基于焦磷酸测序技术的 基因突变检测在精准医疗中的应用研究进展

基因突变检测在肿瘤等疾病的早期诊断、个体化给药指导、疾病治疗进程与耐药监控等方面具有极其重要的意义。随着测序技术的 不断发展,DNA 突变的检测与分析已为病毒感染、血液病和实体瘤等疾病的个体化诊治提供重要参考。焦磷酸测序技术是一种基于生物发 光法测定焦磷酸盐的实时 DNA 测序技术,其用于 DNA 序列分析时不需电泳和荧光标记,定量性能好,结果准确,易于实现自动化,在基 因突变检测分析与肿瘤等疾病诊治中发挥巨大作用。综述基于焦磷酸测序技术的基因突变检测在分子靶向个体化治疗和疾病诊断中的应用 研究进展。     

Analogs of guanine nucleoside triphosphates for sequencing applications

We have synthesized more than 30 different deoxyribonucleosides and triphosphates with modifications either in the base or the phosphate moiety as analogs of 2'-dGTP for DNA sequencing applications. All the modified nucleoside triphosphates were tested as substrates for DNA polymerases, including Sequenase T7 DNA polymerase or Thermo Sequenase DNA polymerase. Two of the analogs, 7-ethyl-7-deaza-dGTP and 7-hydroxymethyl-7-deaza-dGTP meet our requirements as better sequencing reagents.     

Elimination of band compression in sequencing gels by the use of N4-methyl-2'-deoxycytidine 5'-triphosphate.

Taq DNA polymerase, Sequenase, and the large fragment of E.coli polymerase I effectively utilize N4-methyl-2'-deoxycytidine 5'-triphosphate (N4-methyl-dCTP) in the place of dCTP in dideoxynucleotide terminator sequencing reactions on single-stranded templates. When the resulting fragment mixtures are resolved on sequencing gels, they are found to be free of band compressions even in cases where such compressions remain unresolved by the substitution of 7-deaza-dGTP for dGTP. Sequencing reactions using N4-methyl-dCTP instead of dCTP are somewhat more prone to false stops than are sequencing reactions using 7-deaza-dGTP instead of dGTP; this difference is more pronounced when sequencing with Sequenase at 37 degrees C than when sequencing with Taq DNA polymerase at 72 degrees C. For the three polymerases investigated, replacement of dCTP by N4-methyl-dCTP does not fundamentally change the characteristic variations in band intensities seen in the C-lane. N4-methyl-dCTP can also be used for sequencing double-stranded DNA and for DNA amplification by the polymerase chain reaction.     

ANALOGS OF GUANINE NUCLEOSIDE TRIPHOSPHATES FOR SEQUENCING APPLICATIONS

We have synthesized more than 30 different deoxyribonucleosides and triphosphates with modifications either in the base or the phosphate moiety as analogs of 2′-dGTP for DNA sequencing applications. All the modified nucleoside triphosphates were tested as substrates for DNA polymerases, including Sequenase? T7 DNA polymerase or Thermo Sequenase? DNA polymerase. Two of the analogs, 7-ethyl-7-deaza-dGTP and 7-hydroxymethyl-7-deaza- dGTP meet our requirements as better sequencing reagents.     

焦测序技术的研究进展

焦测序技术是一种实时DNA测序技术。它在DNA聚合酶、三磷酸腺苷硫酸化酶、荧光素酶和三磷酸腺苷双磷酸酶4种酶的协同作用下,将焦磷酸转化为等量的荧光信号,通过荧光信号的高低实时检测待测序列,操作简便,可实现高通量、自动化测定,检测不需要电泳,不需要对样品标记和染色,结果准确可靠重复性好。本文综述了焦测序技术的基本原理、历史及其在测序模板制备技术、反应体系和检测仪器三个方面的最新进展,并重点介绍制备单链的Late-PCR技术、高灵敏度反应体系的获得以及454公司超高通量测序技术,并对焦测序技术的发展做了展望。     

Methodological improvements of pyrosequencing technology

Pyrosequencing technology is a rather novel DNA sequencing method based on the sequencing-by-synthesis principle. This bioluminometric, real-time DNA sequencing technique employs a cascade of four enzymatic reactions producing sequence peak signals. The method has been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Although the pyrosequencing procedure is relatively straightforward, users may face challenges due to varying parameters in PCR and sequencing primer design, sample preparation and nucleotide dispensation; such challenges are labor and cost intensive. In this study, these issues have been addressed to increase signal quality and assure sequence accuracy.     

Optimal conditions to use Pfu exo(-) DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA-protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo(-) DNA polymerase (Pfu exo(-)). The relative efficiency of Pfu exo(-) was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo(-) proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo(-), while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo(-) was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo(-) at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.     

Discovery of single nucleotide polymorphisms and mutations by pyrosequencing

Comparative genomics, analyzing variation among individual genomes, is an area of intense investigation. DNA sequencing is usually employed to look for polymorphisms and mutations. Pyrosequencing, a real-time DNA sequencing method, is emerging as a popular platform for comparative genomics. Here we review the use of this technology for mutation scanning, polymorphism discovery and chemical haplotyping. We describe the methodology and accuracy of this technique and discuss how to reduce the cost for large-scale analysis.     

Uniform band intensities in fluorescent dye terminator sequencing.

The use of Cyanine dye (Cy5 and Cy5.5) labeled dideoxy terminators with Thermo Sequenase DNA polymerase in DNA sequencing provides uniform band intensity, improved sequence read-length, and accuracy. It also greatly improves the ability to detect single base heterozygotes with dye-terminator sequencing method.     

Fluorescence resonance energy transfer dye nucleotide terminators: a new synthetic approach for high-throughout DNA sequencing

Fluorescence resonance energy transfer (FRET) based dye-nucleotide terminators (10-13) were designed, synthesized, and formulated with Thermo Sequenase II DNA polymerase into a robust kit for high throughput DNA sequencing. The key energy transfer (ET) rigid and linear linker (2), required for the syntheses of energy transfer cassettes (6-9) was synthesized via Heck coupling reaction on t-Boc-L-4-iodo-phenylalanine (1) with N-TFA-propargylamine.     

Toward pyrosequencing on surface-attached genetic material by use of DNA-binding luciferase fusion proteins

Mutation detection and single-nucleotide polymorphism genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technology based on the sequencing-by-synthesis principle. Currently, the technique is limited to simultaneous analysis of 96 or 384 samples. Earlier, attempts to increase the sample capacity were made using micromachined filter chamber arrays where parallel analyses of nanoliter samples could be monitored in real time. We have developed a strategy for specific immobilization of the light-producing enzyme luciferase to the DNA template within a reaction chamber. By this approach, luciferase is genetically fused to a DNA-binding protein (Klenow polymerase or Escherichia coli single-stranded DNA-binding (SSB) protein) and to a purification handle (Z(basic)). The proteins are produced in E. coli and purified using cation and anion exchange chromatography with removal of Z(basic). The produced proteins have been analyzed using an assay for complete primer extension of DNA templates immobilized on magnetic beads detected by pyrosequencing chemistry. Results from these experiments show that the proteins bind selectively to the immobilized DNA and that their enzymatic domains were active. Z(basic)-SSB-luciferase produced the highest signal in this assay and was further exploited as enzymatic reagent for DNA sequencing.     

Method enabling fast partial sequencing of cDNA clones

Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed.     

Pyrosequencing enhancement for better detection limit and sequencing homopolymers

Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid and real-time sequence determination. Although ample genomic research has been undertaken using pyrosequencing, the requirement of relatively high amount of DNA template and the difficulty in sequencing the homopolymeric regions limit its key advantages in the applications directing towards clinical research. In this study, we demonstrate that pyrosequencing on homopolymeric regions with 10 identical nucleotides can be successfully performed with optimal amount of DNA (0.3125-5 pmol) immobilized on conventional non-porous Sepharose beads. We also validate that by using porous silica beads, the sequencing signal increased 3.5-folds as compared to that produced from same amount of DNA immobilized on solid Sepharose beads. Our results strongly indicate that with optimized quantity of DNA and suitable solid support, the performance of pyrosequencing on homopolymeric regions and its detection limit has been significantly improved.     

Pyrosequencing for microbial typing

Pyrosequencing is a real-time DNA sequencing technique generating short reads rapidly and inexpensively. This technology has the potential advantage of accuracy, ease-of-use, high flexibility and is now emerging as a popular platform for microbial typing. Here, we review the methodology and the use of this technique for viral typing, bacterial typing, and fungal typing. In addition, we describe how to use multiplexing for accurate and rapid typing.     

Use of a chemically modified T7 DNA polymerase for manual and automated sequencing of supercoiled DNA

Procedures are presented for reliable and accurate nucleotide sequence analysis using as template supercoiled DNA prepared by a modified rapid boiling minipreparation protocol. This method yields DNA templates suitable for sequencing within 1 h of bacterial harvest. We describe optimal reaction conditions for supercoiled miniprep DNA sequencing using a modified T7 DNA polymerase (Sequenase) in dideoxynucleotide chain termination reactions. We demonstrate that under these conditions, the sequencing data obtained with miniprep DNA is indistinguishable from that obtained with CsCl purified supercoiled DNA or from that obtained using single stranded DNA templates. We further show that the supercoiled DNA sequencing reactions can be analyzed on a commercially available automated DNA sequencing system that detects 32P labeled DNA during its electrophoretic separation. Taken together, these developments represent a significant improvement in the process of nucleotide sequence analysis.     

A novel method for preparing single-stranded DNA for pyrosequencing

Pyrosequencing technology is a powerful genotyping tool that requires the generation of single stranded DNA. Currently, two simple, solid-phase-based methods are available for this, but they require special equipment, they are not automated, and they are relatively expensive because of the need for biotinylated polymerase chain reaction primers. In this article, an enzymatic liquid-phase method for the generation of high-quality, single-stranded DNA, and its novel use for Pyrosequencing are described. The method has also been fully automated.     

FLUORESCENCE RESONANCE ENERGY TRANSFER DYE NUCLEOTIDE TERMINATORS: A NEW SYNTHETIC APPROACH FOR HIGH-THROUGHOUT DNA SEQUENCING

Fluorescence resonance energy transfer (FRET) based dye-nucleotide terminators (10–13) were designed, synthesized, and formulated with Thermo Sequenase? II DNA polymerase into a robust kit for high throughput DNA sequencing. The key energy transfer (ET) rigid and linear linker (2), required for the syntheses of energy transfer cassettes (6–9) was synthesized via Heck coupling reaction on t-Boc-L-4-iodo-phenylalanine (1) with N-TFA-propargylamine.     

Analysis of read length limiting factors in Pyrosequencing chemistry

Pyrosequencing is a bioluminometric DNA sequencing technique that measures the release of pyrophosphate during DNA synthesis. The amount of pyrophosphate is proportionally converted into visible light by a cascade of enzymatic reactions. Pyrosequencing has heretofore been used for generating short sequence reads (1-100 nucleotides) because certain factors limit the system's ability to perform longer reads accurately. In this study, we have characterized the main read length limiting factors in both three-enzyme and four-enzyme Pyrosequencing systems. A new simulation model was developed to simulate the read length of both systems based on the inhibitory factors in the chemical equations governing each enzymatic cascade. Our results indicate that nonsynchronized extension limits the obtained read length, albeit to a different extent for each system. In the four-enzyme system, nonsynchronized extension due mainly to a decrease in apyrase's efficiency in degrading excess nucleotides proves to be the main limiting factor of read length. Replacing apyrase with a washing step for removal of excess nucleotide proves to be essential in improving the read length of Pyrosequencing. The main limiting factor of the three-enzyme system is shown to be loss of DNA fragments during the washing step. If this loss is minimized to 0.1% per washing cycle, the read length of Pyrosequencing would be well beyond 300 bases.     

Method enabling pyrosequencing on double-stranded DNA

Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis (M. Ronaghi, M. Uhlén, and P. Nyrén, Science 281, 363-365). Pyrosequencing has so far only been performed on single-stranded DNA. In this paper different enzymatic strategies for template preparation enabling pyrosequencing on double-stranded DNA were studied. High quality data were obtained with several different enzyme combinations: (i) shrimp alkaline phosphatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exonuclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuclease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide, single-stranded DNA-binding protein, and Klenow DNA polymerase improved the sequence quality. Apyrase was the fastest and most efficient of the three different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyrosequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content.