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1.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) has been explored widely for DNA sequencing. The major requirement for this method is that the DNA sequencing fragments must be free from alkaline and alkaline earth salts as well as other contaminants for accurately measuring the masses of the DNA fragments. We report here the development of a novel MS DNA sequencing method that generates Sanger-sequencing fragments in one tube using biotinylated dideoxynucleotides. The DNA sequencing fragments that carry a biotin at the 3′-end are made free from salts and other components in the sequencing reaction by capture with streptavidin-coated magnetic beads. Only correctly terminated biotinylated DNA fragments are subsequently released and loaded onto a mass spectrometer to obtain accurate DNA sequencing data. Compared with gel electrophoresis-based sequencing systems, MS produces a very high resolution of DNA-sequencing fragments, fast separation on microsecond time scales, and completely eliminates the compressions associated with gel electrophoresis. The high resolution of MS allows accurate mutation and heterozygote detection. This optimized solid-phase DNA-sequencing chemistry plus future improvements in detector sensitivity for large DNA fragments in MS instrumentation will further improve MS for DNA sequencing.  相似文献   

2.
Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide primers was mixed with biotin-ddNTPs, DNA polymerase and the DNA templates containing the SNPs in a single tube. The nucleotide at the 3′-end of each CFET-labeled oligonucleotide primer was complementary to a particular SNP in the template. Only the CFET-labeled primer that is fully complementary to the DNA template was extended by DNA polymerase with a biotin-ddNTP. We isolated the DNA extension fragments that carry a biotin at the 3′-end by capture with streptavidin-coated magnetic beads, while the unextended primers were eliminated. The biotinylated fluorescent DNA fragments were subsequently analyzed in a multicolor fluorescence electrophoresis system. The distinct fluorescence signature and electrophoretic mobility of each DNA extension product in the electropherogram coded the SNPs without the use of a sizing standard. We simultaneously distinguished six nucleotide variations in synthetic DNA templates and a PCR product from the retinoblastoma tumor suppressor gene. The use of CFET-labeled primers and biotin-ddNTPs coupled with the specificity of DNA polymerase in SBE offered a multiplex method for detecting SNPs.  相似文献   

3.
Kim S  Ulz ME  Nguyen T  Li CM  Sato T  Tycko B  Ju J 《Genomics》2004,83(5):924-931
A mass spectrometry (MS) based multiplex genotyping method using solid phase capturable (SPC) dideoxynucleotides and single base extension (SBE), named the SPC-SBE, has been developed for mutation detection. We report here the simultaneous genotyping of 30 potential point mutation sites in exons 5, 7, and 8 of the human p53 gene in one tube using the SPC-SBE method. The 30 mutation sites, including the most frequently mutated p53 codons, were chosen to explore the high multiplexing scope of the SPC-SBE method. Thirty primers specific to each potential mutation site were designed to yield SBE products with sufficient mass differences. This was achieved by tuning the mass of some primers using modified nucleotides. Genomic DNA was amplified by multiplex PCR to produce amplicons of the three p53 exons. The 30 primers were combined with the PCR products and biotinylated dideoxynucleotides for SBE to generate 3'-biotinylated extension DNA products. These products were then captured by streptavidin-coated magnetic beads, while the unextended primers and other components in the reaction were washed away. The pure extension DNA products were subsequently released from the solid phase and analyzed with MS. We simultaneously genotyped 30 potential mutation sites in the p53 gene from Wilms' tumor, head and neck tumor, and colorectal tumor. Both homozygous and heterozygous genotypes were accurately determined with digital resolution. This is the highest level of multiplex genotyping reported thus far using MS, indicating that the approach might be applicable to screening a repertoire of genotypes in candidate genes as potential disease markers.  相似文献   

4.
We report an approach using solid phase capturable biotinylated dideoxynucleotides (biotin-ddNTPs) in single base extension for multiplex genotyping by mass spectrometry (MS). In this method, oligonucleotide primers that have different molecular weights and that are specific to the polymorphic sites in the DNA template are extended with biotin-ddNTPs by DNA polymerase to generate 3′-biotinylated DNA products. These products are then captured by streptavidin-coated solid phase magnetic beads, while the unextended primers and other components in the reaction are washed away. The pure extension DNA products are subsequently released from the solid phase and analyzed by matrix-assisted laser desorption/ionization time-of-flight MS. The mass of the extension products is determined using a stable oligonucleotide as a common internal mass standard. Since only the pure extension DNA products are introduced to the MS for analysis, the resulting mass spectrum is free of non-extended primer peaks and their associated dimers, which increases the accuracy and scope of multiplexing in single nucleotide polymorphism (SNP) analysis. The solid phase purification approach also facilitates desalting of the captured oligonucleotides, which is essential for accurate mass measurement by MS. We selected four biotin-ddNTPs with distinct molecular weights to generate extension products that have a 2-fold increase in mass difference compared to that with conventional ddNTPs. This increase in mass difference provides improved resolution and accuracy in detecting heterozygotes in the mass spectrum. Using this method, we simultaneously distinguished six nucleotide variations on synthetic DNA templates mimicking mutations in the p53 gene and two disease-associated SNPs in the human hereditary hemochromatosis gene.  相似文献   

5.
In this paper we describe a rapid method for the direct generation of DNA sequencing templates from phage or bacteria. Sequencing of these PCR products can be performed by radioactive and fluorescent methods. The non-radioactive method has been used to sequence a total of approximately 100 kb of human DNA fragments generated by digestion with HpaII and subsequent cloning. The method depends on direct small scale amplification using a biotinylated primer, and the binding of the product to streptavidin coated magnetic beads. All the procedures are carried out in a microtitre plate thus facilitating the handling of large numbers of clones and has potential for automation.  相似文献   

6.
Direct cycle sequencing of double-stranded polymerase chain reaction (PCR) products using thermostable polymerase produces fragments that are shorter than expected when the enzyme prematurely detaches as it approaches the 5′-end of the DNA template. These premature terminations result in a substantially reduced reading length of the DNA sequence. Since some DNA templates spontaneously fold and form stable secondary structures at temperatures that are typically used for primer annealing, one factor that may cause premature terminations to occur is the formation of secondary structures in the template during the annealing step of the cycle sequencing reaction. We describe a simple and effective method for reducing premature terminations in DNA sequences. We demonstrate that maintaining the annealing temperature of the cycle sequencing reaction above a critical temperature reduces premature terminations in DNA sequences that regularly contain premature terminations when the temperature of the annealing step is 60°C. In the method described, annealing and extension of the primer along the template take place at the same temperature (72°C). This procedure for reducing premature terminations can be applied when sequencing with primers that are relatively long (at least 27 mer) and have high optimal annealing temperatures.  相似文献   

7.
根据链霉素磁珠和生物素特异结合的特性,用生物素标记的二聚核苷酸重复序列探针从巴氏蘑菇的基因组中分离微卫星序列。将结合于链霉素磁珠上的标记探针同两端连接已知序列人工接头的巴氏蘑菇DNA酶切片段杂交。洗脱未杂交DNA片段后,用磁珠富集的片段建立微卫星文库。挑取522个菌落用对应重复序列为引物进行PCR筛选,得到48个阳性克隆,经测序有32个菌落含微卫星序列。微卫星富集效率为阳性克隆数的67%,总克隆数的6%。除去重复或无效的微卫星序列,在设计出的12对用于鉴别85个巴氏蘑菇的Co60辐射变异株微卫星引物中,有4对引物总共扩增出明显的变异菌株17个。证明有些微卫星位点可用于巴氏蘑菇辐射变异品种的指纹筛选与鉴别。  相似文献   

8.
We report the site-specific fluorescent labeling of DNA using Staudinger ligation with high efficiency and high selectivity. An oligonucleotide modified at its 5' end by an azido group was selectively reacted with 5-[(N-(3'-diphenylphosphinyl-4'-methoxycarbonyl)phenylcarbonyl)aminoacetamido]fluorescein (Fam) under aqueous conditions to produce a Fam-labeled oligonucleotide with a high yield (approximately 90%). The fluorescent oligonucleotide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Because of the relatively high yield of the Staudinger ligation, simple purification of the product by size-exclusion chromatography and desalting is sufficient for the resulting fluorescent oligonucleotide to be used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which are analyzed by a fluorescent electrophoresis DNA sequencer. The results indicate that the Staudinger ligation can be used successfully and site-specifically to prepare fluorescent oligonucleotides to produce DNA sequencing products, which are detected with single base resolution in a capillary electrophoresis DNA sequencer using laser-induced fluorescence detection.  相似文献   

9.
Sequential DEXAS (direct exponential amplification and sequencing), a one step amplification and sequencing procedure that allows accurate, inexpensive and rapid DNA sequence determination directly from genomic DNA, is described. This method relies on the simultaneous use of two DNA polymerases that differ both in their ability to incorporate dideoxynucleotides and in the time at which they are activated during the reaction. One enzyme, which incorporates deoxynucleotides and performs amplification of the target DNA sequence, is supplied in an active state whereas the other enzyme, which incorporates dideoxynucleotides and performs the sequencing reaction, is supplied in an inactive state but becomes activated by a temperature step during the thermocycling. Thus, in the initial stage of the reaction, target amplification occurs, while in the second stage the sequencing reaction takes place. We show that Sequential DEXAS yields high quality sequencing results directly from genomic DNA as well as directly from human blood without any prior isolation or purification of DNA.  相似文献   

10.
The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods plays an important role in the actual effort to improve the efficiency of large-scale DNA analysis. While dideoxy-terminators labeled with energy-transfer dyes (BigDyes) provide the most versatile method of automated DNA sequencing, premature terminations result in a substantially reduced reading length of the DNA sequence. Premature terminations are usually evidenced by base ambiguities and are often accompanied by diminished signal intensity from that point on in the sequence. I studied a two-step protocol for Taq cycle sequencing using the ABI BigDye terminator for reducing premature terminations in DNA sequences. I demonstrate that combining the annealing step with the extension step at one temperature (60°C) reduces premature terminations in DNA sequences that regularly contain premature terminations when the three temperature steps are used. This modification significantly increases the number of accurately read bases in DNA sequences.  相似文献   

11.
The quality of sequencing results is to a large extent determined by the purity of the template and the purification of the sequencing products. Fragments that can act as unspecific primers and templates are removed before gel analysis, and the background of unspecific signals is highly reduced. Purification of the sequencing products is needed to remove salts, nucleotides, proteins and template DNA that can interfere with the gel separation. We have developed a product, DYNAPURE Dye Terminator Removal, that specifically isolates and purifies the sequencing products in 10 min. The method is based on biotinylated sequencing primers and super-paramagnetic streptavidin beads. A PCR product is sequenced using a biotinylated sequencing primer, and the sequencing products are then bound to streptavidin beads in a 5-min reaction. The bead-DNA complexes are magnetically separated from the rest of the solution, and the remaining buffer constituents are washed away with TE buffer or with 70% ethanol. The whole procedure can be automated on liquid-handling robots fitted with a magnet station. The method eliminates purification of templates before cycle sequencing.  相似文献   

12.
Previously, we established the feasibility of using solid phase capturable (SPC) dideoxynucleotides to generate single base extension (SBE) products which were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for multiplex genotyping, an approach that we refer to as SPC-SBE. We report here the expanding of the SPC-SBE method as a single-tube assay to simultaneously detect 20 single nucleotide variations in a model system and 3 single nucleotide polymorphisms (SNPs) in the human beta2-adrenergic receptor (beta2AR) gene. Twenty primers were designed to have a sufficient mass difference between all extension products for accurate detection of nucleotide variants of the synthetic templates related to the p53 gene. These primers were extended simultaneously in a single tube with biotin-ddNTPs to generate 3(')-biotinylated DNA products, which were first captured by streptavidin-coated magnetic beads and then released from the beads and analyzed with MALDI-TOF MS. This approach generates a mass spectrum free of primer peaks and their associated dimers, increasing the scope of multiplexing SNPs. We also simultaneously genotyped 3 SNPs in the beta2AR gene (5(')LC-Cys19Arg, Gly16Arg, and Gln27Glu) from the genomic DNA of 20 individuals. Comparison of this approach with direct sequencing and the restriction fragment length polymorphism method indicated that the SPC-SBE method is superior for detecting nucleotide variations at known SNP sites.  相似文献   

13.
An approach for DNA sequencing is described that circumvents the need for synthetic oligonucleotide primers, which seriously restrict the progress of DNA sequencing in the commonly used protocol. The method is based on the use of short restriction fragments as primers randomly distributed along single-stranded templates. Premapping of target DNA is eliminated and subcloning manipulation is minimized. This method has been used successfully for sequencing genes in the range of 2 kb, for which about 10 restriction fragment primers per kilobase were sufficient to generate a continuous overlapping sequence in alignment. The approach has also been readily applied for an automated sequencing system with the fluorescent chain-terminating dideoxynucleotides, thus implying its potential for sequencing large genomic DNAs.  相似文献   

14.
Characterization of mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) and mutations is crucial for disease diagnosis, which requires accurate and sensitive detection methods and quantification due to mitochondrial heteroplasmy. We report here the characterization of mutations for myoclonic epilepsy with ragged red fibers syndrome using chemically cleavable biotinylated dideoxynucleotides and a mass spectrometry (MS)-based solid phase capture (SPC) single base extension (SBE) assay. The method effectively eliminates unextended primers and primer dimers, and the presence of cleavable linkers between the base and biotin allows efficient desalting and release of the DNA products from solid phase for MS analysis. This approach is capable of high multiplexing, and the use of different length linkers for each of the purines and each of the pyrimidines permits better discrimination of the four bases by MS. Both homoplasmic and heteroplasmic genotypes were accurately determined on different mtDNA samples. The specificity of the method for mtDNA detection was validated by using mitochondrial DNA-negative cells. The sensitivity of the approach permitted detection of less than 5% mtDNA heteroplasmy levels. This indicates that the SPC-SBE approach based on chemically cleavable biotinylated dideoxynucleotides and MS enables rapid, accurate, and sensitive genotyping of mtDNA and has broad applications for genetic analysis.  相似文献   

15.
Scientific literature describing arrayed primer extension and other array-based minisequencing technologies consistently cite the requirement for four fluorescent dideoxynucleotides (with concomitant absence/inactivation of deoxynucleotides) to ensure single-base extension and thus sequence-specific intensity data that can be interpreted as a base call or genotype. We present compelling evidence that fluorescent deoxynucleotides can reliably be used in microarray minisequencing experiments, generating fluorescent sequence extension intensity profiles that are homologous to the single-base extensions obtained with terminator dideoxynucleotides. Due to the almost 10-fold higher costs (and limited fluorophore choice) of many commercially available fluorescent dideoxynucleotides, compared to fluorescent deoxynucleotides, as well as other potentially constraining intellectual property and licensing issues, this hitherto dismissed microarray chemistry represents an important reevaluation in the field of array-based genotyping and related enzymology.  相似文献   

16.
We have developed a new method for typing single nucleotide polymorphisms (SNPs), MagSNiPer, based on single base extension, magnetic separation, and chemiluminescence. Single base nucleotide extension reaction is performed with a biotinylated primer whose 3' terminus is contiguous to the SNP site with a tag-labeled ddNTP. Then the primers are captured by magnetic-coated beads with streptavidin, and unincorporated labeled ddNTP is removed by magnetic separation. The magnetic beads are incubated with anti-tag antibody conjugated with alkaline phosphatase. After the removal of excess conjugates by magnetic separation, SNP typing is performed by measuring chemiluminescence. The incorporation of labeled ddNTP is monitored by chemiluminescence induced by alkaline phosphatase. MagSNiPer is a simple and robust SNP typing method with a wide dynamic range and high sensitivity. Using MagSNiPer, we could perform SNP typing with as little as 10(-17) mol of template DNA.  相似文献   

17.
It is possible to perform a combined amplification and sequencing reaction ('DEXAS') directly from complex DNA mixtures by using two thermostable DNA polymerases, one that favours the incorporation of deoxynucleotides over dideoxynucleotides, and one which has a decreased ability to discriminate between these two nucleotide forms. During cycles of thermal denaturation, annealing and extension, the former enzyme primarily amplifies the target sequence whereas the latter enzyme primarily performs a sequencing reaction. This method allows the determination of single-copy nuclear DNA sequences from amounts of human genomic DNA comparable to those used to amplify nucleotide sequences by the polymerase chain reaction. Thus, DNA sequences can be easily determined directly from total genomic DNA.  相似文献   

18.
19.
Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.  相似文献   

20.
Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA   总被引:31,自引:0,他引:31  
A solid-phase approach is described for manual and automated sequencing of plasmid DNA obtained directly from bacterial colonies through the polymerase chain reaction. The DNA fragment is selectively immobilized to magnetic beads and after strand-specific elution, the eluted strand, as well as the remaining immobilized strand, is used for bidirectional dideoxy sequencing. The solid-phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The approach is exemplified by fluorescent sequencing of a cloned Streptomyces curacoi gene having a G + C content of more than 70%.  相似文献   

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