to produce DNA chains with the thymine (T) ending by adding dideoxynucleotide triphosphate (ddTTP), an analogue of a normal DNA polymerase substrate that they had synthesised, to the incubation of a template and primer with polymerase (Atkinson, Deutscher, Kornberg, 1969).
Sanger did not expect the dideoxy method to work any better than the plus and minus system, but he was nonetheless eager to try it so wrote to Kornberg to obtain some ddTTP. Kornberg, however, had used up all of the ddTTP he had synthesised. Soon after this, however, Sanger by chance come across Klaus Geider at a meeting in Germany. This was a stroke of luck because Geider had synthesised some ddTTP in his laboratory at the Max Planck Institute and was willing to share a sample. Much to Sanger and Coulson's surprise the addition of Geider's ddTTP in their first experiment produced much better autoradiographs than anything they had seen before. These displayed 'sharp bands of equal intensities extending over a long sequence'. Just as important as the clear results, use of ddTTP rendered preliminary incubations unnecessary (Sanger, 1988; Sanger, Dowding, 1996).
There was one downside, however. The only chain terminator that had been synthesised so far was ddTTP and this was only capable of producing DNA fragments ending with thymine. Unable to persuade anyone else to produce other chain-terminating inhibitors for the three other nucleotides, adenine (A), cytosine (C) and guanine (G), Sanger and Coulson set about preparing them. This was a major challenge because neither of them had any experience in nucleotide synthesis. Sanger's main expertise was in synthetic organic chemistry. Such skills had served him well when making amino acid derivatives thirty years earlier when working on insulin. They, however, were useless for synthesising nucleotides. In the end Sanger and Coulson called upon the support of Mike Gait, an expert on nucleotide synthesis. Gait had joined the LMB in 1975 after spending two years as a postdoctoral researcher in Khorana's laboratory at MIT. Additional advice was sought from Bob Shepherd, head of the LMB's peptide chemistry group from 1971 (Sanger, Dowding, 1996; Finch, 2008).
While new to nucleotide synthesis, Sanger relished the opportunity. In part this was because he thought it would entail doing a lot of crystallisation, something he had enjoyed doing since his days of working alongside Ordish, his schoolmaster at Bryanston. As he recalled, 'the first sign of a pure compound was to obtain pure, well-shaped crystals, and it was always exciting to come into the lab in the morning to find a flask full of beautiful crystals, though more often all one found was an oily mess at the bottom.' (Sanger, Dowding, 1996).
Nucleotide synthesis, however, proved much less fun than Sanger expected. To his great disappointment he discovered that nucleotide products did not crystallise. Instead, he had to run the products on a chromatography system to test their purity, a much less enjoyable exercise. Progress was slow and did not always proceed smoothly. Coulson recalled one occasion where Sanger dropped to the floor one of their synthesised products which they had taken three months to prepare. Fortunately Coulson managed to rescue it by scraping it off the floor. After a year of hard labour Sanger and Coulson had finally succeeded in their objective. Looking back on this work Sanger would comment that what they had done was 'not exactly chemical synthesis' because they had not purified or crystallised the product; nonetheless the impure product 'seemed to do the trick' (Finch, 2008).
Sanger and Coulson's new approach incorporating dideoxys was published in December 1977. It was a much more accurate and rapid approach than the plus and minus system. Now up to 120 nucleotides could be read in one go (Sanger, Nicklen, Coulson, 1977). Following publication, Sanger and Coulson continued refining the method, introducing the use of thinner acrylamide gels and the replacement of the radioactive label 32P with 35S. These changes provided much clearer band patterns than before and higher resolution images. All of this made it easier to read the sequence. Moreover, it allowed 465 nucleotides to be read at a time. Sequencing capacity was also increased through the introduction of gels with narrower lanes. Further improvements were made with the introduction of hand-held repetitive pipetting devices which made it possible to do sequencing reactions in 96-well plates manually. On a single day one person could typically run 8 gels, each with 12 sequence ladders. It was not possible, however, to do such work more than twice a week (Sanger, Coulson, 1978; Finch, 2008).
Diagram showing the key steps in the dideoxy sequencing system.
One of the first applications the dideoxy method was used for was to recheck the phi X 174 bacteriophage DNA sequence previously determined with the plus and minus method. The new results indicated that the team needed to make 30 revisions to their original findings (Sanger, Coulson, 1978 ; Finch, 2008).
Sanger receiving the Nobel Prize, 1980. Credit: Fred Sanger.
In 1980 Sanger was awarded his second Nobel Prize in Chemistry. With his characteristic modesty Sanger would always tell people 'it was much more difficult to get the first prize than to get the second one because if you've already got a prize, then you can get facilities for work, and you can get collaborators, and everything is much easier." (Sanger, 2001a).
Sanger is the only Briton to have won two Nobel Prizes and only scientist to have won the Prize twice for chemistry. Half of the second Nobel Prize went to Walter Gilbert. It was awarded on the basis of 'their contributions concerning the determination of base sequences in nucleic acids'. Paul Berg received the other half of the prize in recognition 'for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant DNA.'
Over the next few years, the dideoxy method, also known as Sanger's method, would quickly overtake the Maxam-Gilbert sequencing technique in terms of popularity. In part this was because it was perceived to be much safer on account of it using fewer toxic chemicals and lower amounts of radioactivity. Sanger's method was also seen to be more natural becasue it shared many similarities with the natural workings of DNA, harnessing the duplicating action of polymerase to produce DNA fragments for analysis as opposed to degrading DNA with chemicals. This was highly attractive to molecular biologists who were at the forefront of adopting the technique. Moreover, it slotted easily into their daily laboratory practices. The rising dominance of the Sanger method would mean that it would subsequently become the technique of choice for automation (Hutchison, 2007; Garcia-Sancho, 2012). Click here to see Sanger celebrating his second Nobel Prize.
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Sanger, F (1980), 'Determination of nucleotide sequences in DNA', Nobel Lecture, 431-47. Back
Sanger, F (1988) 'Sequences, sequences, sequences', Annual Review of Biochemistry, 57: 1-29. Back
Sanger, F (1992) 'A life of research on the sequences of proteins and nucleic acids: Dr Fred Sanger in conversation with George Brownlee, Biochemistry Society archives. Back
Sanger, F (2001) 'The early days of DNA sequences', Nature Medicine, 7/3: 267-8. Back
Sanger, F (2001a), Interview with Harry Kroto, Edward Goldwyn, John Sulston and John Walker, 2001, video. Back
Sanger, F, Air, G M, Barrell, B G, Brown, N L, Coulson, A R, Fiddes, J C, Hutchison, C A, Slocombe, P M, Smith, M (1977), 'Nucleotide sequence of bacteriophage phi X174 DNA', Nature, 265: 687-95. Back
Sanger, F, Coulson A R (1975) 'A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase', Journal Molecular Biology, 94, 441-48. Back
Sanger, F, Coulson A R (1978) 'The use of thin acrylamide gels for DNA sequencing', FEBS Letters, 87/1: 107-10. Back
Sanger, F, Donelson, J E, Coulson, A R, Kossel, H, Fischer D (1973) 'Use of DNA polymerase I primed by a synthetic oligonucleotide to determine a nucleotide sequence in phage f1 DNA', Proc Nat Acad Sci USA, 70/4. 1209-13. Back
Sanger, F, Dowding, M (1996) Selected Papers of Frederick Sanger (with commentaries), London. Back
Sanger, F, Nicklen, S, Coulson, A R (1977), 'DNA sequencing with chain-terminating inhibitors', Proc Natl Acad Sci USA, 74/12: 5463-7. Back
Sinsheimer, R L (1959) 'A single-stranded DNA from bacteriophage phi X174', Journal Molecular Biology, 1:43. Back
Wu, R, Kaiser, A D (1968) 'Structure and base sequence in the cohesive ends of bacteriophage lambda DNA', Journal Molecular Biology, 35: 523-37. Back


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+91 8506017771
+91 8506017772
+91 8506017773
I want Holiday Itinerary in:
International Holidays
Discover Japan ( 11 Days)
( Japan )
Call US: +91- 8506017771/ 8506017774
- Overview
- Inclusions
- Itinerary
- T & C Book Now
Discover Japan
Discover Japan
Discover Japan
Return airfare, Airport taxes, Visa Charges
3 Nights accommodation in Tokyo
2 Nights accommodation in Takayama
1 Night accommodation in Kanazawa
3 Nights accommodation in Kyoto
1 Night accommodation in Osaka
Destinations Covered
Return airfare, Airport taxes, Visa Charges
3 Nights accommodation in Tokyo
2 Nights accommodation in Takayama
1 Night accommodation in Kanazawa
3 Nights accommodation in Kyoto
1 Night accommodation in Osaka
All Sightseeing as per itinerary
American breakfast daily (B)
2 lunches (L), 4 dinners (D)
Including a welcome dinner in Tokyo and a farewell dinner in Kyoto
Airport Tax & Visa Fee included
Travel Insurance, Tips, Porterage
Applicable peak season surcharge
Expenses of Personal Nature
Anything not Mentioned in the package inclusions
Day 1 : Arrival in Tokyo
On your arrival at the Tokyo International Airport, after clearing immigration, you will be met by our local Joy Travels representative, who will escort you to your hotel. On the first day of your Discover Japan tour, to see some of the most famous and historic sights of this beautiful country, you will have some time to relax in Tokyo, the Japanese capital city. Overnight at hotel in Tokyo.
Day 2 : Tokyo
After breakfast, you will be meeting your Tour Director and traveling companions prior to a morning sightseeing of Tokyo. You will be visiting the Meiji Shrine, a magnificent 20th century Shinto monument set in a forested area, before driving down to see from outside the Imperial Palace, its park and moats, from Kokyo Gaien – the large plaza in front of the Palace. This will be followed by a visit to an Artist's studio where a master calligrapher will give a demonstration of his skills, and you can try it out for yourself. You will also be going to a factory making Kimonos, where you can wear a kimono and get yourself photographed in the costume. Later, you will be escorted to the Sensoji or Asakusa Kannon Temple, Tokyo's oldest and most colourful temple, before going to the adjoining Nakamise, an ancient shopping street selling souvenirs and traditional snacks. After this, you may continue with shopping or sightseeing (at your own cost). The Ginza district has several boutiques, shops, art galleries, cafes, and nightclubs. Please remember that you will be given a Welcome Dinner by your Tour Director, and afterwards be taken for a Walking Tour in the city to see the dazzlingly illuminated city. Overnight at hotel in Tokyo.
Day 3 : Tokyo
Following breakfast, you will have the day to yourself. If you wish, you can join a full-day (optional) tour to Kamakura, with a temple containing the second-largest statue of the Great Buddha in Japan, cast in 1252 AD. Then you will go for a scenic drive to Hakone, followed by a ride in the cable car for panoramic views of Mt Fuji (weather permitting). The tour will conclude with a ride on the bullet train back to Tokyo. During the evening, you will be free to continue sightseeing or shopping (on your own). There are several restaurants, bars, pubs, and cafes serving a variety of cuisine to suit all tastes. There are innumerable shops and boutiques selling a variety of merchandise, from designer brands to traditional handicrafts. Overnight at hotel in Tokyo.
Day 4 : Tokyo–Matsumoto–Takayama
Post-breakfast, you will be leaving Tokyo for Matsumoto, the picturesque city nestling amidst the Japanese Alps. You will be visiting the remarkable Matsumoto Castle with a black roof, built more than 4 centuries ago, and the Japan Folklore Museum. Lunch will be served en route, after which, you will be driving through the ruggedly splendid Japanese Alps to Takayama, a beautiful town in the lap of the Japanese Alps, seemingly untouched by time. You will have the rest of the time for your leisure activities, when you can explore the narrow streets crowded with antique shops, and, sake breweries and cellars. Overnight at hotel in Takayama.
Day 5 : Takayama
Following breakfast, you will be taken to see the morning market where local farmers sell flowers, vegetables, and fruits. Following this, you will be visiting the historic region of Sanmachi Suji, abounding with sake breweries and shops plying with their centuries-old trade of selling traditional handicrafts, pottery, porcelain, and lacquer-ware. The Kusakabe Heritage House, a folklore museum with antique furniture. In the afternoon, you will be visiting the Hida Minzoku Mura Folk Village, an "open air museum" with traditional farmhouses where artisans now work and you can watch them practise their crafts – silk dyeing, tie-dyeing, lacquer work, and weaving. You will be able to partake of a Japanese dinner. Overnight at hotel in Takayama.
Day 6 : Takayama–Shirakawa–Kanazawa
After breakfast, you will be leaving Takayama and driving through the charming Shokawa Valley to Shirakawa-go, both being UNESCO World Heritage Sites. The uniqueness of these valleys is the presence of thatched-roof houses with their roofs resembling "gassho zukuri" (hands folded in prayer). You will be travelling further to the scenic Kanazawa, situated on the Sea of Japan, and bordered by beautiful parks and the Japanese Alps. The rest of the day will be for you to roam where you like. Dinner will be served to you at one of the three restaurants of your choice in your hotel. Overnight at hotel in Kanazawa.
Day 7 : Kanazawa–Kyoto
Post-breakfast, you will be visiting one of the three most beautiful Japanese gardens, Kenroku-en. It is also known as the 'Six Attributes Garden', developed in the 17th century. The Ishikawa Prefectural Museum of Art is located in the Kenroku-en Gardens, which showcases antique art objects, Japanese paintings, oil paintings, lacquer and porcelain work, besides several other crafts, including the famous Kanazawa-Haku – gold that is beaten paper-thin, a tradition believed to date back to 800 AD. In the afternoon, you will be traversing along the coast of the Sea of Japan to pretty Kyoto in the Yamoshiro Basin, surrounded by mountains. Here, you will be visiting the Fushimi Inari-taisha (Shrine), a historic Shinto shrine, famed for having thousands of vermilion-coloured torii gates. Following this, you will be treated to a Miko dance performed by girls. Overnight at hotel in Kyoto.
Day 8 : Kyoto
Following breakfast, you will be taken for a visit to the magnificent Nijo Castle, on the UNESCO World Heritage List, and one of the many historic monuments built by Tokugawa shoguns in Kyoto. Following this, you will be visiting the Kinkaku-ji (Temple of the Golden Pavilion), and Hiean Jingu (a Shinto shrine) constructed in 1895. You will be served lunch while you participate in a special Japanese cooking class and will learn to make a popular Japanese dish. After lunch, you will have an option to visit Nara, united Japan's first capital, and see the Todaiji Temple with a large statue of the Buddha; Heian Shrine and Garden; the Kasuga-Taisha (Kasuga Great Shrine) at the foot of two revered mountains, and also famous for having 3,000 lanterns of stone and bronze; and, the Kofukuji Temple, an enchanting 5-storey pagoda structure. Overnight at hotel in Kyoto.
Day 9 : Kyoto
After breakfast, you will have the day free to do shopping or sightseeing (at your own cost). There are several significant temples, shrines, palaces, and museums to be seen in Kyoto. There are shops and malls, while the most popular shopping and amusement district lies in Shijo Kawaramachi. There are many restaurants, and Kyoto excels in the variety of Japanese food it has on offer. Please remember to return in time for a special Farewell Dinner that will be hosted by your Tour Director. Overnight at hotel in Kyoto.
Day 10 : Kyoto–Osaka-Departure for Onward Destination
Transfer to Kansai Airport with a short orientation tour of Osaka. Your homebound flight leaves later this afternoon and arrives the same day.
All prices quoted per person on twin sharing basis in Indian Rupees. Rates are applicable for a minimum of two persons travelling at one time.Rates valid for Indian Nationals only For Enquiries related to the period 1st Oct - 31st March please send Query Rates subject to change without notice depending on currency fluctuation Rates are based on Standard category of rooms Rates not valid during conventions and special events. In case carrier is Air Asia package price does not include charges for checked baggage and meals
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subset: commoncrawl


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Rotary Lift to Require Installers to Receive Certification
March 25, 2014—Rotary Lift on Monday announced that the company now requires each Rotary Authorized Installer (RAI) to have at least one Automotive Lift Institute (ALI) Certified Lift Inspector on staff.
Rotary Lift said there are already more ALI Certified Lift Inspectors in Rotary Lift's RAI network than in any other manufacturer's distribution channel.
"There are nearly 400 RAIs across the United States and 60 percent of them currently have employees enrolled in the ALI Lift Inspector Certification Program," says Ron Lainhart, parts and service manager for Rotary Lift and an ALI factory-designated trainer. "We are requiring the rest of our RAIs to enroll a participant by the end of 2014. As a result, Rotary Lift RAIs will be fully equipped to meet the changing needs of the vehicle service industry."
The ALI Lift Inspector Certification Program provides third-party verification that an inspector is qualified to inspect any vehicle lift. All ALI Certified Lift Inspectors must meet prerequisites and pass two exams to complete the program.
Rotary Lift said RAI companies are located in all 50 states, so the new requirement will put an ALI Certified Lift Inspector close to most lift users.


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If you know the Excel G-Series, you are probably familiar with the Excel G-Lite Pro. The Excel G-Logic is a perfect continuation of this chair. Like the G-Lite pro, the G-Logic is very lightweight (less than 12 kg!) and super convenient to take with you on travels, thanks to the fold-down back, detachable footrests, removable wheels, detachable upholstery and folding frame.
The unique thing about the G-Logic is that it can be assembled using three different frame colors and four different seat and back upholstery sets. The G-Logic is available in the frame colors black, silver and white and with upholstery colors blue-black, red-black, orange-black and brown-black.
Why choose the Excel G-Logic?
The Excel G-Logic can only be ordered as a complete package, meaning that it is not possible to order the wheelchair without seat / back upholstery. However, it is possible to order additional seat / back upholstery sets.
The Excel G-Logic is equipped with lightweight rear wheels with a new generation of polyurethane leak-proof tyres. This means lower rolling resistance and this makes the G-Logic a lot lighter. The rear wheels are also removable.
With the design of the G-Logic, eye for detail certainly comes forward. For example, the G-Logic arm rests are height-adjustable. This is useful when the G-Logic has to be driven at the table. The backrest is half folded down, which is not only useful when carrying, but also when a jacket needs to be pulled on or off by the occupant.
Total height: 95 cm (height push handles)
Total width: Seat widht + 20 cm
Total length: 108 cm (incl. footrests)
Total Weight: 11,5 kg
User Weight: 113 kg
Available colors: Framecolour: silver, white and black.
Upholstery colour: orange-black, blue-black, red-black and brownblack.
Seat width: 40, 45 and 50 cm
Seat depth: 42 cm
Seat height: 54 cm
Back height: 42 cm


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Bibione was the first tourist resort in Europe to obtain EMAS environmental certification, an extraordinary achievement which recognises the constant improvement in environmental and area quality of recent years.
Certification guarantees sensitivity to the preservation of the natural heritage and top quality bathing water thanks to controlled management of the water treatment plant, improved beach facility quality, effective waste recycling, careful urban development policies involving action aimed at safeguarding the environment, building safe cycle tracks and environmental awareness programmes.
Bibione's is a prestigious achievement which is important to its future and one that it undertook in the belief that the community will pay ever greater attention to the environmental quality of the resort.
Obtaining environmental certification is just a starting point: the true challenge and commitment is reaching the short and long term objectives which can ensure that Bibione will continue to improve.


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About me
Thanks for visiting my site and hopefully considering me as your videographer.
I hail from a little town called Palmer, Alaska (born and raised), but have been in Austin since 2011. My wife and I absolutely love it here. Warm weather, live music, and great bbq and tacos, whats not to love?! For 6 years I was co-owner of the award winning videography duo known as FG FILMHOUSE LLC, where we worked with clients like Circuit of the Americas, Austin FC, IBM, Formula 1, and Keller Williams. When we decided to go solo in 2019, I took on a full time job with a local startup as their marketing and media guy. I still love to do freelance photo and video on the side, mostly through referrals from professionals I've built relationships with over the years. I'm laid back and easy to work with, so if you're interested in teaming up please feel free to reach out.
Let's talk
Randy Gorman, Freelance Videographer
Austin, Texas
Tel: 509-475-0768


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By making sure you choose the right piece of equipment you can avoid making an expensive mistake.
Waterjet features to look out for
Before investing in a waterjet cutter, think about what features you need. Whilst cutting accuracy is important, it is more important to buy a machine to suit the majority of the cutting work you do. Catering for the most detailed work will make the machine a more expensive investment.
- Heads – Waterjets can run with one or two heads, Consider how long your cutting cycle is and work out in advance whether one head is more cost effective than two heads.
- Stock plates – Instead of buying a waterjet to match your stock plate consider cutting a large plate down to size. Alternatively, let an overly large stock plate hang out beyond the catcher.
Operator costs
Skilled operators and programmers are required to run waterjet machines. As part of the procurement process, it is essential that you take into account whether you will find it easy to recruit operators and programmers for your waterjet machine. If you are likely to find it difficult recruiting skilled operators and programmers, think about whether a machine with an intelligent control system could be a more economic choice. These are easier to run than CAM and CNC machines, which are a lot more complex in their operation.
Another issue to consider is whether your operator will be running more than one machine at a time. If this is the case, some level of automation will be necessary. Look at features such as stack cutting and cutting performance monitors.
Maintenance issues
It is perfectly reasonable to expect a trained operator to undertake routine maintenance on the machine, but if this is the case, you need to ensure your operators have received sufficient training. Maintenance training is normally available for waterjet cutter operators, if required.
Do you require a specific nesting package?
There are several different nesting packages you can use with a waterjet cutter. Generic nesting packages are fine for many users, but if you use a specific process, a customised nesting package will be easier to use.
Do you have the right facilities?
A number of key facilities are required for the installation of waterjet cutters. Adequate floor space is essential for these are large pieces of equipment. You will also need power, water, drainage, and compressed air. Further to this, make sure you also have enough room for materials to be stored and loaded, and a clean area for maintenance tasks to be carried out. Your operators will also require a programming office.
Be practical when considering what features you need in your waterjet cutter. If necessary, ask for advice from the manufacturers before you buy because investing in the wrong machine will cost you dearly.


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Date: 1715
"Or can they ought that's mean, when God has set / A Jewel in their earthly Cabinet?"
preview | full record
— Wesley, Samuel, The Elder (bap. 1662, d. 1735)
Date: 1735, 1745
"Only to trifle sev'nty Years away / In this frail Flesh, this Tenement of Clay, / In Doubt, in Fear, in Sorrow, in Despair, / Then cease to be, and vanish into Air?"
preview | full record
— Trapp, Joseph (1679-1747)


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CDOM is developing a network of Catholic-owned businesses as a resource for you.
Place your business or services ad on the CDOM Business Directory for everyone to see! It's easy – simply click the Register Business link (below) to start the process, or click HERE to contact the webmaster.
It can be tremendously important to know that, when you need something, there are businesses out there you can trust. Not because they have a cool logo, a catchy tagline, or a slick website, but because you join together with them each week for mass.We will have the CDOM Business Directory available in print form in December, however the Business Directory is best utilized online for the most up to date business additions.
With this Directory, it is our sincere desire to connect our parishioners across our diocese who own businesses or provide services that you may need. The Catholic Diocese of Memphis in no way endorses or vets these businesses. However, we encourage our member business owners to use this directory to bless the Church through exceptional service and we encourage our parishioners to use this business directory to bless these member businesses with consistent patronage.
The listings in this directory are provided as a courtesy to parishioners of the Diocese of Memphis and visitors, and should not be relied upon as having any affiliation, sponsorship, approval and/or recommendation from CDOM.
CDOM neither endorses any listed business, guarantee or warranty performance of any listed business, nor is liable for the results of any business transaction between members. Should a dispute occur, it must be resolved solely between the parties of the transaction in a biblical manner.
You can Click one of the buttons below to either view our directory, or register your business.
You may also contact the Office of Communications if you have any questions or to register your business without using the online form. Contact info is email@example.com or 901-373-1252.


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5 x musthave sandals Any favorites? June 11, 2015 Share on Previous articlePacking for Marrakech Next articleUrban Outfitters Rotterdam You may also like Wandler Penelope Mini bag – Must-have summer bag Trend – Floral dresses Office Friendly Items That Will Take You From Day To Night 5 x Velvet items you must invest in 2017 Muroexe and its functionality and aesthetics Falling for knitwear Heeled Trends for Summer 2016 10 Items You Need In Your Wardrobe This Season Peach colors for Spring Summer 2016 1 Comment Shanah says: June 16, 2015 at 11:29 am Such a great selection! I'm especially fond of the snake print and the golden ones Reply Leave a Reply Cancel reply Your email address will not be published. Required fields are marked * Name * Email * Website Comment You may use these HTML tags and attributes:   <acronym title="">
      <q cite="">


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British Cypriot Pedro Evangelou who is serving a nine-year sentence for raping a 19 year old woman claims that he is being discriminated against in jail for his religious beliefs, the Mirror reported.
Evangelou, 44, says he is being harassed by other prisoners about his occult beliefs and satanic tattoos, the Mirror writes.
A prison source told the Mirror: "He stands out like a sore thumb with an upside down pentagram tattoo on his forehead. It's made him a target.
"Rapists are generally the lowest of the low in the prison, but add in a person who believes in ritual magic and pretending to be evil and it creates problems with other prisoners just trying to get on with serving their sentence.
"Other prisoners don't want to deal with his spouting nonsense about Satan. But he's been complaining he's being treated unfairly because of his beliefs and appearance."
Evangelou, who describes himself as a Luciferian, was sentenced in 2017 for raping a 19 year old woman, which he had turned into a shrine to Satan.
According to the Irish Times, Evangelou met the woman at a nightclub in Islington, north London in 2016.
The pair travelled back to his flat on his Harley Davidson motorbike at 6 am the following morning.
He told police he had taken two helmets to the club "in case he met someone".
Scotland Yard said the victim had been subjected to "a prolonged and harrowing sexual assault".
Police were alerted when the woman managed to get to a window and mouthed "help" and "phone police" to a member of the public.
She escaped a short while afterwards and ran out into the street "visibly shaking and upset".
Evangelou was arrested at the scene.


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Great Home-based Business Tips That Can Create A Difference
As with all business, running a online business wants a good layout concerning how it will probably be run, what plans need to be put forth and precisely what do when things aren't going right. Read on to understand the basic principles of planning your business and having all you need to put that plan into actionhttps://www.youtube.com/embed/EcL3IRBhB3k
If you wish to "wine and dine" your clients, make sure that you deduct the price of these meetings during tax time. These events are considered business expenses. However, make certain that these meetings are with potential or paying clients.
Or even, they aren't tax-deductible.
Make certain you're capable of say what your home-based business is around in a short sentence. Whenever you can succinctly explain everything you business does in some words, you will probably impress clients within a meeting and attract customers. This statement also provides you with a ready-made base for any slogan, as it will already cover the main points of the business.
Be sure to put real efforts into making your current customers delighted. It can be much easier to target people who have purchased from you before and were satisfied, rather than selling to a new customer. Make sure your existing consumers are happy!
Keep two phone lines, one for home then one for the business. Using the same phone line for is unprofessional. Your organization calls could be answered by your family, who know nothing in regards to the customer. Furthermore, you ought to have a message on your answering machine which is specific for your business.
Take some breaks through the day, but don't get trapped by the television or any other distractions. Long calls about personal things should be avoided, along with projects that require some time, like home remodeling. Keep your body and mind fresh by taking breaks to do other stuff like exercising or going for a short walk.
Produce a schedule that separates your own time from your work time, and stay with it. Set a period of time for your business to seal, and quit working in those days each day. Allow sufficient time to enjoy with the family and friends, in addition to time and energy to relax and pursue your interests.
Research anyone you need to hire for your own home business. Employees which do not support your vision along with your policies will simply bring your organization down.
Be sure to gives customers notice associated with a item that is certainly out of stock. Back-ordered merchandise is an annoyance to customers, and it can really upset them. Give your clients the possibility to chose other products and inform them every time a item is on back-order.
Whenever you launch your own home-based business, email your family members, friends and colleagues to let them know relating to your new venture. Offer discounts or freebies to your business get going. Also, it is advisable to tell them to spread the word. It is no secret that word of mouth marketing advertising is very helpful.
Given that you've gotten some info from those people who are already successfully running their house businesses, you have to be able to take your upcoming step towards using a successful business of your very own. It will require dedication and persistance from you, but it can be done..


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International List of Highly Cited Researchers in 2019 Includes Rensselaer Researcher
Related Articles
International List of Highly Cited Researchers in 2019 Includes Rensselaer Researcher
Physics professor Humberto Terrones among the world's most highly cited researchers in 2019
Humberto Terrones, a professor in the Department of Physics, Applied Physics, and Astronomy at Rensselaer Polytechnic Institute, is among the most highly cited researchers in the world in 2019, according to a list released by the Web of Science Group.
The Highly Cited Researchers 2019 list identifies scientists and social scientists who produced multiple papers ranking in the top 1% by citations for their field and year of publication, demonstrating significant research influence among their peers. This year, the list includes 6,217 Highly Cited Researchers in various fields from nearly 60 nations.
"The Highly Cited Researchers list contributes to the identification of that small fraction of the researcher population that contributes disproportionately to extending the frontiers of knowledge," said David Pendlebury, senior citation analyst at the Institute for Scientific Information. "These researchers create gains for society, innovation and knowledge that make the world healthier, richer, more sustainable and more secure."
Terrones' research interests are in the electronic, optical, mechanical, and chemical properties of few-layered 2D materials, as well as in using them as building blocks to form new 3D nanostructures. He is a member of the World Academy of Sciences for the Advancement of Science in Developing Countries (TWAS) and the Mexican Academy of Sciences.
"Rensselaer is leading the drive to develop new materials with the specialized properties needed to create the next generation of electronic and optical devices," said Curt Breneman, dean of the School of Science. "It comes as no surprise that Humberto's computational and experimental research in nanostructures is highly cited by other researchers in the field. We are proud of him for achieving this milestone, and secure in the knowledge that his work is critical for enabling further advances in functional materials that are truly transformational."
On the 2019 list, Terrones was recognized in the cross-field category, which recognizes researchers with substantial influence in several fields. The cross-disciplinary work of these researchers exemplifies the entrepreneurial and collaborative spirit of The New Polytechnic, the driving model for teaching and research at Rensselaer.
Web of Science Group, a Clarivate Analytics company, organizes the world's research information to enable academia, corporations, publishers, and governments to accelerate the pace of research. It is powered by Web of Science — the world's largest publisher-neutral citation index and research intelligence platform.
The full 2019 Highly Cited Researchers list and executive summary can be found here, and the methodology can be found here.
For general inquiries: email@example.com
About Rensselaer Polytechnic Institute
Founded in 1824, Rensselaer Polytechnic Institute is America's first technological research university. Rensselaer encompasses five schools, over 30 research centers, more than 140 academic programs including 25 new programs, and a dynamic community made up of over 6,800 students and 104,000 living alumni. Rensselaer faculty and alumni include upwards of 155 National Academy members, six members of the National Inventors Hall of Fame, six National Medal of Technology winners, five National Medal of Science winners, and a Nobel Prize winner in Physics. With nearly 200 years of experience advancing scientific and technological knowledge, Rensselaer remains focused on addressing global challenges with a spirit of ingenuity and collaboration. To learn more, please visit www.rpi.edu.


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|(File Photo)
Researchers at the National University of Singapore (NUS) has found that a component in green tea may help protect against Parkinson's disease, a local news channel reported on Tuesday.
What mentality does 'Chinese-style street crossing' reflect?
The researchers at Yong Loo Lin School of Medicine identified an activation process involving a component in green tea and a protein in the human body that can provide cellular protection against the disease, Channel NewsAsia reported.
The green tea component, known as EGCG, is found to trigger AMP kinase, a protein in the body, to help prevent brain cells from dying under stress.
The EGCG is found in red wine, too.
The researchers experimented on engineered fruit flies which had developed Parkinson's and found that the EGCG-treated flies exhibit much better movement ability and show significant preservation of their brain neurons, compared to untreated flies.
The team hopes to partner with pharmaceutical companies to formulate a drug which can be administered to Parkinson's disease patients.
The potential medication would offer neuro-protection. Current treatment for Parkinson's is usually in the form of a drug known as L-DOPA, which only offers symptomatic therapy, said Lim Kah Leong, an associate professor from the school's department of physiology.
"There are benefits of drinking green tea but in this case we have something even more potent than drinking green tea. So hopefully medication centred around AMPK activation can provide real and faster benefit to Parkinson's patients than green tea could at that stage," he said.
What mentality does 'Chinese-style street crossing' reflect?
All IDs to be checked on trains to Beijing


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Walking Problems in Elderly-What Can You Do About It?
Walking is something very simple and easy what we do almost without noticing it-yet it can become such a difficult task at an old age. The list of possible causes is long: seniors' walking problems might be a result of pain in one or more joints involved in walking, either caused by prior injuries, bone … [Read more…]


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Functional Medicine Doctor Morristown, NJ
What is Functional Medicine?
Functional medicine is a medical specialty that is devoted to discovering the underlying causes of chronic medical conditions.
Viewing your body as an integrated system, functional medicine doctors use a systems-oriented approach to patient-centered care.
Medical Conditions Treated by Functional Medicine Doctors in Morristown
Functional medicine can be used to treat a wide variety of medical conditions including:
How is Functional Medicine Different from Conventional Medicine?
While conventional medicine focuses on suppressing the symptoms of a medical condition, Functional Medicine discovers and addresses its underlying causes.
Unlike conventional medicine, Functional Medicine addresses the patient as a whole - not as an isolated set of systems and symptoms.
Functional Medicine looks at both external (environmental) and internal (lifestyle) factors to determine the best course of patient care.
Consultation with Functional Medicine Physician in Morristown
To request more information or to schedule your consultation with Morristown functional medicine specialist, call (908) 206-7202 or contact Metabolic Wellness and Longevity Institute online.
Metabolic Wellness and Longevity Institute
Address266 King George Rd
Suite H
Warren, NJ 07059
(908) 206-7202
9:00 am - 4:00 pm
Tue: 11:00 am - 5:00 pm
Wed: Closed
Thu: 9:00 am - 4:00 pm
Fri: 9:00 am - 4:00 pm
Areas We Service:Bernards, NJ, Newark, NJ, Edison, NJ, Middlesex, NJ, New Brunswick, NJ, Woodbridge, NJ, Gillette, NJ, Morristown, NJ, Parsippany, NJ, Bridgewater, NJ, Somerville, NJ, Elizabeth, NJ, Plainfield, NJ, Summit, NJ, Westfield, NJ
Read More+


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Spoon , Red
Regular price
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Silicone spoon for easier and safer cooking. Ergonomic handle that adapts to your wrist movements. Cook without scratching or damaging your frying pan thanks to the silicone blade. Resistant to high temperatures. Flexible and non-stick. Dishwasher-friendly. BPA free.
All plastics and silicone used in the manufacture of Lékué products are very easy to clean. This can be done by hand with a neutral soap, without using abrasives.
A dishwasher is only recommended if it can be programmed at temperatures that do not exceed those specified on the individual product's packaging and if they are placed on the top shelf of the dishwasher, unless they are products made from SBS plastic or Tyvek® in which case they are not suitable for dishwashers.
10-Year Guarantee
Lékué offers a 10 year warranty for manufacturer's defects. We will replace any item with the same or similar item, if the item fails during use. Warranty not effective for commercial use.


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The 2015 Tony Awards: The winners and performances
Broadway may have had the night off last night but the stars of the stage were out in force to attend the 69th annual Tony Awards at New York's Radio City Music Hall, where hosts Alan Cumming and Kristen Chenoweth held the evening together as the cheeky Tony winning duo.
And there was plenty to celebrate as this year has seen the Broadway box office take record breaking sales...
As ever there were plenty of quips at the expense of the film and television industries, and Harvey Weinstein, who this year produced the Broadway production of Finding Neverland, took some ribbing, in song form, from Chenoweth and Cumming who joked about his box office wealth.
The big winner of the night was Fun Home, which picked up five trophies in key musical categories like 'Best Musical', 'Best Actor in a Musical', for Michael Cerveris, 'Best Director of a Musical', and 'Best Book of a Musical'. The Curious Case of the Dog in the Night-Time, a book adaptation that transferred from London's West End, tied with five as well, including 'Best Play' and the 'Best Actor' prize for young Alex Sharp. The King and I followed closely on their heels with four Tonys, including one for Broadway veteran, Kelli O'Hara who won 'Best Leading Actress in a Musical.'
Dame Helen Mirren held onto her crown wining 'Best Leading Actress in a Play' for yet another depiction of Queen Elizabeth II in The Audience, which also transferred from London's West End. She opened her acceptance speech with: "Your Majesty, you've done it again," expressing thanks to the Queen, whose role has earned Mirren a myriad of accolades and awards. The Tony Award means that Mirren is now only a Grammy Award away from gaining legendary EGOT status – as she already has an Emmy, Oscar and now a Tony under her belt.
Performances came from the cast of Finding Neverland, a solo performance from Chita Rivera who sang a song from The Visit, Lisa Howard and Tyne Daly performed a song from It Shoulda Been You, and the show closed with a big number from Jersey Boys.
Kristin Chenoweth and Alan Cumming
Kiefer Sutherland and Rita Wilson
Larry David, Jason Alexander
Jennifer Lopez, Nick Jonas and Kiesza
Thomas Sadoski and Amanda Seyfried
Rose Byrne and Bobby Cannavale
Jim Parsons, Taylor Schilling
John Cameron Mitchell
Natasha Katz
Tommy Tune


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¿Le gustaría tener ExpressVPN en su iOS?Comprar la app para iOS
If you see a message that says "Your internet traffic is exposed" message on the ExpressVPN iOS app, the VPN has unexpectedly disconnected. One common cause is another VPN app or VPN profile trying to connect.
Two VPN services of the same type cannot be used simultaneously on iOS devices. If the other VPN app or VPN profile is prioritized over the ExpressVPN app, the ExpressVPN app will disconnect.
To resolve the issue:
- Remove the other VPN apps.
- Remove the other VPN profiles:
- On your device, tap Settings > VPN.
- Tap next to the name of your other VPN profiles.
- Tap Delete VPN.
- Tap Delete.
- Connect to the ExpressVPN app again.
Need help? Contact the ExpressVPN Support Team for immediate assistance.


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Bunkie woman pleads in fatal case of man hit by truck
A woman accused of killing a man by running him over in a truck took a deal Wednesday, pleading guilty to a reduced charge of manslaughter.
Shalynne Lacomalechie Johnson, 27, also pleaded guilty to a felony count of obstruction of justice. She will be sentenced on Sept. 11.
Johnson had been in ad hoc Judge Harry Randow's courtroom Wednesday morning for a hearing on a motion filed by her attorney, Mark Jeansonne. But instead of hearing arguments on the motion for a mistrial, Jeansonne and Rapides Parish Assistant District Attorney Numa V. Metoyer III conferred with Randow.
More:Police: Bunkie woman hit, killed man after fight
More:Bunkie woman accused of running down man has bond reduced
Randow then said the matter would come back before him later that day after a meeting between Jeansonne and Metoyer. The two met with family of victim Shontherean Ramond Williams before Johnson appeared again before the judge.
Johnson, a Bunkie resident, was arrested by the Alexandria Police Department in February 2017 after she was accused of running Williams over in a truck and trying to blame it on a hit-and-run driver.
Johnson and Williams had been staying at a motel in the 1100 block of MacArthur Drive, but had a disagreement. Police alleged that, after the disagreement, Johnson hit Williams with her truck and then parked it before calling for help.
She initially was charged with second-degree murder. Manslaughter carries a maximum penalty of 40 years in prison.


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Cleansing spray to refresh and clean the hair without using water. It absorbs the excess sebum, it gives immediate body to the hair that looks airy and scented. It cleans and refreshes hair without using water. It gives body and volume without weighing the hair down. Rice starch absorbs the excess serum.
How to use:
After shaking well, spray the product to the dried hair from a distance of about 20cm in small amounts. Leave the product on for about 1 minute, then brush. To increase the hair volume, use a cold blow of air.


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All That
The new kids of comedy bring the funny as All That, America's #1 kid's sketch comedy show, returns with a new cast and a few familiar faces. From Executive Producers Kenan Thompson and Kel Mitchell.
Released: 1994-04-16
Genre: Comedy
Duration: 30 min
Country: United States of America
Production: N/A


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Well done to all the children in the Senior room for their hard work and preparation for...
Junior Room
Take a look at our Junior Room Classroom Activities. Check out our Learning Links! …Read More >
Senior Room
Have a look at what we have been doing in the Senior Room. Check out our Learning Links!…Read More >
Here are some of the Faugher National School pupil's achievements in extra curricular activities…Read More >
Our School News
Check out our weekly Faugher School News and Blog written by our teachers and students…Read More >
Welcome to Faugher National School
As principal it gives me great pleasure to welcome you to our school website which aims to provide you with a glimpse of the welcoming, fun and happy environment that Faugher National School is known for and where we aim to help all our pupils achieve their fullest potential. As you navigate through the pages, you will find lots of information about our school, our ethos, our achievements, news updates and much more.
Please do not hesitate to contact us if you require further information about our school. I look forward to meeting you in the near future.
The children and staff had great fun in school today making scrumptious pancakes for Shrove Tuesday in...
On Saturday the 29th of September, we went to Croke Park. We got the bus at ten...


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An Essential Guide to Blondie
Published Mar 16, 2017Blondie are a new wave group from when that was actually new; a punk group with clear vocals and pop hits; a rock'n'roll group whose best-known track is disco and who have been credited with the first single to hit #1 featuring a rap vocal.
Of the many important acts to emerge from late 1970s New York, Blondie are the commercially successful one — songs like "Heart of Glass," "Call Me" and that rapping "Rapture" helped them sell over 40 million records and eventually earn a spot in the Rock and Roll Hall of Fame. Their creative peak was short lived, from 1977 to 1980, and after breaking up in 1982, they went away for 17 years before reuniting, but their influence has endured, most notably on the punk-disco explosion of the early 2000s.
Singer Debbie Harry is a force unto herself — a perpetually cool style icon, actor and solo artist often mistakenly referred to as "Blondie" — but Blondie have always been a band; currently, they feature Harry, co-founding guitarist Chris Stein, original drummer Clem Burke and long-time band members Leigh Foxx (bass), Tommy Kessler (guitar) and Matt Katz-Bohen (keyboards).
They are about to release their 11th studio album, Pollinator (featuring songs written by TV on the Radio's David Sitek, Johnny Marr, Sia, Nick Valensi from the Strokes and Charli XCX), on May 5, and tour North America with Garbage, one of the bands most obviously influenced by them. To get you in the mood, here's Exclaim!'s guide to Blondie's essential albums.
Essential Albums:
5. Greatest Hits
You can't have an Essential Guide to Blondie without "Call Me," but the band's biggest hit was not from any of their own records. Written by Giorgio Moroder for his soundtrack to American Gigolo (with lyrics by Harry), the Grammy-nominated track about a working girl who can be called "anytime, any place, anywhere" (with lyrics cooed in French and Italian, plus an entire alternate version in Spanish) appears on several Blondie compilations.
However, this particular one, from 2005, gets you all the usual early singles plus 1999's "Maria," a '60s girl group-style number and UK hit that brought the band back in the spotlight after a long hiatus — as well as a DVD of their videos, in case you need to know how to dress like a bad-ass art punk after Google and YouTube go down.
4. Autoamerican
An experiment that was underappreciated at the time despite including two smash singles, "The Tide Is High" and "Rapture," the band's fifth album does remain a curiosity in their catalogue. Superstar producer Mike Chapman famously decamped the crew to L.A. for the recording, which might explain why it sounds like the soundtrack to a bizarre Hollywood film.
Opening with an instrumental called "Europa" that starts with an orchestral overture and ends with a Debbie Harry spoken word bit about cars, and closing with a cover of "Follow Me" from the 1960s musical Camelot, Autoamerican is not unlike a cult B-movie: trash to some, but really fun if you're in the right mood and don't take it all too seriously. (In fairness to its critics, it's Harry's least powerful vocal performance. But remember: Blondie are a group.)
3. Blondie
It all started with "X Offender," a song about a prostitute in love with a cop, set to the innocent sound of 1960s girl-group pop. This 33-minute debut, initially released on indie Private Stock and rereleased in 1977 after the band signed to Chrysalis, didn't initially set the world on fire, but it introduced Blondie's particular take on punk — melodic, rhythmic, cheeky — and Debbie Harry's detached yet mesmerizing, prominent, performative vocals.
The aggressive synth-rock of single "Rip Her to Shreds" is an anomaly here: most of the songs are retro fun, from the wistful ballad "In the Flesh" to the silly "The Attack of the Giant Ants." If you're into handclaps and harmonies, this is the Blondie record for you.
2. Eat to the Beat
This is the sound of a band that, having broken through with 1978's million-selling Parallel Lines, could do whatever they wanted. And what they wanted was to spread confidently in as many directions as possible without losing their core melodicism.
It's a fast-paced record propelled by Clem Burke's confident drumming, from the first beat of "Dreaming" (the source of that bathroom graffiti-favourite lyric, "Dreaming is free.") onwards, through the frenetic title track, the punk-disco blast of "Atomic" and Caribbean rhythms of "Die Young, Stay Pretty," which foreshadowed the band's future reggae-based hit "The Tide is High." The label released an entire video album, with clips for every song.
1. Parallel Lines
All killer, no filler. Sometimes the best album is simply the one with the most songs you love, and Parallel Lines has "Heart of Glass," "Sunday Girl," "One Way or Another" and "Hanging on the Telephone." It's quintessentially first wave new wave but also perfectly timeless, amalgamating the best sounds of American radio from the '50s, '60s and '70s into pure, shiny pop-rock goodness.
Debbie Harry is on fire here, whether sneering on "One Way or Another" (which, let's face it, in any other hands would be straight-up creepy) or getting her ya-yas out on "Pretty Baby" or Buddy Holly cover "I'm Going To Love You Too." Buried near the end: "Heart of Glass," the ultimate essential Blondie song.
What to Avoid:
There's a reason Blondie broke up in 1982 — they were all out of ideas. Too bad they didn't do it before releasing The Hunter, an album as cringe-worthy as its cover art, featuring Debbie as a glam rocker cavewoman. She didn't fare much better on her solo debut, 1981's KooKoo, which, despite production by Nile Rodgers and Bernard Edwards of Chic, sounds painfully dated. Cool cover by H.R. Giger, though.
While it would have been nice to unearth a hidden gem from the latter half of Blondie's career here, the band's LPs since 1999's No Exit have tended to sound like Blondie trying to keep up with the bands that were influenced by Blondie — and failing.
Further Listening:
Falling just short of "essential" is Blondie's second album, 1978's perky Plastic Letters, which contains their first charting singles: the synth-meets-doo-wop French-English cover "Denis" and "(I'm Always Touched by Your) Presence, Dear."
And Debbie Harry's non-Blondie discography contains some interesting collaborations. Check out her bizarre theme song to 1997's Texas Chainsaw Massacre: The Next Generation, "Der Einziger Weg," and her eerie interpretation of Edgar Allen Poe's poem "The City and the Sea," recorded with the Jazz Passengers. Then, lend an ear to her vocal contributions to the 1996 album No Talking, Just Head by the Heads, a David Byrne-less Talking Heads.