Genomic amplification with transcript sequencing.
A sequencing method called genomic amplification with transcript sequencing (GAWTS) is described that is based on amplification with the polymerase chain reaction (PCR). GAWTS bypasses cloning and increases the rate of sequence acquisition by at least fivefold. The method involves the attachment of a phage promoter onto at least one of the PCR primers. The segments amplified by PCR are transcribed to further increase the signal and to provide an abundance of single-stranded template for reverse transcriptase-mediated dideoxy sequencing. An end-labeled reverse transcriptase primer complementary to the desired sequence generates the additional specificity required to generate unambiguous sequence data. GAWTS can be performed on as little as a nanogram of genomic DNA. The rate of GAWTS can be increased by coamplification and cotranscription of multiple regions as illustrated by two regions of the factor IX gene. Since GAWTS lends itself well to automation, further increases in the rate of sequence acquisition can be expected.
Stoflet
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ES
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905.
Koeberl
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DD
Sarkar
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Sommer
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SS
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases; D004587:Electrophoresis, Agar Gel; D005091:Exons; D005164:Factor IX / Q000235:genetics*; D006467:Hemophilia A / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques; D013604:T-Phages / Q000201:enzymology; D014158:Transcription, Genetic
D005164:Factor IX; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase