Nanocolonies and diagnostics of oncological diseases associated with chromosomal translocations.

This paper reviews chromosomal abnormalities observed in oncological diseases, the history of discovery of chromosomal translocations (a widespread type of chromosomal abnormalities), and statistical data showing a correlation between translocations and emergence of oncological diseases (in particular leukemia). The importance of detection of minimal residual disease (MRD) in treatment of leukemia associated with translocations is discussed along with methods of MRD diagnosis, followed by description of a novel diagnostic procedure for the detection of single chimeric mRNA molecules serving as MRD markers. This procedure includes a number of improvements, of which the most important is the use of a PCR version of the method of nanocolonies (other names are molecular colonies, polonies) that provides for the determination of the absolute titer of RNA tumor markers, excludes false positive results in the detection of chimeric molecules, and significantly exceeds other methods in the sensitivity of MRD detection.

[Use of nanocolonies for detection of minimal residual disease in patients with leukemia t(8;21)].

A complete diagnostic procedure was developed that allows single molecules of mRNA AML1-ETO to be detected in samples of whole blood and bone marrow of the leukemia t(8;21)(q22;q22) patients. The procedure includes: a method for preservation of biological samples ensuring the RNA integrity; an improved method for isolation of RNA from the unfractionated whole blood and bone marrow; an optimized reverse transcription; and the use of nanocolonies for detection and enumeration of RNA target molecules. The developed procedure is the first one that provides for determination of the absolute titer of an RNA target without reference to a control (housekeeping) gene, and significantly increases sensitivity, precision and reliability of detection of the minimal residual disease at a leukemia associated with known chromosomal translocation.

[Express hybridization of molecular colonies with fluorescent probes].

DNA colonies formed during PCR in a polyacrylamide gel and RNA colonies grown in an agarose gel containing Qbeta replicase can be identified using the procedure of transfer of molecular colonies onto a nylon membrane followed by membrane hybridization with fluorescent oligonucleotide probes. The suggested improvements significantly simplify and accelerate the procedure. By the example of a chimeric AML1-ETO sequence, a marker of frequently occurring leukemia, the express hybridization method was shown to allow the rapid identification of single molecules and the determination of titers of DNA and RNA targets.

[Scientific and practical applications of molecular colonies].

Reviewed are the history of invention of the molecular colony technique, also known under name "polony technology", applications of this method to studies of reactions between single RNA molecules, ultrasensitive diagnostics, gene cloning and screening in vitro, and also concepts on the origin of life that consider molecular colonies as a prototype of living organisms.

[Retention of nucleic acid integrity in guanidine thiocyanate lysates of whole blood].

High-molecular-mass RNA and DNA have been shown to retain their integrity for three days at room temperature, no less than two weeks at +4 degrees C, and more than a year at -20 degrees C when whole blood samples are stored as lysates containing 4 M guanidine thiocyanate. Storage time at room temperature can be prolonged at least up to 14 days if nucleic acids were precipitated by two volumes of isopropanol. This preservation technique allows storage and transportation of samples at ambient temperature and is completely compatible with the procedure of subsequent isolation of nucleic acids.