Direct DNA sequence determination from total genomic DNA

Kilger, Christian

doi:10.1093/nar/25.10.2032

Cited by 10 publications

(13 citation statements)

References 7 publications

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“…A method for resequencing called direct amplification and sequencing (DEXAS) is nominally simpler than our protocol because the PCR and sequencing steps are performed in the same tube at the same time using two kinds of DNA polymerases (Kilger and Paabo 1997). However, with competing polymerases, single optimized conditions that yield consistent results for high-throughput reaction using a variety of GC content DNAs may not be possible for DEXAS.…”

Section: Resultsmentioning

confidence: 99%

Efficient High-Throughput Resequencing of Genomic DNA

Miller

Duan²,

Lovins³

et al. 2003

Genome Res.

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Targeted resequencing of genomic DNA from organisms such as humans is an important tool enabling experimental access to variation within the species and between similar species. Taking full advantage of the reference genome sequences in designing robust, specific PCR assays and using stringent conditions, resequencing can be done efficiently without purification of the PCR product. By using a 10-fold greater amount of one primer when setting up the PCR initially in a new version of asymmetric PCR, one simply adds the rest of the sequencing reagents at the end of PCR and allows the sequencing reaction to proceed, with the excess PCR primer serving as the sequencing primer. We demonstrated that this streamlined protocol can be used with PCR products up to 1300 bp and had up to a 97% success rate in high-throughput analysis of allele frequencies for >30,000 single-nucleotide polymorphisms (SNPs). SNP primers and characterization results are provided athttp://snp.wustl.edu.

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Section: Resultsmentioning

confidence: 99%

Efficient High-Throughput Resequencing of Genomic DNA

Miller

Duan²,

Lovins³

et al. 2003

Genome Res.

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“…Kilger and Pääbo (1997b) suggested to use two different DNA polymerases in the same PCR, one of them strongly preferring dNs and the other with a relatively higher affinity to ddNs (the latter possessing the F667Y mutation). Kilger and Pääbo (1997b) suggested to use two different DNA polymerases in the same PCR, one of them strongly preferring dNs and the other with a relatively higher affinity to ddNs (the latter possessing the F667Y mutation).…”

Section: Resultsmentioning

confidence: 99%

Theoretical Description of the Direct Exponential Amplification and Sequencing (DEXAS) Method

Ingr

Konvalinka²

2000

Biological Chemistry

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We present a theoretical description of the method of DNA sequencing with simultaneous exponential PCR amplification of the template (DEXAS). Based on the theory of probability, the formula determining the optimal ratio of concentrations of deoxy- and dideoxynucleotides in the reaction mixture is derived, as well as the length distribution of sequenced DNA fragments. The prediction of the number of mutations is given and the theoretically determined aspects of DEXAS are compared with the corresponding quantities of classical sequencing methods. Some other experimentally observed effects are also discussed.

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“…One previously described method, coupled amplification and sequencing, used a two-stage protocol in which samples were manually partitioned after several cycles of amplification so that labeled primers and ddNTPs could be added to eight individual reactions (10,11 ). A second previously described method, termed DEXAS (direct exponential amplification and sequencing), was based on dye-primer sequencing chemistry (12,13 ). DEXAS required four separate sequencing reactions and used two separate DNA polymerases; one for amplification (incorporation of dNTPs) and one for sequencing (incorporation of ddNTPs) (14 ).…”

Section: Discussionmentioning

confidence: 99%

Sequencing of Genomic DNA by Combined Amplification and Cycle Sequencing Reaction

2005

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Background: Despite considerable advances, DNA sequencing has remained somewhat time-consuming and expensive, requiring three separate steps to generate sequencing products from a template: amplification of the target sequence; purification of the amplified product; and a sequencing reaction. Our aim was to develop a method to routinely combine PCR amplification and cycle sequencing into one single reaction, enabling direct sequencing of genomic DNA. Methods: Combined amplification and sequencing reactions were set up with Big Dye TM sequencing reagents (Applied Biosystems) supplemented with variable amounts of forward and reverse primers, deoxynucleotide triphosphates (dNTPs), and input DNA. Reactions were thermal-cycled for 35 or 45 cycles. Products were analyzed by capillary electrophoresis to detect sequencing products. Results: Reactions using two oligonucleotide primers at a ratio of 5:1 (500 nM primer 1 and 100 nM primer 2), 125 M supplemental dNTPs, and 35-45 thermal cycles optimally supported combined amplification and cycle sequencing reactions. Our results suggest that these reactions are dominated by PCR during early cycles and convert to cycle sequencing in later cycles. We used this technique for a variety of sequencing applications, including the identification of germline mutations/polymorphisms in the Factor V and BRCA2 genes, sequencing of tumor DNA to identify somatic mutations in the DPC4/SMADH4 and FLT3 genes, and sequencing of 16S ribosomal DNA for bacterial speciation. Conclusions: PCR amplification and cycle sequencing can be combined into a single reaction using the conditions described. This technique allows direct sequenc-

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Direct DNA sequence determination from total genomic DNA

Cited by 10 publications

References 7 publications

Efficient High-Throughput Resequencing of Genomic DNA

Efficient High-Throughput Resequencing of Genomic DNA

Theoretical Description of the Direct Exponential Amplification and Sequencing (DEXAS) Method

Sequencing of Genomic DNA by Combined Amplification and Cycle Sequencing Reaction

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