The result is obtained based on a pre-prepared standard curve and an internal, positive and negative control that need to be run; i. The method is based on the fact that the dye only binds to double-stranded DNA, which is accompanied with an increase in fluorescence. Thus, the signal intensity correlates with the amount of amplified DNA fragment and, respectively, with the initial sample input amounts Figure 2C.
In , Hubert et al. In general, real-time PCR is more sensitive than conventional PCR and needs the target sequences to be shorter than those used in conventional PCR, maximum — bp in length; results are obtained in real time and it is not necessary to use gel electrophoresis. The cost of a single reaction excluding the controls is much higher than that of conventional PCR. For example, in conventional PCR, the minimum reaction cost is 0. This cDNA is then used in conventional or real-time PCR, either in one step the reaction directly proceeds from reverse transcription to subsequent amplification steps in the same tube or in two steps the RT reaction is run separately and a new reaction mixture is prepared for conventional or real-time PCR.
These methods are also applied for multiplex reactions, i. What is important for the primer pairs used in the reaction is for them not to be complementary to each other so that they do not form dimers. Other methods that are also based on amplification of a target nucleic acid sequence are ligase chain reaction LCR , nucleic acid sequence-based amplification NASBA , and strand displacement amplification SDA. Ligase chain reaction LCR was first described by Barany [ 49 ].
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It combines a ligase reaction with amplification and is particularly suitable for differentiation of single-base substitutions Figure 4. The thermostable ligase, then, proceeds to ligate only those primers that share perfect complementarity to the target sequence and hybridize immediately next to each other.
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Thus, if two primers bear a single base-pair mismatch at the junction, they will not ligate effectively enough and, in turn, there will be no product amplification. To avoid ligation of the 3' ends, the discriminating primers contain a 2-bp non-complementary AA tail at their 5' ends [ 49 , 50 ]. Next, reverse transcription yields a cDNA — strand. Strand displacement amplification SDA combines the principles of isothermal DNA amplification with those of restriction enzyme digestion [ 52 ].
Thus, the specific HincII cleavage site is regenerated, which closes the cycle. There are various modifications and versions of these methods, even some commercial kits; however, it is not possible for all of them to be discussed here. There are different sequencing methods and variations, as the methodology evolves based on successful adaptation and application of the properties of nucleic acids. Sanger method — in , Sanger et al.
The polymerase enzyme can incorporate either dNTPs or ddNTPs depending on their relative concentration at each elongation step. The resulting fragments are of different size length and, in gel electrophoresis, will migrate toward the positive electrode at a rate of migration inversely proportional to their molecular weight. The method can differentiate fragments that are only 1 bp different in length.
The reaction components are the same as those in the Sanger method, but are mixed in such a way as to allow thermal cycling: denaturation, annealing, elongation, and generation of a balanced population of short and long fragments, using the same principle as the Sanger method Figure 5A. A limitation of the method is the size of the fragments that can be sequenced: maximum — nucleotides per run. The sequences obtained using forward and reverse primers are analyzed by programs available either as freeware [ 55 , 56 ] or as commercial software.
What is important to remember is to always check the sequencing results in the file generated by the software against the electropherogram: there may often be discrepancies between the nucleotide sequence in the file and that in the electropherogram. In such cases, the electropherogram should be considered more reliable but the background effect should also be accounted for.
In the alignment of sequences first, between the F and R primers of a sample and, second, between different samples , it is important for them to be equal in length. The next step is sequence analysis — phylogenetic analysis, genetic distance analysis, etc. For example, in phylogenetic analysis, it is particularly important to choose the mathematical model that is most appropriate for each particular case.
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Instead, there are software programs especially designed to determine the most appropriate model depending on the sequence length, the number of sequences, potential substitutions, etc. For example, jModelTest [ 57 ] analyzes and selects among 89 different models. The way is now open for nucleic acid research using NGS technologies. The Illumina technology is based on the principle described above, i. The identification of incorporated nucleotides is based on fluorophore excitation. The whole process includes the following steps:. Thus, each strand in the DNA double helix is adapter-ligated.
Fragmentation and ligation may be performed in a single step, which, according to 9 , markedly increases the effectiveness. The adapter-ligated fragments are then amplified by PCR and purified in a gel. Cluster generation takes place on the surface of a flow cell. The flow cell is, in fact, a glass slide with channels on whose surface two types of oligonucleotides are attached.
These oligonucleotides are complementary to one of the ends of the library fragments the adapter and are identical with the other end. This ensures cluster generation is done by hybridization of the adapter-ligated amplified fragments from the library with synthetic oligonucleotides on the surface of the flow cell that are complementary to the adapters. Each single-stranded fragment binds with the complementary end, while its other end remains free, perpendicular to the surface of the flow cell The free end is identical with the oligonucleotide in the flow cell to provide synthesis of a complementary sequence in the new strand.
Then complementary strand synthesis can begin, displacing the original strand when the synthesis is completed. The fragments thus bound act as templates for the so-called bridge amplification of discrete clonal clusters. Thus, the templates generated by bridge amplification form clusters of single-stranded fragments bound at one end and the number of DNA templates grows.
Then, the reverse templates are removed and only the forward strands remain — clones identical to the original templates. Sequencing — The samples are ready for the sequencing step when the clusters are complete. A proprietary reversible terminator-based method is used: a sequencing primer is annealed it is complementary to the part of the adapter that is adjacent to the DNA fragment of interest , followed by cycles of incorporation of terminator-bound dNTPs.
In each cycle, there is a mixture of the four different dNTPs and they naturally compete for incorporation based on complementarity. As a result, only one complementary dNTP per cycle is incorporated in the growing strand.
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In each cycle, a labeled terminator-bound dNTP is incorporated in each chain in each cluster and is detected based on emission wavelength and intensity. The signal emitted by each nucleotide incorporated in each strand at the end of a cycle is recorded in real time in each cluster. The cycle is repeated n times to obtain an n -base long sequence. When the new strand is synthesized and its sequence is recorded, it is removed. This approach of base-by-base sequencing achieves very high precision, as errors are eliminated even in repetitive sequence regions and homopolymers. Data processing and analysis — The fluorescence emissions from different clusters are digitally processed in parallel and are visualized as nucleotide sequences for each individual cluster.
Following sequence alignment, the data are compared against referent sequences to perform phylogenetic analysis, single-nucleotide polymorphism analysis, distance determination, and a range of other analyses. This technique allows multiplex analysis to be carried out by creating distinct libraries based on the so-called index sequences short nucleotide sequences specific for each library that can be used like a barcode , which are attached during library preparation step.
Different libraries are first individually prepared and are then combined together and loaded in one and the same flow cell lane. The labeled libraries are sequenced simultaneously, in a single run of the equipment, and at the end of the process, the sequences are exported in a single file. Next, a demultiplexing algorithm is used to separate the sequences in different files based on their barcode. This is followed by alignment with referent sequences of interest.
The platform is compatible with different library preparation methods depending on the purpose of the sequencing analysis whole genome sequencing, target sequencing, mRNA sequencing, 16s RNA gene sequencing, etc. This is possible because the sequencing steps that come after the library preparation step are fundamental and do not depend on the library preparation method. There are ready-to-use, standardized library preparation kits designed for different sequencing purposes and more and more new approaches and modifications are being developed [ 58 ].
Nanopore technology is an innovative NGS platform that also includes sequence library preparation. This is achieved by magnetic particle purification It is probably possible to use other methods as well, e. Bayley sequencing technology is used. In this method, the DNA molecule is basically passed through a system consisting of a processive exonuclease enzyme bound to a protein nanopore Figure 6A.
This continues until the hairpin loop at the end of the first strand is reached and then the enzyme simply proceeds on to push the ligated reverse strand through the nanopore as well. The system is designed so that a constant ion current flows through the aperture of the free protein nanopore. This current is specifically disrupted when each base enters the aperture. These specific disruptions in the ion current are recorded by an electronic device and are interpreted to identify each base.
The disruption that each of the four bases causes in the ion current is different due to differences in their chemical structure and chemical characteristics. Recordings from multiple channels in parallel allows high-throughput sequencing of DNA. Protein nanopores are inserted into an electrically resistant polymer membrane so that a membrane potential can be created driving the ion current through the pore aperture.
This ion current is disrupted in a specific way when a nucleotide passes through the aperture of the protein nanopore, which causes a change in the membrane potential. An array of microscaffolds holds the membrane in which the nanopores are embedded, giving stability to the structure during operation.
Each microscaffold on the sensor array chip contains an individual electrode, allowing for multiple nanopore experiments to be performed in parallel. Each nanopore channel is controlled and measured by an individual channel on a corresponding, bespoke application specific integrated circuit ASIC Figure 6B [ 59 ]. In this method, following library preparation, the sequencing step is actually performed in wells that contain the DNA template, an underlying sensor, and electronics. This leads to changes in the surface potential of a metal-oxide sensing layer and to changes in the potential of the source terminal of the underlying field-effect transistor.
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1 Introduction and Scope of this Review
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