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Pyrosequencing Technology for Short DNA Sequencing and Whole 生物物理 47（2），129-132（2007） 理論／実験 技術 Pyrosequencing Technology for Short DNA Sequencing and Whole Genome Sequencing Baback Gharizadeh1, Mehran Ghaderi2 and Pål Nyrén3 1Stanford Genome Technology Center, Stanford University 2Clinical Pathology/Cytology, Karolinska University Hospital 3Department of Biotechnology, Royal Institute of Technology 1．Introduction 2．Pyrosequencing chemistry Recent impressive advances in DNA sequencing technolo- Pyrosequencing technique is based on sequencing-by-syn- gies have accelerated the detailed analysis of genomes from thesis principle8), 9) and employs a series of four enzymes to ac- many organisms. We have been observing reports of complete curately detect short nucleic acid sequences during DNA syn- or draft versions of the genome sequence of several well-stud- thesis. In Pyrosequencing10), the sequencing primer is ied, multicellular organisms. Human biology and medicine hybridized to a single-stranded DNA biotin-labeled template are in the midst of a revolution by Human Genome Project1) (post-PCR alkali treated) and mixed with the enzymes; DNA as the main catalyst. polymerase, ATP sulfurylase, luciferase and apyrase, and the The chain termination sequencing method, also known as substrates adenosine 5′ phosphosulfate (APS) and luciferin. Sanger sequencing, developed by Frederick Sanger and col- Fig. 1 illustrates schematically the chemistry of Pyrose- leagues2), has been the most widely used sequencing method quencing method. Cycles of four deoxynucleotide triphos- since its advent in 1977 and still is in use after more than 29 phates (dNTPs) are separately added to the reaction mixture years since its advent. The remarkable advances in chemistry iteratively. After each nucleotide dispensation, DNA poly- and automation to the Sanger sequencing method has made it merase catalyzes the incorporation of complementary dNTP to a simple and elegant technique, central to almost all past into the template strand. Each nucleotide incorporation event and current genome-sequencing projects of any significant is followed by release of inorganic pyrophosphate (PPi) in a scale. Despite all these grand advantages, there are limitations quantity equimolar to the amount of incorporated nucleotide. in this method, which could be complemented with other ATP sulfurylase quantitatively converts PPi to ATP in the techniques. presence of APS. The generated ATP drives the luciferase-me- Among the current state-of-art DNA sequencing tech- diated conversion of luciferin to oxyluciferin, producing visi- niques, Pyrosequencing3) has emerged, which is being used for ble light in amounts that are proportional to the amount of a wide variety of applications. In the beginning, the method ATP. The light in the luciferase-catalyzed reaction is then de- was only restricted to single nucleotide polymorphisms (SNP) tected by a photon detection device such a charge coupled de- genotyping4), 5) and short reads6), 7) when it was introduced in vice (CCD) camera or photomultiplier. The generated light is 1997 but it is now being used for broader applications. observed as a peak signal in the pyrogram (corresponding to electropherogram in dideoxy sequencing). Each signal peak is 1 分子微弱発光を利用したパイロシークエンス法による実時間ゲノム配列計測技術 Baback GHARIZADEH1, Mehran GHADERI2 and Pål NYRÉN3 1Stanford Genome Technology Center, Stanford University 2Clinical Pathology/Cytology, Karolinska University Hospital 3Department of Biotechnology, Royal Institute of Technology 129 生 物 物 理 Vol.47 No.2（2007） Fig. 1 Schematic illustration of Pyrosequencing method. Pyrosequencing is the energy to firefly luciferase to oxidize luciferin and generate light a non-electrophoretic real-time DNA sequencing method that uses (hυ). The light is detected by a photon detection device and moni- the luciferase-luciferin light release as the detection signal for nucle- tored in real time by integrated software in a format called pyro- otide incorporation into target DNA. The four different nucleotides gram. Finally, apyrase catalyzes degradation of nucleotides that are are dispensed iteratively to a four-enzyme mixture. The pyrophos- not incorporated and the sequencing reaction will be ready for the phate (PPi) released in the DNA polymerase-catalyzed reaction is next nucleotide addition. quantitatively converted to ATP by ATP sulfurylase, which provides proportional to the number of nucleotides incorporated (a tri- inhibitory effect on apyrase catalytic activity, causing non-syn- ple dGTP nucleotide incorporation generates a triple higher chronized extension12). It was found out later that the dATP-α-S peak). Apyrase is a nucleotide-degrading enzyme, which con- consisted of two isomers Sp and Rp. The Rp isomer was not in- tinuously degrades ATP and non-incorporated dNTPs in the corporated in the DNA template as it was not a substrate for reaction mixture. There is a certain time interval (usually 65 DNA polymerase, and its presence in the sequencing reaction seconds) between each nucleotide dispensation to allow com- simply inhibited apyrase activity. By introducing the dATP-α-S plete degradation. For this reason, dNTP addition is per- Sp isomer, substantial longer reads were achieved. This improve- formed one at a time. ment had a major impact on Pyrosequencing read length and al- During this synthesis process, the DNA strand is extended lowed sequencing of up to one hundred bases12) and opened many by complementary nucleotides, and the DNA sequence is avenues for numerous applications. demonstrated by the signal peaks in the pyrogram on a screen. Currently, the Pyrosequencing method is broadly being applied Base-callings are performed with integrated software, which for many applications such as SNP genotyping4), 5), 13) and for has many features for related SNP and sequencing analysis. identification of bacteria14)-16), fungal17)-19) and viral typing6), 20)-23). 3．Applications of Pyrosequencing Moreover, the method has shown the ability for determination of difficult secondary structures24), mutation detection25), 26), DNA Pyrosequnecing is well suited for de novo sequencing and rese- methylation analysis27)-29), multiplex sequencing30), 31), tag sequenc- quencing. Pyrosequencing was earlier limited to sequencing of ing of cDNA library7) and clone checking32). short stretches of DNA, due to the inhibition of apyrase. The nat- Another highly significant application is whole genome se- ural dATP was a substrate for luciferase, resulting in false se- quencing33). Pyrosequencing is now being applied in microfluidic quence signals. dATP was substituted by dATP-α-S11). Our exper- format commercially by 454 Life Sciences Corporation (Branford, iments showed that higher concentrations of this nucleotide had CT, USA). The microfluidic pyroseqeuncing has been integrated 130 Pyrosequencing Technology for Short DNA Sequencing and Whole Genome Sequencing with emulsion PCR and DNA sequencing assembly software. The polymerase caused the template extensions to go out of phase and DNA sequence signal peaks are presented in flowgrams. Sequence cause asynchronous and ambiguous sequence peak signals that reads of up to 100 bases are generated, allowing massive parallel could be demonstrated in the pyrograms as uniformly reduced se- fashion sequencing in picoliter volumes. The new platform has quence signal peak-height. Therefore, efforts were made to com- the capacity to sequence up to 300,000 samples and generate up pensate for this in the reading of those sections. By employing Se- to 20-40 million bases at an accuracy of 99% per 4 hours se- quenase35), an exonuclease deficient T7 DNA polymerase, the quencing run. poly-T homopolymer strings reads were significantly improved In brief, the entire genomic DNA is sheared, and the fragments and generated significantly more synchronized sequence and uni- are ligated to adapters. The fragments are bound to the beads that form signal peaks after homopolymeric T regions. favor one fragment per bead by limiting dilution approach. The An important factor in Pyrosequencing is primer design for beads are captured in the droplets of a PCR-reaction-mixture-in- PCR and sequencing. Sequencing primers should be investigated oil33). The DNA fragments are clonally amplified and the emul- for self-looping, primer-dimers (primer-primer hybridizations) and sions are broken after complete amplification. After single strand cross-hybridizations (when more than one sequencing primer is treatments, the beads are deposited into the picotiter wells (etched used). Single-stranded DNA binding protein (SSB)36) is very effec- in a fiber-optic slide). Each well has the capacity for one single tive for primer and template complications in Pyrosequencing. bead. Smaller beads carrying immobilized enzymes for Pyrose- Homopolymeric regions especially homopolymer T (complemen- quencing reaction are added to the wells and the regents flow cy- tary nucleotide A in nucleotide dispensation) are challenging due clically through the wells. The generated photons are detected by to Klenow polymerase and apyrase inefficiencies to incorporate ful- a CCD camera and presented in flowgrams in integrated software. ly and degrade the analogue dATP-α-S, resulting in non-synchro- 4．Advantages and challenges nized extension and non-uniform peaks. Furthermore, it is highly recommended that sequencing primers to be designed before PCR Pyrosequencing has emerged as an alternate method. Although it primers in order to biotin-label the suitable DNA strand. This fa- is relatively facing read-length limitations
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