Direct polymerase chain reaction from whole blood without DNA isolation.
Blood and other animal fluids contain a variety of substances that inhibit the polymerase chain reaction (PCR), so that isolation of DNA is generally necessary prior to PCR. We have developed a novel reagent cocktail that effectively suppresses these inhibitory substances and makes DNA isolation from blood unnecessary for PCR. When this reagent was included in the PCR mixture, DNA fragments of the beta-globin gene could be efficiently amplified directly from human blood samples treated with various anticoagulants or PCR-inhibitory substances. We confirmed the usefulness of this cocktail by examining a large number of blood samples with various PCR primer sets. In addition to fresh blood, this method enabled PCR amplification from blood samples stored at 4 degrees C, -20 degrees C or -80 degrees C for a minimum of 1 year.
Nishimura
N
N
Technology Research Laboratory, Shimadzu Corp, Tsukuba City, Ibaraki, Japan. nisimura@shimadzu.co.jp
Nakayama
T
T
Tonoike
H
H
Kojima
K
K
Kato
S
S
D000818:Animals; D004247:DNA / Q000097:blood* / Q000737:chemistry; D004586:Electrophoresis; D005914:Globins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D007202:Indicators and Reagents; D005914:Globins; D004247:DNA