DNA MICROCHIPS TECHNOLOGY FOR HLA CLASS I TYPING

G.B. Ferrara°,C. Pera°, L. Delfino°, G. Salani*, C. Consolandi*, C. Battaglia*, R. Cubeddu^, P. Dario §, L. Rossi Bernardi* & G.L. De Bellis#

° National Cancer Institute c/o Advanced Biotechnology Center, Genoa ITALY

# ITBA CNR, Milano ITALY. * Universita' degli Studi di Milano, Dip di Scienze e Tecnologie Biomediche Avanzate Milano ITALY. ^Politecnico, Milano ITALY  § Scuola Sup. S. Anna, Pisa ITALY 

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The aim of this study was to set up a fast methodology that could give a strong discrimination among hundreds of HLA class I alleles in the field of HLA typing. Our approach has been performed selecting oligos that contain the mismatch at the 3’ end, and spotting them onto a particularly treated glass surface, in order to hybridize themwith the DNA-SEQUENCE-TARGET and to performe a ligation reaction. This strategy allows the identification of known sequence variants differing by as little as a single nucleotide. The approach combines the capability of oligonucleotides to hybridize to the sequence of interest and the discriminating power of a DNA-specific enzyme, T4 DNA ligase,towards mismatched nucleotides in a DNA double helix. Two specially designed oligonucleotides probes are allowed to hybridize to a denatured DNA (target) in such a way that the 3’ end of one oligo is hybridized immediately adjacent to the 5’ end of the other. The ligase enzyme can join the two juxtaposed oligos by the formation of a phosphodiester bond, provided that the nucleotides at the junction are correctly base-paired with the target strand. In case of mismatch the reaction will be not possible.In this study we present a typing technology that can be performed in few hours. From adrop (0.5 ML) of blood,we obtained a sufficient quantity of DNA to put on a microchip. A locus specific amplification is performed for each sample analyzed. After PCR the denatured DNA target is added to the microarray, consisting of a glass slide, on which all probes (the positive and the negative probe for each allele) are nano-spotted,and hybridized with the CY3-labelled common probe. The ligation will bewhere a perfect match will be present, and a specific fluorescent signal will be detectable.The intensity of the fluorescence on spotted oligonucleotides probes can be analyzed by an in-house developed software program.This software automatically detects spots, remove the background, design the virtual array and then calculates the integral of the processed spots, generating matrixes containing the integral, the noise the average signal per pixel and the integral noise. These numerical matrixes, in excel format, can be used for further processing, for example to obtain the complete HLA typing result.