An efficient and simple method of DNA extraction from whole blood and cell lines to identify infectious agents.
Routine methods of extraction of DNA from blood involve the enrichment of cells by Ficoll-Hypaque gradient centrifugation followed by lysis of the cells with extraction buffer, proteinase K digestion of the lysate, and phenol:chloroform-isoamyl alcohol extraction. These methods generally require large amounts of blood, which poses a problem with pediatric patients. To overcome this, we developed a new method of extracting DNA directly from whole blood. This method involves the treatment of whole blood with an equal volume of NaI (3 M final concentration) followed by chloroform:isoamyl alcohol extraction to clear hemoglobin and cell debris. The clear aqueous layer is then mixed with isopropanol to obtain DNA. A large number of samples can easily be handled by this extraction procedure, as it can be carried out in 30 min and requires only a microcentrifuge.
Loparev
V N
VN
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Cartas
M A
MA
Monken
C E
CE
Velpandi
A
A
Srinivasan
A
A
D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D004279:DNA, Viral / Q000097:blood / Q000302:isolation & purification*; D006678:HIV / Q000235:genetics / Q000302:isolation & purification; D015658:HIV Infections / Q000382:microbiology; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012974:Sodium Iodide
D004279:DNA, Viral; D012974:Sodium Iodide