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Tiling arrays are a subtype of
microarray A microarray is a multiplex (assay), multiplex lab-on-a-chip. Its purpose is to simultaneously detect the expression of thousands of biological interactions. It is a two-dimensional array on a Substrate (materials science), solid substrate—usu ...
chips. Like traditional microarrays, they function by hybridizing labeled
DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
or
RNA Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...
target molecules to probes fixed onto a solid surface. Tiling arrays differ from traditional microarrays in the nature of the probes. Instead of probing for
sequences In mathematics, a sequence is an enumerated collection of objects in which repetitions are allowed and order matters. Like a set, it contains members (also called ''elements'', or ''terms''). The number of elements (possibly infinite) is call ...
of known or predicted genes that may be dispersed throughout the
genome A genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding genes, other functional regions of the genome such as ...
, tiling arrays probe intensively for sequences which are known to exist in a contiguous region. This is useful for characterizing regions that are sequenced, but whose local functions are largely unknown. Tiling arrays aid in
transcriptome The transcriptome is the set of all RNA transcripts, including coding and non-coding, in an individual or a population of cells. The term can also sometimes be used to refer to all RNAs, or just mRNA, depending on the particular experiment. The ...
mapping as well as in discovering sites of DNA/
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...
interaction (
ChIP-chip ChIP-on-chip (also known as ChIP-chip) is a technology that combines chromatin immunoprecipitation ('ChIP') with DNA microarray (''"chip"''). Like regular Ch-IP, ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA ''in ...
, DamID), of DNA
methylation Methylation, in the chemistry, chemical sciences, is the addition of a methyl group on a substrate (chemistry), substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replac ...
(MeDIP-chip) and of sensitivity to DNase (DNase Chip) and array CGH. In addition to detecting previously unidentified genes and regulatory sequences, improved quantification of transcription products is possible. Specific probes are present in millions of copies (as opposed to only several in traditional arrays) within an array unit called a feature, with anywhere from 10,000 to more than 6,000,000 different features per array. Variable mapping resolutions are obtainable by adjusting the amount of sequence overlap between probes, or the amount of known
base pair A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...
s between probe sequences, as well as probe length. For smaller genomes such as ''
Arabidopsis ''Arabidopsis'' (rockcress) is a genus in the family Brassicaceae. They are small flowering plants related to cabbage and mustard. This genus is of great interest since it contains thale cress (''Arabidopsis thaliana''), one of the model organ ...
'', whole genomes can be examined. Tiling arrays are a useful tool in
genome-wide association studies In genomics, a genome-wide association study (GWA study, or GWAS), is an observational study of a genome-wide set of genetic variants in different individuals to see if any variant is associated with a trait. GWA studies typically focus on assoc ...
.


Synthesis and manufacturers

The two main ways of synthesizing tiling arrays are
photolithographic Photolithography (also known as optical lithography) is a process used in the manufacturing of integrated circuits. It involves using light to transfer a pattern onto a substrate, typically a silicon wafer. The process begins with a photosensit ...
manufacturing and mechanical spotting or printing. The first method involves ''
in situ is a Latin phrase meaning 'in place' or 'on site', derived from ' ('in') and ' ( ablative of ''situs'', ). The term typically refers to the examination or occurrence of a process within its original context, without relocation. The term is use ...
'' synthesis where probes, approximately 25bp, are built on the surface of the chip. These arrays can hold up to 6 million discrete features, each of which contains millions of copies of one probe. The other way of synthesizing tiling array chips is via mechanically printing probes onto the chip. This is done by using automated machines with pins that place the previously synthesized probes onto the surface. Due to the size restriction of the pins, these chips can hold up to nearly 400,000 features. Three manufacturers of tiling arrays are
Affymetrix Affymetrix is now Applied Biosystems, a brand of DNA microarray products sold by Thermo Fisher Scientific that originated with an American biotechnology research and development and manufacturing company of the same name. The Santa Clara, Calif ...
, NimbleGen and Agilent. Their products vary in probe length and spacing. ArrayExplorer.com is a free web-server to compare tiling arrays.


Applications and types


ChIP-chip

ChIP-chip is one of the most popular usages of tiling arrays.
Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genom ...
allows binding sites of
proteins Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, re ...
to be identified. A genome-wide variation of this is known as ChIP-on-chip. Proteins that bind to
chromatin Chromatin is a complex of DNA and protein found in eukaryote, eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important r ...
are cross-linked
in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, an ...
, usually via fixation with
formaldehyde Formaldehyde ( , ) (systematic name methanal) is an organic compound with the chemical formula and structure , more precisely . The compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde. It is stored as ...
. The chromatin is then fragmented and exposed to
antibodies An antibody (Ab) or immunoglobulin (Ig) is a large, Y-shaped protein belonging to the immunoglobulin superfamily which is used by the immune system to identify and neutralize antigens such as bacteria and viruses, including those that caus ...
specific to the protein of interest. These complexes are then precipitated. The DNA is then isolated and purified. With traditional DNA microarrays, the immunoprecipitated DNA is hybridized to the chip, which contains probes that are designed to cover representative genome regions. Overlapping probes or probes in very close proximity can be used. This gives an unbiased analysis with high resolution. Besides these advantages, tiling arrays show high reproducibility and with overlapping probes spanning large segments of the genome, tiling arrays can interrogate protein binding sites, which harbor repeats. ChIP-chip experiments have been able to identify binding sites of transcription factors across the genome in yeast, drosophila and a few mammalian species.


Transcriptome mapping

Another popular use of tiling arrays is in finding expressed genes. Traditional methods of gene prediction for annotation of genomic sequences have had problems when used to map the transcriptome, such as not producing an accurate structure of the genes and also missing transcripts entirely. The method of sequencing cDNA to find transcribed genes also runs into problems, such as failing to detect rare or very short RNA molecules, and so do not detect genes that are active only in response to signals or specific to a time frame. Tiling arrays can solve these issues. Due to the high resolution and sensitivity, even small and rare molecules can be detected. The overlapping nature of the probes also allows detection of non-polyadenylated RNA and can produce a more precise picture of gene structure. Earlier studies on chromosome 21 and 22 showed the power of tiling arrays for identifying transcription units. The authors used 25-mer probes that were 35bp apart, spanning the entire chromosomes. Labeled targets were made from polyadenylated RNA. They found many more transcripts than predicted and 90% were outside of annotated
exon An exon is any part of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term ''exon'' refers to both the DNA sequence within a gene and to the corresponding sequence ...
s. Another study with Arabidopsis used high-density
oligonucleotide Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, Recombinant DNA, research, and Forensic DNA, forensics. Commonly made in the laboratory by Oligonucleotide synthesis, solid-phase ...
arrays that cover the entire genome. More than 10 times more transcripts were found than predicted by ESTs and other prediction tools. Also found were novel transcripts in the centromeric regions where it was thought that no genes are actively expressed. Many noncoding and natural
antisense RNA Antisense RNA (asRNA), also referred to as antisense transcript, natural antisense transcript (NAT) or antisense oligonucleotide, is a single stranded RNA that is complementary to a protein coding messenger RNA (mRNA) with which it hybridizes, and ...
have been identified using tiling arrays.


MeDIP-chip

Methyl-DNA immunoprecipitation followed by tiling array allows DNA methylation mapping and measurement across the genome. DNA is methylated on
cytosine Cytosine () (symbol C or Cyt) is one of the four nucleotide bases found in DNA and RNA, along with adenine, guanine, and thymine ( uracil in RNA). It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attac ...
in CG di-nucleotides in many places in the genome. This modification is one of the best-understood inherited
epigenetic In biology, epigenetics is the study of changes in gene expression that happen without changes to the DNA sequence. The Greek prefix ''epi-'' (ἐπι- "over, outside of, around") in ''epigenetics'' implies features that are "on top of" or "in ...
changes and is shown to affect gene expression. Mapping these sites can add to the knowledge of expressed genes and also epigenetic regulation on a genome-wide level. Tiling array studies have generated high-resolution methylation maps for the Arabidopsis genome to generate the first "methylome".


DNase-chip

DNase chip is an application of tiling arrays to identify hypersensitive sites, segments of open chromatin that are more readily cleaved by DNaseI. DNaseI cleaving produces larger fragments of around 1.2kb in size. These hypersensitive sites have been shown to accurately predict regulatory elements such as promoter regions, enhancers and silencers. Historically, the method uses Southern blotting to find digested fragments. Tiling arrays have allowed researchers to apply the technique on a genome-wide scale.


Comparative genomic hybridization (CGH)

Array-based CGH is a technique often used in diagnostics to compare differences between types of DNA, such as normal cells vs. cancer cells. Two types of tiling arrays are commonly used for array CGH, whole genome and fine tiled. The whole genome approach would be useful in identifying copy number variations with high resolution. On the other hand, fine-tiled array CGH would produce ultrahigh resolution to find other abnormalities such as breakpoints.


Procedure

Several different methods exist for tiling an array. One protocol for analyzing gene expression involves first isolating total RNA. This is then purified of rRNA molecules. The RNA is copied into double stranded DNA, which is subsequently amplified and in vitro transcribed to cRNA. The product is split into triplicates to produce dsDNA, which is then fragmented and labeled. Finally, the samples are hybridized to the tiling array chip. The signals from the chip are scanned and interpreted by computers. Various software and algorithms are available for data analysis and vary in benefits depending on the manufacturer of the chip. For Affymetrix chips, the model-based analysis of tiling array (MAT) or hypergeometric analysis of tiling-arrays (HAT) are effective peak-seeking algorithms. For NimbleGen chips, TAMAL is more suitable for locating binding sites. Alternative algorithms include MA2C and TileScope, which are less complicated to operate. The Joint binding deconvolution algorithm is commonly used for Agilent chips. If sequence analysis of binding site or annotation of the genome is required then programs like MEME, Gibbs Motif Sampler, Cis-regulatory element annotation system and
Galaxy A galaxy is a Physical system, system of stars, stellar remnants, interstellar medium, interstellar gas, cosmic dust, dust, and dark matter bound together by gravity. The word is derived from the Ancient Greek, Greek ' (), literally 'milky', ...
are used.


Advantages and disadvantages

Tiling arrays provide an unbiased tool to investigate protein binding, gene expression and gene structure on a genome-wide scope. They allow a new level of insight in studying the transcriptome and methylome. Drawbacks include the cost of tiling array kits. Although prices have fallen in the last several years, the price makes it impractical to use genome-wide tiling arrays for mammalian and other large genomes. Another issue is the "transcriptional noise" produced by its ultra-sensitive detection capability. Furthermore, the approach provides no clearly defined start or stop to regions of interest identified by the array. Finally, arrays usually give only chromosome and position numbers, often necessitating sequencing as a separate step (although some modern arrays do give sequence information.)


References

{{Reflist, 30em Microarrays Computational biology