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Selecting and amplifying one fragment from a DNA fragment mixture by polymerase chain reaction with a pair of selective primers
Author(s) -
Matsunaga Hiroko,
Kohara Yoshinobu,
Okano Kazunori,
Kambara Hideki
Publication year - 1996
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150171207
Subject(s) - electropherogram , subcloning , fragment (logic) , primer (cosmetics) , dna , microbiology and biotechnology , polymerase chain reaction , primer dimer , restriction enzyme , chemistry , base pair , capillary electrophoresis , biology , biochemistry , multiplex polymerase chain reaction , plasmid , gene , organic chemistry , computer science , programming language
A new method for selecting and amplifying a single DNA fragment from a mixture is proposed. This method is applicable for the rapid classification of DNA fragments from a mixture and for preparation of sequencing templates. DNAs of several to tens of kilobases (kb) are digested with a four‐base recognition restriction enzyme to produce smaller fragments. The complementary strand extension reactions are then carried out to produce fluorophore‐labeled DNA fragments from the digestion products. These fragments can be rapidly classified accrording to their terminal‐base sequences and their sizes are analyzed by capillary‐array gel electrophoresis (CAGE). Electropherograms are used to characterize the fragments and to select polymerase chain reaction (PCR) primers. Any fragment in a digestion mixture can be amplified by PCR with a pair of primers selected from a primer pool by referring to the electropherograms of the fragments. This method was successfully used to compare the electropherograms of two different DNA strands and to sequence a several‐kb DNA fragment without subcloning. Combined with CAGE, this method could be used to dramatically simplify DNA fragment analysis.