Biochips for Individual Genetic Identification
Study of Applicability of Biochip Technology for Individual Genetic Characterization in Forensic Genetic Testing and Formation of DNA Databases
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
8 Project completed
Senior Project Manager
Komashko B A
Engelhardt Institute of Molecular Biology, Russia, Moscow
- Forensic Science Center of the Ministry of Home Affairs of the Russian Federation, Russia, Moscow\nInstitute of Genetics and Selection of Industrial Microorganisms, Russia, Moscow
- US Department of Energy, USA, DC, Washington
Project summaryThe aim of the project is the development of biochip technology for inpidual genetic characterization using human tissues and biological traces in forensic genetic testing and formation of DNA databases.
At the present time the problem of inpidual genetic characterization and formation of DNA databases is carried out using the analysis of human polymorphous microsatellite loci, so-called Short Tandem Repeats (STR). This technology makes possible the practical solution of identification problems and the DNA database formation, however it is a durable procedure, which requires expensive equipment and expendable materials, as well as special laboratory conditions.
In accordance with the instructions №№ BМ-П4-11781 of 10.08.2002, ВМ-П4-16760 of 30. 11.2002, ВМ-П4-01431 of 06.02.2003, and П4-5890 of 22.03.04 of the Government of Russian Federation, the Ministry of the Interior of Russia together with interested Ministries and Departments is preparing the project of federal law “On the State of Genomic Registration in the Russian Federation.” The realization of the suggested law is based on using STR-typing technology with expensive foreign equipment and continuous purchasing of imported reagents. In addition, highly experienced staff is needed to perform the testing.
Principally different approach to solve the problems of inpidual genetic characterization using biological traces is the analysis of highly polymorphous regions of human genes using the biological microchip technology. In the case of positive results demonstrating the forensic applicability of biochip technology for genetic characterization of human tissues, fluids and biological traces, it will be possible to consider this approach as an alternative technology for the formation of federal databases, obtained with the use of the DNA analysis technique.
The development of biological microchips is based on new technologies that are widely used for detection and identification of different infectious pathogens, in diagnostics of inborn genetic diseases, for determination of compatibility of organ and tissue transplants, as well as for solution of other important medical problems. The main advantage of the biochip technology is the possibility to obtain rapidly the highly reliable information using relatively inexpensive equipment.
The project is suggested to carry out in two Phases. The total cost of the project is estimated as 630,8 K; in this case 80,8 K should be spent at the first Phase and 550,0 K at the second Phase.
The realization of the project during the first Phase suggests, first, the development of biochips for allelic characterization of highly polymorphous locus HLA-DQA1(chromosome 6p21), as well as amelogenin locus (AMELX of chromosome Xp22.3-p22.1 and AMELY of chromosome Yp11) which is used for the sex determination, and, second, the evaluation of forensic applicability of the technology for inpidual characterization and formation of DNA databases.
The following problems will be solved during the first phase:
1. Design of oligonucleotide probes for the amelogenin locus analysis (AMELX of chromosome Xp22.3-p22.1 and AMELY of chromosome Yp11) that is used for sex determination. Design and synthesis of primers for polymerase chain reaction.
2. Cloning of DNA fragments containing highly polymorphous sequences of the 2 exon of HLA-DQA1 gene. The determination of cloned fragment nucleotide sequences and identification of the HLA-DQA1 gene alleles. The establishment of a collection of recombinant plasmids containing major allele variants of the HLA-DQA1 gene exon 2, which will be used to optimize the parameters of the HLA-DQA1 gene allele identification using the biochip technology.
3. Design of oligonucleotide probes for the analysis of HLA-DQA1 gene, exon 2 polymorphism. The design and synthesis of primers for polymerase chain reaction.
4. Development of a model biochip for inpidual genetic characterization, which will enable to define the affiliation of an inpidual to one of ninety groups formed upon combination of allele variants of two above-mentioned loci. The biochip preparation will be carried out using photo-induced co-polymerization of oligonucleotide probes complementary to polymorphism regions in polyacrylamide gel. Gels of different composition and different polymerization conditions will be tested to provide the highest efficiency of hybridization and the best reproducibility of the results.
5. Testing of oligonucleotide probes. Each oligonucleotide probe will be tested for efficient binding to a complementary sequence in experiments with control samples from the collection of recombinant plasmids.
6. Carrying out the pilot experiments on biological analysis of samples originated from different sources using a portable biochip analyzer. The development of software for the analysis of such kind biochips. The determination of the biochip analysis sensitivity. The evaluation of forensic applicability of this approach for the inpidual genetic characterization.
In the case of positive results obtain at the first Phase, it is planned to launch the second Phase of the Project. This Phase suggests further extension of biochip, involvement in the analysis of other polymorphous loci located on different human chromosomes. The use of such extended biochip will allow one to discriminate between no less than 1012 genomes, differing by selected loci. The DNA database, that is prepared in parallel, will allow one to evaluate the frequency of different alleles in each locus and thus the possibility of unambiguous identification using an appropriate biochip.
The following problems will be solved during the second Phase:
- The development of biochips for identification of alleles of the highly polymorphous loci HLA-DRB1 (chromosome 6p21, about 160 alleles), ABO (chromosome 9q34), CYP2D6 (chromosome 22q13.1), CYP3A4 (chromosome 7q22.1), NAT-2 (chromosome 8p23.1-p21.3), ADRB2 (chromosome 5q33-34), etc.;
- The improvement of methodological approaches enabling simultaneously precise analysis on several loci;
- The optimization of isolation techniques and preliminary amplification of DNA from minor biological traces;
- The Construction and manufacturing of an analyzer with appropriate software;
- The testing of the model biochip;
- The up-date of the model biochip and analyzer;
- The testing of up-dated model and analyzer using expert DNA samples.