ReviewPyrosequencing: History, biochemistry and future
Introduction
During the latter part of the 20th century, the innovation of a range of DNA sequencing techniques enabled a revolution in the field of molecular biology. In the 1970s, technologies for sequence determination of DNA were invented, both by Maxam-Gilbert [1] and Sanger [2], and these techniques enormously increased the possibilities of genetic research. The complete DNA sequences of whole genomes are currently known for an increasing number of organisms including the human [3], [4]. Detection of genetic variations in a large number of samples representing a broad range of biological material give insight into genetic mechanisms of different diseases. Even with an increasing number of genomes already sequenced, the importance of technical developments in the field of DNA analysis is evident. The number of DNA sequencing technologies is currently high. Different techniques are advantageous over others depending on the application and therefore, a general ranking of the technologies may be incorporated or misleading. The invention of the Sanger DNA sequencing technique in 1977 [2] revolutionized DNA sequencing technology. This sequencing technology is undoubtedly, by far the most frequently used, exemplified by sequencing of various genomes such as the human. The Sanger DNA sequencing technique is based on DNA synthesis with incorporation of normal dNTPs as well as ddNTPs causing a termination of the newly synthesized DNA molecule. Thus, the prematurely ended fragments can be analyzed with regard to size. The size separation of the Sanger fragments are usually performed by electrophoretic separation although mass spectrometry analysis has also been described [5], [6]. Since the different dideoxy nucleotides are used in different tubes or alternatively marked with different fluorophores the DNA sequence can be deduced from these results. Another DNA sequencing technique presented by Maxam and Gilbert in 1977 is based on sequencing by chemical cleavage [1]. In this technique, the DNA fragments are generated either by digestion of the sequencing template by restriction enzymes or PCR amplification with the ends of the fragments labeled, traditionally by radioactivity. Single stranded DNA fragments radioactively labeled at one end are isolated and subjected to chemical cleavage of base positions. Four parallel cleavage reactions are performed, each one resulting in cleavage after one specific base. The sequence is deduced from the gel separation pattern like in the Sanger DNA sequencing method. In 1975, Ed Southern [7] presented a technique for detection of specific DNA sequences using hybridization of complementary probes. This principle lays the foundation for the sequencing by hybridization (SBH) technology presented in 1988 [8], [9]. Sequencing by hybridization utilizes a large number of short nested oligonucleotides immobilized on a solid support to which the labeled sequencing template is hybridized. The target sequence is deduced by computer analysis of the hybridization pattern of the sample DNA. DNA sequences can also be analyzed by sequencing by synthesis. Pyrosequencing is a sequencing method based on real-time monitoring of the DNA synthesis. It is a four-enzyme DNA sequencing technology monitoring the DNA synthesis detected by bioluminescence [10]. The system is thoroughly described in this paper.
Sequencing technologies like Sanger, Maxam and Gilbert and pyrosequencing have the ability to determine unknown DNA sequence, de novo sequence determination. In contrast, sequencing by hybridization (SBH) is mainly suitable for detection of genetic variations within known DNA sequences, re-sequencing. SBH may also be employed for certain applications such as genotyping samples with well-characterized genetic variations such as single nucleotide polymorphisms (SNPs). However, the extremely small differences in duplex stability between a perfect match and a one-base mismatch duplex may limit the reliability and applicability of this technology [11]. This difficulties can be relieved by use of probes made of Peptide Nucleic Acid, PNA, or Locked Nucleic Acids, LNA, which forms duplexes with DNA with higher melting point than the corresponding DNA–DNA duplex [12], [13], [14]. However, currently the price of these molecules is significantly higher compared to DNA.
The read length and accuracy of the obtained sequences is of crucial importance for the choice of sequencing technology. In the case of the Maxam and Gilbert technique read length up to 500 bp has been achieved [15]. Nevertheless, the occurrence of incomplete reactions usually decreases the read length. Using Sanger sequencing followed by separation by capillary gel electrophoresis, the average read-length obtained is typically between five hundred and thousand bases. Several commercial systems are available for this technology and development in capillary electrophoretic equipment has enabled rapid and accurate determination of up to significantly above thousand bases [16]. However, when using sequence technology for identification of genetic variants such as SNP genotyping, bacterial- or virus typing, detection of specific mutations, gene identification in transcript analysis, etc., the read-length required is much shorter. In these cases, running a several hour experiment for obtaining sequences of several hundred bases is not meaningful. In such cases, faster sequence analysis methods like pyrosequencing are very attractive and have been successfully used [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32]. Moreover, the use of directed base dispension in pyrosequencing analysis of SNPs in close proximity to each other enables haplotype profiling that is not possible using Sanger DNA sequencing.
One important argument for the choice of sequencing technique is the amount of work and time required as well the possibility for automation of different steps. In the sequencing methods described above a step of template amplification performed by Polymerase Chain Reaction, PCR [33] is generally required. A PCR clean up prior to sequence analysis is necessary and a vast number of commercial solutions are available for this purpose. If using the pyrosequencing technology, the purified PCR samples are directly analyzed by real-time monitoring of the DNA synthesis. In turn, when using Sanger DNA sequencing, the sequencing reaction is followed by purification and thereafter separation of the Sanger DNA fragments. The fragment purification has mainly been performed by ethanol precipitation which includes several manual operations and therefore does not readily lend itself to automation. However, alternative techniques such as separation using magnetic beads are available [34]. Although the Sanger DNA sequencing methods can be highly automated, the higher analysis time compared to pyrosequencing decreases its suitability when only shorter sequences are required. The chemical reactions in the Maxam and Gilbert technique are slow and involve hazardous chemicals that require special handling in the DNA cleavage reactions. Therefore, this technology has not been suitable for large-scale investigations. Sequencing by hybridization would, if the accuracy and reliability of the technique were sufficient, provide a very fast analysis of a specific sequence.
Section snippets
History
The real time monitoring of DNA synthesis, the sequencing-by-synthesis principle, was first described in 1985 [35]. The technique is based on sequential addition of nucleotides to a primed template and the sequence of the template is deduced from the order different nucleotides are incorporated into the growing DNA chain which is complementary to the target template. In 1987, P. Nyrén described how DNA polymerase activity can be monitored by bioluminescence [36], [37]. Recently fluorescently
Analytical performance
In order to increase the use of the pyrosequencing technology platform, further improvements were made. One problem addressed was the limited read length obtained. The use of pyrosequencing technology is currently restricted to analyses of short DNA sequences exemplified by mutation detection [24], [25], [26], [27] and single nucleotide polymorphism, SNP, analysis [17], [18], [19], [20], [22], [23]. The factors limiting read length in pyrosequencing can be divided into three major groups:
Applications
An important feature of the pyrosequencing technique is its ability to sequence at least 20 bases. This characteristic allows numerous applications such as sequencing and determining known as well as unknown polymorphic positions, microbial typing and tag sequencing.
Future
The pyrosequencing technology is currently used in 96 or 384 plate format but to be a high throughput technology, an improved sample capacity would be beneficial. One way of doing this would be to use micromachined filter-chamber arrays where parallel analyses of nano-liter samples can be monitored in real-time. In this experimental setup, the DNA sample is immobilized on beads trapped in a filter chamber that allows for injection of solutions into the chamber and transport through the cell to
Concluding remarks
Pyrosequencing is a DNA sequencing technology based on real-time detection of DNA synthesis monitored by bioluminescence. Four enzymes are exploited by the technology and a fifth protein, SSB, can be included to enhance the quality of the obtained sequences and thereby prolong the read length. The pyrosequencing technology has been used in a broad range of applications such as SNP genotyping, de novo mutation detection, gene identification and microbial genotyping. We believe that the
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