Coupled amplification and sequencing of genomic DNA.

Ruaño, Gualberto; Kídd, Kenneth K.

doi:10.1073/pnas.88.7.2815

Cited by 78 publications

(22 citation statements)

References 22 publications

(16 reference statements)

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“…The improvements that have been described include separating the labeling step from the termination step (1 l), altering the concentrations of specific deoxynucleoside triphosphates (40), utilizing low numbers of cells (27,39), sequencing DNA PCR amplicons (6) with an additional 70°C extension step (4), and coupling DNA amplification with thermal cycle sequencing (30,33). None of the methods that have been described combined all of the characteristics for developing sequence information desired by us.…”

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confidence: 99%

Comparison of Mycobacterium 23S rRNA Sequences by High-Temperature Reverse Transcription and PCR

Stone

Nietupski

Breton

et al. 1995

International Journal of Systematic Bacteriology

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We describe a modified rRNA sequence analysis method which we used to determine the phylogenetic relationships among 58 species belonging to the genus Mycobacterium. We combined the sensitivity of the reverse transcriptase PCR for amplifying nanogram amounts of template rRNA material with the elevated extension temperatures used for the thermostable DNA polymerase from Thermus thermophilus. A 70°C reverse transcription extension step permitted improved read-through of highly structured rRNA templates from members of the genus Mycobacterium, which have G+C contents of 66 to 71 mol%. The nucleic acid sequences of the amplified material were then determined by performing thermal cycle sequencing with a-"P-labeled primers, again with extension at 70°C. Nonspecifically terminated bands were chased by using terminal deoxynucleotidyl transferase. Our method had a template requirement of nanogram amounts or less of purified RNA or 2,000 CFU of intact cells and had sufficient sensitivity so that lyophils obtained from the American Type Culture Collection could be used as source material. Sequences from a 250-nucleotide stretch of the 23s rRNA were aligned, and phylogenetic trees were evaluated by using the De Soete distance treeing algorithm and Rhodococcus bronchialis as the outgroup. Our 23s rRNA trees were compared with previously published 16s rRNA trees, including the comprehensive trees developed by the University of Illinois Ribosomal Database Project, and included 15 species not evaluated previously. Most of the groups were in general agreement and were consistent with relationships determined on the basis of biochemical characteristics, but some new relationships were also observed.

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confidence: 99%

Comparison of Mycobacterium 23S rRNA Sequences by High-Temperature Reverse Transcription and PCR

Stone

Nietupski

Breton

et al. 1995

International Journal of Systematic Bacteriology

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“…Previously attempts require that the samples be partitioned after several cycles of amplification so that radioactively labeled primers and dideoxynucleotides can be added to eight individual reactions. 18,19 We anticipate that SimulSeq and AmpliSeq can be combined to simultaneously amplify and sequence multiple genes at the same time. Although AmpliSeq and SimulSeq require attention to primer design, they require no additional steps, sample manipulations, or reagents such that any lab currently performing DNA sequencing reactions can perform either of these techniques.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions

Murphy

Eshleman

2002

The American Journal of Pathology

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DNA sequencing is considered the gold standard for nucleic acid identification and mutation detection. However, sequencing is labor intensive because it requires previous amplification and only a single sequence is analyzed at a time. We developed two novel strategies that substantially improve DNA sequencing. The first allows multiple polymerase chain reaction (PCR) products to be sequenced in a single sequencing reaction and analyzed simultaneously in a single lane or capillary. Simultaneous sequencing by this method, designated "SimulSeq," can provide either simultaneous single-direction sequencing of multiple genes or simultaneous forward and reverse sequencing from a single gene. In the second approach, designated "AmpliSeq," we demonstrate a technique combining PCR amplification and sequencing in a single reaction that is analyzed in a single lane or capillary. We demonstrate combined PCR with short bidirectional sequencing, and combined PCR with unidirectional sequencing. We anticipate that these methods will have utility in research and clinical settings where panels of mutations or large numbers of samples are be analyzed and/or when turnaround time is critical. (Am J Pathol 2002, 161:27-33) DNA sequencing 1 has been the standard against which other types of DNA testing is compared. Major advances in DNA sequencing include the development of automated sequencers, 2 discovery of fluorescent terminator chemistry, 3 and cycle sequencing. 4 These developments have made sequencing easier to perform and therefore more widely used. Currently, sequencing is used to identify microbial drug resistance mutations, 5 cancer predisposition, somatic mutations, 6 and genetic diseases. 7With the cloning and sequencing of the human genome 8,9 and the new era of molecular medicine, one can only expect the use of DNA sequencing to increase.Despite advances in sequencing technology, significant limitations remain. First, most applications require polymerase chain reaction (PCR) amplification of the target sequence, and purification of the product before sequencing. Second, standard Sanger-sequencing reactions are performed with a single primer and therefore yield only a single sequence. These limitations are costly and have somewhat hindered the widespread application of DNA sequencing in clinical and research settings.In this article we report two novel sequencing strategies that directly address these limitations. In the first, we engineered sequencing reactions to permit simultaneous sequencing of multiple PCR products in a single lane. Under normal conditions, multiple sequencing reactions run simultaneously are superimposed on each other because the sequencing products overlap in size. SimulSeq prevents this because of two principles: sequencing products stop when the end of a PCR product is reached, and long oligonucleotide primers can be used to prevent otherwise overlapping short sequencing products. In the second sequencing strategy, we designed conditions and primer modifications to permit combined PCR and sequenc...

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“…The first 70 bp could not be read because of overlap with the residual dye-terminators. Finally, direct exponential amplification and sequencing (DEXAS) uses sequential activation of PCR and sequencing polymerases to combine PCR and sequencing reactions (28,29 ). DEXAS also uses dye-primer chemistry, requiring multiple sequencing reactions per sample.…”

Section: Discussionmentioning

confidence: 99%

Single-Tube Method for Nucleic Acid Extraction, Amplification, Purification, and Sequencing

et al. 2004

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Background: The hepatitis C virus (HCV) genotype determines patient prognosis and duration of treatment, but sequencing of the gene is lengthy and labor-intensive. We used a commercially available nucleic acid extraction system to develop a single-tube extraction-tosequencing (STETS) method for HCV genotyping. Methods: HCV RNA was purified and amplified in tubes coated with a solid-phase matrix that irreversibly bound nucleic acid during the extraction step. After reverse transcription-PCR, the amplicon was adsorbed to the original extraction matrix for purification and use in the subsequent sequencing reactions. Results: The STETS method generated genotypingquality sequence for a range of HCV titers from 500 to 6 000 000 IU/mL. If a viral sample was detected during real-time reverse transcription-PCR, it could be sequenced and genotyped. Read lengths >600 bases were observed with the STETS method. Mixed infections were detected and genotyped if at least 15% of the minor species was present. Combining the STETS method with consecutive sequencing provided a means of performing both forward and reverse sequencing in a single tube. Conclusions: A single-tube nucleic acid extraction-tosequencing method, which requires less time and labor than conventional methods, generates HCV sequence data that are equivalent to conventional methods and can be used to genotype HCV.

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Coupled amplification and sequencing of genomic DNA.

Cited by 78 publications

References 22 publications

Comparison of Mycobacterium 23S rRNA Sequences by High-Temperature Reverse Transcription and PCR

Comparison of Mycobacterium 23S rRNA Sequences by High-Temperature Reverse Transcription and PCR

Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions

Single-Tube Method for Nucleic Acid Extraction, Amplification, Purification, and Sequencing

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