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Coupled amplification and sequencing of genomic DNA.

Authors
Type
Published Article
Journal
Proceedings of the National Academy of Sciences of the United States of America
Publication Date
Volume
88
Issue
7
Pages
2815–2819
Identifiers
PMID: 1672768
Source
Medline
License
Unknown

Abstract

Addition of dideoxyribonucleotides during the exponential phase of the PCR should result in the synthesis of two complementary sequence ladders. We have explored this hypothesis to develop coupled amplification and sequencing of genomic DNA. Coupled amplification and sequencing is a biphasic method for sequencing both strands of template as they are amplified. Stage I selects and amplifies a single target from the genomic DNA sample. Stage II accomplishes the sequencing as well as additional amplification of the target using aliquots from the stage I reaction mixed with end-labeled primer and dideoxynucleotides. We have successfully applied coupled amplification and sequencing to a 300-base-pair fragment 4 kilobases upstream from HOX2B directly from human whole genomic DNA.

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