In this modern world, we are very enthusiastic to create synthetic life, as a part of which intensive research is going in the related fields. This leads to the field of biology, and discussion about the gene synthesis which will help in creating the synthetic life. Basically, synthetic life is created using biochemicals in vitro, the principle of synthetic life is modelling of the living systems i.e. to try to simulate the chemical processes so that it gives the required functionality. Creating artificial life is not a bread-butter task; it is like being next to god, which is incredible. In fact, going back to history there was a person named Frankenstein, who tried to create human of bring back life into dead bodies, he made several attempts to figure out the mystery of life. Though the approach was not right, but the intention was great. Even though now we humans are trying to find the essence of life, we are not trying the same mad attempt as was done by Frankenstein. The authors express that in order to construct good synthetic biological systems we need very good quality synthetic DNA, so that is why good research is being done in order to create high-quality artificial DNA, this in turn requires improvements in the scale and accuracy of chemical DNA synthesis because in order to get high quality synthetic DNA should be able to process longer DNA which is tacked up using shorter fragments. Also, this requires improvements in methods in order to process longer DNA which is tacked up from small fragments. The authors present an automated method which is termed as megacloning, in order to give high quality synthetic DNA which will be highly useful for gene synthesis, even though it is stated as automated it requires some manual intervention. They have processed both micro-array derived and chemically synthesized oligonucleotides using their method. The method designed by the authors is a parallel method, so it will be able to run more than one step at a time, which saves lot of time and is very much helpful for researchers since they get to test many times. The method that the authors framed, is used to get high quality synthetic DNA from microarray derived nucleotides and also from chemical synthesis of oligonucleotides. The computing capability of this method is very high, it can sequence around millions of DNA in just one run, which is highly advantageous. Also this method reduces the error rate around 500 times when compared with that of the pool of nucleotide rendered by the microarray method. Their method uses the next-generation sequencing (NGS) as a step in order to produce high quality DNA, this DNA obtained from the megacloning is used for making the synthetic or man-made artificial genes. Before we discuss about the method, let us learn about the terminology and the previous techniques. In this, they use the DNA sequencing which refers to finding out the arrangement of the nucleotide bases (thymine, cytosine, guanine, and adenine). This information about the DNA is very important; sometimes it lets us to identify the behaviours, in fact this has lead the research on to a great path towards the mystery of life, also this has increased the research in the field of biology. This has also lead to the initiation of Human genome project. Many advances were in the methods of DNA sequencing, a popular method of them was the Sanger method also called as the dideoxy method. The inventor of this method received a noble prize for this accomplishment. Dideoxynucleotide triphosphates (ddNTPs) were used as chain terminators; this was the basis for the Sanger method. This method requires the following ingredients, a template of single-stranded DNA, DNA primer, DNA polymerase, deoxynucleotide phosphates (dNTPs), dideoxy nucleotide triphosphates. A DNA primer is a strand of nucleic acid which serves as the initial point for the synthesis of DNA. DNA polymerase is an enzyme that will help in catalysing the formation of new DNA from already existing DNA, every time the cell divides this will twin extra DNA from the existing one and this is passed to the new cell. The accuracy of a DNA polymerase is very high; it is really astonishing to believe its accuracy, this accuracy is achieved because it will check for errors after it replicates DNA. Dideoxynucleotide triphosphates are used as chain terminators for terminating the extension DNA strand. The methods is performed as follows, before the actual DNA sequencing is carried the DNA must be first be changed naturally by heat into single stranded DNA. Then the DNA primer is tempered onto a strand of DNA template, this should be tagged or marked radioactively which will be useful in detection in gel electrophoresis. The reaction is carried in four separate tubes as per separate nucleotide bases i.e. corresponding deoxynucleotide phosphate was added to each tube, also corresponding chain terminators and DNA polymerase are added to each tube. Nucleotides will be added each tube where the chain keeps growing and these nucleotides will be incorporated into DNA, once when the dideoxynucleotide is integrated instead of a normal nucleotide then the chain terminates because these act as chain terminators, this might result in various lengths of DNA fragments. Once we obtain these fragments, they are heat denatured in order to separate them by sizes and prepare for the electrophoresis. Before we continue let us know about the process of gel electrophoresis, earlier very laborious techniques were used to separate the fragments of DNA, but this method is simple to use. It uses the electricity in order to separate the fragments of DNA as per their size through gel matrix. In this, the gel is immersed into a salt solution which will help in passing electricity, then with the use of a pipette, the sample of DNA are then arranged onto the matrix made by the gel, once the power is switched on, the negatively charged DNA will shift to the positive end of the electrophoresis setup or enclosure, in this way the DNA fragments are separated by size by gel electrophoresis. Continuing the discussion about Sanger method, the matter of the four tubes is put on different lanes on a gel, then this gel is exposed to ultraviolet light or x-ray, then the sequence is read from bottom to top. This method was accurate, and it was comparatively much efficient, used less toxic materials and the level of radioactivity used was less compared to previously designed methods for sequencing DNA. The authors compare their method with this Sanger method and they found that it was very advantageous in many ways, first to mention is that the method discussed by the authors is highly parallel, also it is automated and requires less manual intervention. Next generation sequencing (NGS) is what the authors try to implement as step of their method. It refers to the techniques used for sequencing which are highly parallel i.e. the run multiple step in just one go, low-cost techniques i.e. these techniques are comparatively of less cost when compared with that of other sequencing methods, these techniques can give rise to millions of sequences in just one run, also most of the next generation sequencing technologies are automated. These technologies are completely inspiring genomics and helping it to be widely spread. There are many next generation sequencing techniques, of which �massively parallel signature sequencing� (MPSS) was the first NGS technique; the other good sequencing methods that followed were the pyrosequencing and the advance versions of pyrosequencing etc. Next generation sequencing is helping humans to explore in new areas and is widely being used in surprising areas. Since the cost of the next generation sequencing technologies is decreasing, the number of applications is growing apace and will continue to be used in different and various aspects of biological sciences. The basic principle of pyrosequencing is sequencing by synthesis, in this method depends on the detection of pyrophosphate when compared to that of the dideoxynucleotide triphosphates as chain terminators in Sanger method. It is a not so complicated to use technique for the perfect analysis of the DNA sequences. The pyrosequencing techniques require a single-strand DNA which is rendered from PCR (polymerase chain reaction) amplification. The reaction is carried out as follows, a primer is added to the template DNA, then four enzymes are added and those are apyrase, ATP sulfurylase, DNA polymerase, and luciferase then the nucleotides are added. The DNA polymerase is an enzyme which will help in catalysing the reaction, it will help in incorporating the nucleotides to the DNA strand, and once the nucleotide is incorporated pyrophosphate (PPi) is released. The released PPi is then converted to ATP with the help of ATP sulfurylase. The ATP which is produced will help in converting luciferin to oxyferin which will help in producing light, the light produced will be proportional to the amount of ATP produced. The light produces is detected, and is observed as a peak in the program. We can determine the number of nucleotides integrated, because the number of nucleotides integrated is proportional to the peak length i.e. if just one nucleotide is incorporated then there would be just one peak, if two nucleotides were integrated the there will be two peaks. The enzyme apyrase, which is in the reaction, will come into action and continuously removes the nucleotides which were not incorporated and it also removes the ATPs. Once this is done, more nucleotides are added and the process is repeated as said above. The light is produced only when the nucleotides are integrated else they are simply removed by apyrase. As the process is continued the pyrogram is generated which gives the sequence. The accuracy of the pyrosequencing is pretty high when compared
to that of earlier methods; this is because the DNA polymerase rarely makes an error. Later advanced version of pyrosequencing was designed, which is parallel version of pyrosequecing. Megacloning uses the GS Flx (Genome Sequencer Flx) workflow, which basically has the following steps, the steps include propagation of collection of DNA with single strands, then the amplification is carried out by the emulsion process, after that sequencing of DNA is done which would be based on sequencing-by-synthesis, then the analysis is done using various tools, we discuss these steps in detail. The GS Flx is the latest pyrosequencing platform, it is cost effective, it yields good amount of accuracy. The method is carried out by taking a sample input from different materials; these inputs are divided into smaller fragments. After this, some biological techniques are used to add short adaptors to the fragments, and are used in sequencing, purification and amplification steps. The specially designed capture beads are used to capture the DNA strand, and then the beads are captured in water droplets inside the oil, this will help in the amplification process and many copies are created per bead. These are then poured onto a PicoTiterPlate in order to proceed for sequencing. The PicoTiterPlate consists of many wells, where each well is used only for one bead, this is then fed to the machine, where the sequencing is performed like the pyrosequencing, where nucleotides are added and every time they are incorporated a light signal is produced which is recorded by a camera and this recording will help is getting the sequence of DNA. This has great accuracy, and also the cost would be around 5000 to 7000 US dollars per run and it can generate around four hundred million nucleotide information in around ten hours run. This sequencer is used as a step in the megaclonig process described by the authors in order to obtain high quality DNA. The technique of the PCR is to duplicate the DNA from a given sample to a huge number of copies of that sample sequence of DNA. This techniques is used for research purposes for several applications, one such application could be the duplication of DNA which will be used for sequencing etc. So basically the PCR helps in DNA amplification, where DNA amplification implies creating multiple copies of a particular DNA sequence. The megacloning process basically has the following steps, firstly the required sequence is fed from the computer, and then that is followed by the oligonucleotide synthesis, this is carried out using various methods like the conventional nucleotide synthesis, microarrays, natural sources etc., after the microarray synthesis then comes the step where next generation sequencing is applied using the Roche/454 or Illumina or the ABI/SOLiD, after this the required sequence is selected and put onto a PicoTiterPlate and then amplify PCR is used as the final step to get the perfect plant of DNA. Presently the construction of gene depends on the older technologies for the chemical synthesis of nucleotides i.e. people use older techniques for building the gene; these old techniques are generally costly and will have much error rate. Recently, as a result of good research, array technology has come has come into existence and that will help is synthesizing millions of sequences with the lengths commensurable to that derived from the conventional synthesis. This provides a great source of nucleotides which will yield high throughput and is cost efficient. The number of errors produced in conventional method is lower when compared to that of the microarray method. The method presented by the authors, start with DNA which is taken from different sources, they used oligonucleotides synthesized from conventional sources and also from the microarrays. In order to read and distinguish the nucleotides with the wanted sequences they used the next generation sequencing. In order to perform then next generation sequencing they used the GS Flx platform which implements the advanced version of pyrosequencing, this was the DNA sequencing is done. The DNA was then sorted by the use of the PicoTiterPlate. They used micro actuators in order to control the micropipettes which were used to sort and select the DNA retrieved from the next generation sequencing; microscopic cameras were used in order to guide the micropipette. The selected DNA beads are placed in multiwell plate, such that each bed is placed in each well of the multiwell plate. The last step of the process is to perform the amplification using the PCR. In this way by the use of pyrosequencing, we get the sequence verified DNA which is highly useful for gene synthesis. In order to verify the faithfulness of their setup, they took a natural sample of DNA and extracted using the micropipette and compared that with that of the sequences amplified using the Sanger method, and they found that the match was 99.9%. Later in order to make sure that the reads from the GS Flx matched the initial pool, 319 beads were collected which were derived from the microarray. The Genome Analyzer II was used in order to obtain the sequenced DNA from both treated and untreated pool, when these were compared around 3.1% reads of 319 sequences matched without any errors with the initial pool. When the enriched or treated pool was mapped to initial pool around 84.3% of the sequences were mapped correctly.
