Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-10-22
1999-06-15
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
536 243, C12Q 168
Patent
active
059121262
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to the field of DNA detection for basic research, medical diagnostic testing, and forensic testing. More specifically, the invention relates to methods of detecting DNA by attaching labeling groups to the ends of DNA strands, and then detecting the labeling groups by using antibodies specific for those labeling groups to attach a secondary label that can be observed, for example, by eye or by a fluorescence or spectrophotometric detector.
The present invention has particular utility in detecting the occurrence of programmed cell death, which is known as "apoptosis." It also has utility as a method for detecting DNA replication and repair, and as a general method for DNA end labeling.
BACKGROUND ART
There is currently considerable interest in the process of programmed cell death, known as "apoptosis." This process is considered to be a normal part of an organism's biological defense system. For example, when cells become infected with certain viruses, the resulting overstimulation of the cellular machinery appears to trigger apoptosis; the infected cells die, and thus protect the remaining cells from infection. It is also believed that apoptosis is an important weapon in the body's defense against cancerous growths, and that cancer results at least in part from a loss of the ability of affected cells to trigger their own death.
Researchers often wish to determine whether cells have died because of apoptosis, or because of some other cause. This is generally determined by examining the DNA of the cells. The DNA of cells that have died from apoptosis is typically broken into quite small and uniquely sized fragments, which is generally not the case when cells die from other causes. The DNA fragmentation that is characteristic of apoptosis is caused by enzymes in the cells known as endonucleases, and results in fragments of approximately 300 kilobases and 50 kilobases in length. Often, breaks in the DNA occur in sections between the nucleosome proteins, leading to broken pieces of DNA 180-200 base pairs long (Arends, Morris & Wyllie 1990, Compton 1992, Oberhammer et al. 1993, Walker et al. 1994, Wyllie, Kerr & Curie 1980).
To determine whether apoptosis has occurred, the DNA strand breaks need to be labeled in some manner so that they can be detected. Cell samples are generally first fixed with a crosslinking fixative, so that the small fragments of DNA are not lost from the sample cells in subsequent washing steps. The cells are then treated to make them permeable to further reagents. In two prior art methods, the permeabilized cells are then reacted with deoxynucleotides that have been labeled with either biotin or digoxygenin, using the enzymes DNA polymerase (the "nick translation" enzyme) or terminal deoxynucleotidyl transferase (TdT) to attach the labeled nucleotides to the 3'OH ends of the DNA fragments (Darzynkiewicz et al. 1992, Gorczyca et al. 1992; Gavrieli et al. 1992, Gorczyca, Gong & Darzynkiewicz 1993, Wijksman et al. 1993). The biotin or digoxygenin nucleotides themselves are not readily detectable; however, biotin can be specifically bound by the lectin avidin, and antibodies that can specifically bind digoxygenin are also available. By binding avidin or digoxygenin antibodies to the fluorescent compound fluorescein, which can be done by reacting them with fluorescein-5-isothiocyanate (FITC), and then using these as secondary labels, the DNA strand breaks can be visualized by observing the fluorescence of the labeled DNA strand ends. A single step method utilizing deoxynucleotides directly labeled with fluorochromes, which is simpler but less sensitive than the above-described indirect methodology, has also been recently described (Gold et al. 1993, Li, et al. 1995).
Another area of active research is the study of DNA replication and repair. One method for detecting the cellular replication and/or repair of DNA is known as Strand Breaks Induced by Photolysis, or SBIP. In this method, the living cells are first supplied with halogenated DNA precu
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Darzynkiewicz Zbigniew
Li Xun
Traganos Frank
Horlick Kenneth R.
Siew Jeffrey
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