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Sequential DEXAS: a method for obtaining DNA sequences from genomic DNA and blood in one reaction.

ABSTRACT: 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.

SUBMITTER: Motz M

PROVIDER: S-EPMC219489 | biostudies-literature | 2003 Oct

REPOSITORIES: biostudies-literature

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Sequential DEXAS: a method for obtaining DNA sequences from genomic DNA and blood in one reaction.

Motz Michael M Sagner Gregor G Pääbo Svante S Kilger Christian C

Nucleic acids research 20031001 20

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 amplificat ...[more]

PMID: 14530454

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Project description:Retrotransposons with long terminal repeats (LTR-RTs) are widespread mobile elements in eukaryotic genomes. We obtained a total of 81 partial LTR-RT sequences from lentil corresponding to internal retrotransposon components and LTRs. Sequences were obtained by PCR from genomic DNA. Approximately 37% of the LTR-RT internal sequences presented premature stop codons, pointing out that these elements must be non-autonomous. LTR sequences were obtained using the iPBS technique which amplifies sequences between LTR-RTs. A total of 193 retrotransposon-derived genetic markers, mainly iPBS, were used to obtain a genetic linkage map from 94 F7 inbred recombinant lines derived from the cross between the cultivar Lupa and the wild ancestor L. culinaris subsp. orientalis. The genetic map included 136 markers located in eight linkage groups. Clusters of tightly linked retrotransposon-derived markers were detected in linkage groups LG1, LG2, and LG6, hence denoting a non-random genomic distribution. Phylogenetic analyses identified the LTR-RT families in which internal and LTR sequences are included. Ty3-gypsy elements were more frequent than Ty1-copia, mainly due to the high Ogre element frequency in lentil, as also occurs in other species of the tribe Vicieae. LTR and internal sequences were used to analyze in silico their distribution among the contigs of the lentil draft genome. Up to 8.8% of the lentil contigs evidenced the presence of at least one LTR-RT similar sequence. A statistical analysis suggested a non-random distribution of these elements within of the lentil genome. In most cases (between 97% and 72%, depending on the LTR-RT type) none of the internal sequences flanked by the LTR sequence pair was detected, suggesting that defective and non-autonomous LTR-RTs are very frequent in lentil. Results support that LTR-RTs are abundant and widespread throughout of the lentil genome and that they are a suitable source of genetic markers useful to carry out further genetic analyses.

Dataset Information

Sequential DEXAS: a method for obtaining DNA sequences from genomic DNA and blood in one reaction.

Publications

Sequential DEXAS: a method for obtaining DNA sequences from genomic DNA and blood in one reaction.

Similar Datasets

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