Selecting and amplifying one fragment from a DNA fragment mixture by polymerase chain reaction with a pair of selective primers.
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 according 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.
Matsunaga
H
H
Central Research Laboratory, Hitachi Ltd., Tokyo, Japan.
Kohara
Y
Y
Okano
K
K
Kambara
H
H
D004247:DNA / Q000032:analysis* / Q000378:metabolism; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods; D006801:Humans; D008433:Mathematics; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004247:DNA; C024669:endodeoxyribonuclease AluI; C048422:endodeoxyribonuclease NlaIII; D015252:Deoxyribonucleases, Type II Site-Specific