Various applications of direct PCR using blood samples.
Samples of blood or other animal fluids contain a variety of substances that inhibit the polymerase chain reaction (PCR), meaning that isolation of DNA, involving multiple labor-intensive steps, is generally necessary prior to PCR. We have developed a novel reagent cocktail that effectively suppresses these inhibitory substances. Using this reagent cocktail, DNA from various targets can be efficiently amplified directly from various forms of blood samples without DNA isolation. 1. DNA sequences within the beta-globin gene could be amplified directly from human blood samples treated with various anticoagulants. Either fresh blood or blood samples stored frozen for up to 4 years could be used for PCR. 2. DNA sequences of up to 2056 bp within the beta-globin gene could be amplified directly from human blood samples. 3. Human chromosomal and mitochondrial DNA from different individuals could be amplified directly from blood samples. 4. Low titers of hepatitis B virus could be amplified directly from human blood samples. 5. DNA could be amplified directly from various target sequences using dried blood in a PCR tube or on a filter paper. 6. Transgenes could be detected directly in blood samples from transgenic mice.
Nishimura
Naoyuki
N
Analytical and Measuring Instruments Division, Shimadzu Corporation, Ibaraki, Japan. nisimura@shimadzu.co.jp
Nakayama
Tomoko
T
Tonoike
Hiroshi
H
Kojima
Kouichi
K
Shirasaki
Yoshinari
Y
Kondoh
Kensuke
K
Yamada
Taketo
T
D000818:Animals; D001800:Blood Specimen Collection; D004247:DNA / Q000097:blood*; D004272:DNA, Mitochondrial / Q000097:blood; D004279:DNA, Viral / Q000097:blood; D005914:Globins / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics; D006801:Humans; D007202:Indicators and Reagents; D051379:Mice; D008822:Mice, Transgenic; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012016:Reference Values; D012680:Sensitivity and Specificity; D019076:Transgenes
D004272:DNA, Mitochondrial; D004279:DNA, Viral; D007202:Indicators and Reagents; D005914:Globins; D004247:DNA