MicroArray Technology possibile application in the diagnostic virology Dott. ssa Maria Concetta Bellocchi Prof Carlo Federico Perno UNIVERSITA' DEGLI STUDI DI ROMA "TOR VERGATA"DIPARTIMENTO DI MEDICINA SPERIMENTALE E SCIENZE BIOCHIMICHE VIA MONTPELLIER ROMA TEL FAX

Uses for MicroArrays  Identification of sequence (gene / gene mutation) Sequencing Arrays - tests for nucleotide sequence in a fragment of DNA (sequencing by hybridization - ideal for detection of single nucleotide polymorphisms[snps]). Mutation Analysis  Determination of expression level (abundance) of genes. Expression Arrays : tests for mRNA expressed in a tissue or cells Expression Analysis  Identification of presence viral contamination Diagnostic Analysis

HYBRIDIZATION Base-pairing or hybridization is the underlining principle of DNA microarray. (i.e., A-T and G-C for DNA; A-U and G-C for RNA)

Traditional methods in molecular biology generally work on a "one gene in one experiment" basis, which means that the throughput is very limited and the "whole picture" of gene function is hard to obtain. This technology promises to monitor the whole genome on a single chip so that researchers can have a better picture of the interactions among thousands of genes simultaneously.

An array is an orderly arrangement of samples. It provides a medium for matching known and “unknown” DNA samples based on base-pairing rules and automating the process of identifying the unknowns. An array experiment can make use of common assay systems such as microplates or standard blotting membranes, and can be created by hand or make use of robotics to deposit the sample. Terminologies that have been used in the literature to describe this technology include, but not limited to: biochip, DNA chip, DNA microarray and gene array

Overview of Array Technology  MicroArrays  Macroarrays In general, arrays are described as macroarrays or microarrays, the difference being the size of the sample spots.

Macroarrays contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scanners. Microarrays require specialized robotics and imaging equipment. The sample spot sizes in microarray are typically less than 200 microns in diameter and these arrays usually contains thousands of spots.

RT-PCR Ribonuclease Protection Assays Northern blot Compared to:

Microarray Technology There are two variants of the DNA, in terms of the property of arrayed DNA sequence with known identity:

Format I : probe cDNA (500~5,000 bases long) is immobilized to a solid surface such as glass using robot spotting and exposed to a set of targets either separately or in a mixture. This method, "traditionally“ is called DNA microarray is widely considered as developed at Stanford University

Format II : an array of oligonucleotide (20~80-mer oligos) or peptide nucleic acid (PNA) probes is synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. The array is exposed to labeled sample DNA, hybridized, and the identity/abundance of complementary sequences are determined. This method, "historically" is called DNA chips

It was developed at Affymetrix, Inc., which sells its photolithographically fabricated products under the GeneChip ® trademark.Affymetrix, Inc. GeneChip ® Many companies are manufacturing oligonucleotide based chips using alternative in-situ synthesis or depositioning technologies.

"DNA microarray(s)" and "DNA chip(s)" are used interchangeably. But viewers should aware this technical difference.

cDNA microarrays mechanical placement of pre-assembled genes on a glass slide suited for gene expression analysis and novel gene discovery allow two mRNA populations to be compared on the same chip, a technique known as ratiometric gene expression analysis.

DNA chips synthesis of oligonucleotide sequences on silicon chips by light activation well suited for high-efficiency analysis for gene expression and mutation not suited for the discovery of novel genes

What Are MicroArrays  Arrangements of DNA on matrix supports  Cloned DNAs, PCR products, oligos  Usually glass slides or silicon chips  DNA is spotted in regular arrays  Typically ≤ 1nl of ~1µg/µl DNA  Spots can be µm dia  100µM spots every 130µm = ~70,000 on slide

Typical Arrays  All ~4400 coding regions of E. coli  All ~6300 coding regions of yeast  ~30,000 human genes & eSTs  ~20,000 mouse or rat genes & eSTs  Sets for other mainstream organisms  Custom sets of eg ~400 “cytokine” human genes

What to Spot cDNAs – cDNA libraries already exist for many species – Amplified inserts available for eg humans, mouse – Significant contamination problems – Cross talk between clones for multigene families Long oligos – Does not require manipulation of libraries – Can be gene or splice variant specific – Require extensive high quality sequence information – Probably to be 50-70mers for consistent TMs

Arrayers Spotters Growers

Spotters  Pins transfer DNA from reservoir to matrix  Spot size very consistent ± 10% Growers Affymetrix synthesise oligos on matrix (photolithography) Size limited - ~25 nucleotides (median 18mer) Densely packed ~10µm dia

Detection - Array Readers Radioactive Detection Fluorescence Detection Electrical Detection

Fluorescence Detection  Label DNA/RNA with fluor  Hybridise to DNA spots on matrix  Detect bound DNA by scanning

LABELLING  RNA/DNA labelling with fluor- nucleotides  eg Cy3-dNTP and Cy5-dNTP  poor, uneven incorporation  Amino-allyl Labelling  incorporate amino-allyl nucleotides  chemically couple to eg Cy3 and Cy5  Hybridise control & test sample on same slide

Scanner Process DyePhotons Electrons Signal Laser PMT A/D Convertor excitation amplification Filtering Time-space averaging Laser Detection Laser passes over the slide Excites fluor which releases photon Photon hits PMT and converted to signal Digital signal summed over eg 10µm2 area Image built up from these “pixel” values

Image Quantification Alignment Quantification – Intensity – Background – Area – Variance of pixel intensity

Alignment

Cy3 Cy5

Registration composite

Quantified Data Thousands of cDNA sequences spotted on a glass array Hybridise and scan array Image Analysis

Tests for the presence of a nucleic acid sequence by hybridizing a probe bound to a matrix to the target sequence. Many different probes can be bound to the same matrix. Therefore, a single sample can be evaluated for many different target sequences simultaneously.

No virus+ Virus Annealing cDNA synthesis Degratation of mRNA template Purification of cDNA Coupling cDNA with CyDye NHS ester Purification of CyDye labelled cDNA

APPLICATION MICROARRAY IN VIROLOGY

Performing a Microarray Study Normal Infected /tumor/ blood Extract RNA Extract RNA Make cDNA Amplify by PCR PCR Product Labeled with Green Dye PCR Product Labeled with Red Dye Mix Hybridize on Micro Array Green Signal RNA Expressed in Normal Tissue Red Signal RNA Expressed in Infected /Tumor Tissue or Blood

HIV DIAGNOSTIC: Analysis of viral resistance by microarrays

HIV-1 genome

DRUGS NRTI (n = 11): –AZT,ddI, ddC, d4T, 3TC, Abacavir (1592U89), Lodenosine (FddA), FTC, Adefovir (PMEA), bisPOC PMPA, BCH10652 NNRTI (n = 6): –Atevirdine, Delavirdine, Loviride, Nevirapine, Efavirenz (DMP266), MKC442 PI (n = 8): –Ritonavir, Indinavir, Saquinavir, Nelfinavir, Amprenavir, ABT378, PNU140690, PD Foscarnet

Viral resistance

Mutations in protease

Mutations in RT: NRTI NNRTI

MUTATIONS NRTI: 49 mutations NNRTI: 40 mutations Foscarnet: 8 mutations PI: 69 mutations gag cleavage sites: 2 mutations TOTAL168

HIV Chip Prot RT gag 1 Amplicon Includes 2 Gag cleavage sites: p7/p1 (2086) and p1/p6 (2134) I ncludes the cleavage site at start of protease (2252) and between protease and RT (2546) Includes all known mutations in protease 3 Amplicons Includes all known mutations in RT Includes the cleavage site between RT and RNAseH (3866) 2 Amplicons Includes the cleavage site between p17 and p24 (1186) Includes 2 cleavage sites: p24/p2 (1879) and p2/p7 (1921)

HIV PRT Mutant Analysis T215 T215Y T215F F214L / T215F

The next step in microarrays The next step in microarrays would then be the introduction of a more global analysis of the proteome, where at least 5,000 to 10,000 proteins could be studied using this principle. The probes used for such approach would have to be based on recombinant antibodies and phage display libraries with several billions of antibody members. As the antibodies cannot be synthesized on the surface of the chips, as is possible for DNA arrays, the probes have to be spotted on the chips in an array format and coupled to a downstream high-throughput detection system. Some obvious choices for detection include: fluorescent tags, nano-electrodes, and in the case of smaller arrays, MS.

Protein chips architecture Protein chips use a “probe” on the surface of a silicon chip to be able to catch native and post-translationally modified proteins. One of the more obvious choices for such probes is antibodies because of the exquisite specificity of the their molecular design. In fact, antibodies have already been used in a limited analysis of cellular changes occurring in cultured human cells, using a very small array and detected by SELDI (surface enhanced laser desorption ionization) MS.

Analysis of the proteome

Reversing the problem In addition to developments in the use of microarrays of capture antibodies to measure protein levels, protein chips have been printed with antigens that are used for the detection of circulating antibodies in clinical specimens.

“Catcher” molecules In contrast to DNA arrays, in which binding molecules may be defined by sequence and synthesized onto the surface of the array, protein-based catcher molecules with defined and predetermined specificities cannot be produced in such a way. Instead, protein-based “catcher” molecules need to be developed for each ligand. Arrays can be seen as miniaturized variants of assay formats having existed for many years. Typically, such formats include enzyme-linked immunosorbent assay (ELISA) and other types of immunometric assays utilizing antibodies as catcher molecules.

Until now, the few protein-based arrays that have been presented have utilized monoclonal and full-length antibodies produced by conventional hybridoma technology and obtained from commercial or in-house sources. These arrays have consisted of antibodies with specificities against known antigens, e.g., cytokines, cell signal, or cell matrix proteins, but also other proteins, and have counted as many as 250 different antibody specificities. These antibodies may be developed through immunization of animals yielding monoclonal or polyclonal formats or may be developed using recombinant technologies using libraries of randomly recombined antibody fragments.

Disadvantages of Microarray Testing Use of Complex 'Research' Procedures Labor Intensive High Cost

Multiple arrays. Multiple spots containing the same DNA oligo sequence/ protein on the same microarray. Multiple spots containing different oligo sequences/protein that assay the same gene RNA on the same microarray. Multiple spots containing different oligo sequences that assay different RNA products from the same gene on the same microarray. Replication of total experiment Replication of just the hybridization step Replication of only some other set of steps. Levels of Replication