1997
DOI: 10.1093/nar/25.10.2032
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Direct DNA sequence determination from total genomic DNA

Abstract: 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 re… Show more

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Cited by 10 publications
(13 citation statements)
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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%
“…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%
“…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%
“…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%