Microarray

Lecture Notes for BIO702

Manjusha Verma

DNA Microarray is a collection of microscopic DNA spots attached to a solid surface

Also called as Gene chip, DNA chip, or Biochip

DNA microarrays are used to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome

Each DNA spot contains picomoles (1012 moles) of a specific DNA sequence, known as probes (or reporters)

short section of a gene

Other DNA element

synthetic DNA probes

Hybridized to a cDNA or cRNA sample (called target) under high-stringency conditions

Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target In standard microarrays, the probes are synthesized and then attached via surface engineering to a solid surface by a covalent bond to a chemical matrix (via epoxy-silane, amino-silane, lysine, polyacrylamide or others).

Glass

silicon chip

Illumina -microscopic beads

Uses : to measure changes in expression levels to detect single nucleotide polymorphisms to genotype or resequence mutant genomes Microarrays differ in:
Fabrication
methods of analyzing the data cost experimental design Efficiency

Workings

accuracy

Microarray evolved from Southern blotting

First reported use of this approach was the analysis of 378 arrayed lysed bacterial colonies each harboring a different sequence which were assayed in multiple replicas for expression of the genes in multiple normal and tumor tissue in 1982. 4000 human sequences in1987 gene arrays were made by spotting cDNAs onto filter paper with a pinspotting device

Principle
Hybridization between two DNA strands, the property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs After washing off of non-specific bonding sequences, only strongly paired strands will remain hybridized

Fluorescently labelled target sequences that bind to a probe sequence generate a signal strength of the hybridization

number of paired bases

hybridization conditions

washing after hybridization

Microarrays use relative quantization in which the intensity of a feature is compared to the intensity of the same feature under a different condition, and the identity of the feature is known by its position.

Microarray workflow

Types

Traditional solid-phase array is a collection of orderly microscopic "spots", called features with a specific probe attached to a solid surface in known locations Bead array is a collection of microscopic polystyrene beads with a specific probe and a ratio of two or more dyes

Spotted microarray
The probes are synthesized prior to deposition on the array surface and are then "spotted" onto glass.
Utilizes an array of fine pins or needles controlled by a robotic arm that is dipped into wells containing DNA probes and then depositing each probe at designated locations on the array surface

Oligonucleotide microarray
Produced by printing short oligonucleotide sequences designed to represent a single gene or family of gene splice-variants by synthesizing this sequence directly onto the array surface

One-channel microarray
Arrays provide intensity data for each probe or probe set indicating a relative level of hybridization with the labelled target

Two-channel microarray
Hybridized with cDNA prepared from two samples to be compared Cy3, which has a fluorescence emission wavelength of 570 nm (corresponding to the green part of the light spectrum), and Cy5 with a fluorescence emission wavelength of 670 nm (corresponding to the red part of the light spectrum).

The degree of hybridization between the spike-ins and the control probes is used to normalize the hybridization measurements for the target probes

Replication of the biological samples is essential for drawing conclusions from the experiment

Technical replicates (two RNA samples obtained from each experimental unit) help to ensure precision and allow for testing differences within treatment groups.

Spots of each cDNA clone or oligonucleotide are present as replicates (at least duplicates) on the microarray slide,

Applications
Gene expression profiling Expression levels of thousands of genes are simultaneously monitored to study the effects of certain treatments, diseases, and developmental stages on gene expression Assessing genome content in different cells or closely related organisms Small microarrays to check IDs of organisms in food and feed DNA sequences bound to a particular protein can be isolated by immunoprecipitating that protein Genomic regions bound by a protein of interest can be isolated and used to probe a microarray to determine binding site occupancy

Comparative genomic hybridization GeneID Chromatin immunoprecipitatio n on Chip DamID

Applications
SNP detection Used for genotyping, forensic analysis, measuring predisposition to disease, identifying drugcandidates, evaluating germline mutations in individuals or somatic mutations in cancers, assessing loss of heterozygosity, or genetic linkage analysis An exon junction array design uses probes specific to the expected or potential splice sites of predicted exons for a gene to assay the expression of alternative splice forms of a gene Enables combined measurements of chimeric transcript junctions with exon-wise measurements of individual fusion partners Overlapping probes designed to densely represent a genomic region of interest to detect expression of transcripts or alternatively splice forms not previously known

Alternative splicing detection

Fusion genes microarray Tiling array

A protein microarray, sometimes referred to as a protein binding microarray, provides a multiplex approach to identify proteinprotein interactions, to identify the substrates of protein kinases, to identify transcription factor protein-activation, to identify the targets of biologically active small molecules

The array is a piece of glass on which different molecules of protein or specific DNA binding sequences (as capture probes for the proteins) have been affixed at separate locations in an ordered manner thus forming a microscopic array

The most common protein microarray is the antibody microarray, where antibodies are spotted onto the protein chip and are used as capture molecules to detect proteins from cell lysate solutions

ProteinProtein array: The proteins can be externally synthesised, purified and attached to the array
Alternatively they can be synthesised in-situ and directly attached to the array The proteins can be synthesised through biosynthesis, cell-free DNA expression or chemical synthesis With cell-free DNA expression, proteins are attached to the support right after their production

Peptides chemically procured by solid phase peptide synthesis are already attached to the support
Selective deprotection is carried out through lithographic methods or by the so-called SPOT-synthesis.

DNA-Protein array: Double-stranded DNA (the exact binding sequence of the protein)is attached/ spotted on the array.

Artifacts To avoid variability in results, use efficient lysis buffer and maintain consistent sample processing conditions Many antibodies don't work well as capture reagents, Some antibodies often bind poorly to intact proteins in a cell extract; Different proteins like different solution conditions, so if no binding is seen, it doesn't mean that there is no binding between the two partners in physiological conditions; Adjust the solute conditions to avoid non-specific association: change salt concentration, pH, add 1% alignate; On the array's surface the conjugated protein should be in the right conformation (i.e., folded, NOT denatured), anchored by the same amino acid (in the same orientation), and be kept away from the surface by a linker to avoid steric hindrance.

An antibody microarray is a specific form of protein microarrays

A collection of capture antibodies are spotted and fixed on a solid surface such as glass, plastic or silicon chip, for the purpose of detecting antigens Used for detecting protein expressions from cell lysates in general research and special biomarkers from serum or urine for diagnostic applications

Cell-free protein array technology produces protein microarrays by performing in vitro synthesis of the target proteins from their DNA templates Used for testing proteinprotein interactions, as well as protein interactions with other cellular molecules such as DNA and lipids, enzymatic inhibition assays and screenings of antibody specificity Simplifies protein microarray construction by bypassing the need to express the proteins in bacteria cells and the subsequent need to purify them

Nucleic acid programmable protein array (NAPPA)

Protein in situ array (PISA)

In situ puromycin-capture

Nano-well array format

DNA array to protein array (DAPA)

Rapid and cost-effective Avoids DNA cloning (with the exception of NAPPA) reduced steps in production saves on reagent consumption and cuts production costs Improves protein availability Many proteins, including antibodies, are difficult to express in host cells Enables long term storage proteins are a heterogeneous class of molecules with different stability and physiochemical properties. option to quickly obtaining protein microarrays on demand Flexible Amenable to a range of different templates: PCR products, plasmids and mRNA Additional components can be included during synthesis to adjust the environment for protein folding, disulfide bond formation, modification or protein activity

Advantages

Peptide microarray (also commonly known as peptide chip or peptide epitope microarray) is a collection of peptides displayed on a solid surface, usually a glass or plastic chip USES

to study binding properties and functionality and kinetics of protein-protein interactions in general
profile an enzyme map an antibody epitope find key residues for protein binding

Peptide microarrays show several advantages over protein microarrays: Detection of binding events on epitope level, enabling study of i.e. epitope spreading Flexible design (i.e. posttranslational modifications, sequence diversity, non-natural amino acids )

Assays rely on detection of robust linear epitope interactions

Extended shelf stability Cost efficiency High batch-to-batch reproducibility

Immunology Mapping of immunodominant regions in antigens or whole proteomes Seromarker discovery Monitoring of clinical trials Profiling of antibody signatures Finding neutralizing antibodies Enzyme profiling Identification of substrates for orphan enzymes Optimization of known enzyme substrates Elucidation of signal transduction pathways Detection of contaminating enzyme activities Consensus sequence and key residues determination

Applications of peptide microarrays