[Development of a biochip for quantitative determination of two forms of prostate specific antigen using internal calibration curve].

Three-dimensional gel-based microchip allowing simultaneous quantitative detection of total (PSAtot) and free (PSAfree) forms of prostate specific antigen in human serum (in a format "one patient-one biochip") was developed. A method, which doesn't require preliminary construction of calibration curves when performing an assay, was applied for quantitative determination of PSAtot and PSAfree. Gel elements with immobilized antigen (PSA) in different concentration, forming an internal calibration curve, were included in a structure of the microchip, in addition to the elements with immobilized antibodies specific against PSAtot and PSAfree. The specialized software "ImaGelAssay" was used for data processing and interpretation. The sensitivity of the assay performed on biochips was 0.3 ng/ml for PSAtot and 0.2 ng/ml for PSAfree. Variation coefficient for the measurements inside one series of microchips didn't exceed 10%. Correlation coefficient between the results of measurements in human sera obtained on biochips and by the standard ELISA method was 0.988 for PSAtot and 0.987 for PSAfree.

[Detection of single base polymorphism in p53 gene by ligase detection reaction and rolling circle amplification on microarrays].

We combined three modern technologies of single base polymorphism detection in human genome: ligase detection reaction, rolling circle amplification and IMAGE hydro-gel microarrays. Polymorphism in target DNA was tested by selective ligation on microarray. Product of the ligase reaction was determined in microarray gel pads by rolling circle amplification. Two different methods were compared. In first, selective ligation of short oligonucleotides immobilized on microarray was used with subsequent amplification on preformed circle probe ("common circle"). The circle probe was designed especially for human genome research. In second variant, allele-specific padlock probes that may be circularized by selective ligation were immobilized on microarray. Polymorphism of codon 72 in human p53 gene was used as a biological model. It was shown that LDR/RCA on microarray is a quantitative reaction and gives high discrimination of alleles. Principles and perspectives of selective ligation and rolling circle amplification are being discussed.

[Analysis of chromosome translocations involving MML by hybridization with an oligonucleotide microarray]. / Analiz khromosomnykh translokatsii s uchastiem gena MLL metodom gibridizatsii s oligonukleotidnym mikrochipami.

Identification of chromosome rearrangements is of importance for exact diagnosis, risk assessment, and therapy in blood malignancies. A new method was proposed for rapid and accurate identification of leukemia forms caused by chromosome rearrangements involving MLL (11q23). The method combines reverse transcription-multiplex PCR and hybridization with an oligonucleotide microarray. The microarray was designed to detect the five most common MLL rearrangements: t(4;11) MLL/AF4, t(9;11) MLL/AF9, t(11;19) MLL/ELL, t(11;19) MLL/ENL, and dup(11) MLL/MLL. With clinical specimens, the method was shown to efficiently identify the chromosome translocations in leukemia patients.

[Microchips based on three dimensional gel cells: history and perspective]. / Mikrochipy na osnove trekhmernykh iacheek gelia: istoriia i perspektivy.

The review describes the history of creation and development of the microchip technology and its role in the human genome project in Russia. The emphasis is placed on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous versions. The microchips are manufactured by photo-initiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical strength and stability, which allowed one to work with the DNA fragments of up to 500 nt in length, as well as with rather large protein molecules. At present, the gel-based microchips are widely applied to address different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA-protein interactions. The oligonucleotide microchips are a cheap and reliable tool of diagnostics designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; for diagnostics of orthopoxviruses, including the smallpox virus; for diagnostics of the anthrax pathogen; and for identification of chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteomics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which open a wide potential for creation biosensors on the basis of microchips.