Use of magnetic beads for tissue DNA extraction and IS6110 Mycobacterium tuberculosis PCR.
Polymerase chain reaction (PCR) techniques are used increasingly for the diagnosis of Mycobacterium tuberculosis infection and can be used on the DNA obtained from both frozen and formalin fixed, paraffin wax embedded tissues. However, the extraction of DNA by means of the conventional phenol/chloroform method is time consuming and requires the use of potentially dangerous chemical reagents. This paper describes a method based upon the use of magnetic beads for the extraction of M tuberculosis DNA from both routinely formalin fixed, paraffin wax embedded tissues and frozen tissues. Magnetic bead extracted DNA from brain, lymph node, and lung tissues collected from patients with human immunodeficiency virus and tuberculosis was compared with that extracted using the phenol/chloroform method. The magnetic bead extraction procedure requires less than two hours, including the time necessary to dewax the tissue sections. In all cases, the DNA extracted with both methods was amplified successfully by PCR for the M tuberculosis IS6110 sequence. Magnetic bead DNA extraction can be used on both frozen and archival tissues: the method is reliable, simple, sensitive, and rapid; in addition, it does not use hazardous procedures or specialised laboratory equipment and can be used for routine DNA isolation from various human tissues.
Caldarelli-Stefano
R
R
Pathology Unit, L. Sacco Hospital, Institute of Biomedical Sciences, University of Milan, Italy.
Vago
L
L
Bonetto
S
S
Nebuloni
M
M
Costanzi
G
G
D001921:Brain / Q000382:microbiology; D015925:Cryopreservation; D004269:DNA, Bacterial / Q000302:isolation & purification*; D006801:Humans; D008168:Lung / Q000382:microbiology; D008198:Lymph Nodes / Q000382:microbiology; D008280:Magnetics; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D014376:Tuberculosis / Q000175:diagnosis*
D004269:DNA, Bacterial