The DNA can be 'spotted' as intact pieces onto the membrane. An alternative method is one inspired by the computer chip industry - leading to the "DNA Chip" terminology. This technique is oligonucleotide arraying.

Oligonucleotide arrays are, in effect, miniaturised DNA microarrays and have evolved as an important tool in research into genomes. They are small, high-density arrays, containing tens of thousands of synthetic oligonucleotides

Oligonucleotide arrays differ in many details but do share the essential simplicity of the experimental design of DNA microarrays.

GeneChips

One type of chip, containing high density arrays of short oligonucleotides, was developed by Affymetrix and makes it possible to array as many as 300,000 oligonucleotides in an area of less than 2cm2.

These types of chip are known as 'GeneChips'. These are produced by adapting the technique of semiconductor photolithography to synthesise oligonucleotide probe sequences in situ. The DNA is synthesised, directly onto the substrate, one base at a time.

These strategies generate microarrays of high density using relatively few steps in the synthesis process. The number of probes actually increases exponentially with a linear increase in the number of repetitions of the synthesis cycle.

There have been GeneChips made with 96,000 probes per chip. Photolithography has been used to define individual elements of 5 - 10 micrometres. This corresponds to an array density of 106 probes cm-2.

This high array density is the main advantage of the technique, but the main disadvantage is the absolute requirement for the arrayed sequence to be known. In many cases, particularly the human genome, this just is not possible yet. However, DNA chip technology is rapidly advancing, with many potential applications to diagnostics, gene discovery, gene expression and mapping.

Disadvantages of photolithography for producing arrays

There are also disadvantages associated with the method for producing oligonucleotide arrays.

Uses of Oligonucleotide Arrays

Oligonucleotide arrays have a wide range of possible applications, and in a wide range of species. In many cases they have been employed for the same purposes as DNA microarrays.

The main areas of application in which oligonucleotide arrays can speed up genomics studies are:

  1. Genotyping

    2. Oligonucleotides representing all the possible variations of a certain gene sequence are present in an array used for genotyping.

    This can have a number of applications, particularly in the identification of polymorphisms. With this type of array, it is possible to rapidly screen a gene of unknown sequence, determining whether a large number of deleterious changes have occurred. This allows identification of polymorphisms in gene whose sequences have been determined for the first time. It is important to understand the impact of these polymorphisms on biological process, such as in disease.

    An example of the use of this is the GeneChip HuSNP Mapping Assay .This array contains almost 1,500 human SNPs. The hybridisation image can be analysed, and the genotypes for each polymorphism reported.

    Advantages of Genotyping Arrays
    Affymetrix's GeneChips used in single nucleotide polymorphism mapping assays have a number of advantages, including speeding up the process of genetic analysis by decreasing labour, time to run an assay and time to analyse the data.
    These types of assays allow a better understanding of the connections between these polymorphisms or mutations and disease (See Research into Diseases: Mutations relevant to disease). This will lead to improved knowledge of the mechanisms that induce disease, and also the responses of patients to treatment. The intention of this is obviously better medical care.

    Drawbacks of Genotyping Arrays
    Unfortunately genotyping using an oligonucleotide array requires the complete sequencing of the oligonucleotides involved. Associated with this problem is that the longer the target DNA sequence, the longer the nucleotide sequence must be to eliminate any possible inaccuracies or ambiguities. The size of the fragment to be analysed is limited by the fact that any alteration in the length of the fragment results in a change in the number of probe cells four-fold.
    It is also well known that this technique is weak for those sequences which contain direct repeats or inverted repeats i.e. non-random sequences.

  2. Gene Expression

    3. Oligonucleotide arrays can be effectively used to generate accurate data concerning the expression of certain gene sequences. This has implications for a number of biological assays, and also improving knowledge of cellular pathways.

    They can be used to study thousands of mRNA molecules, whether genes or ESTs, quantitatively and simultaneously. This greatly increases the ease with which large genomic analysis can be carried out, simplifying genomic research.

    Oligonucleotide arrays have the advantage of being very specific and very sensitive. This enables the detection of mRNA that is only present in a few copies per cell, as well as in several hundred thousand copies. Each probe cell is made to contain millions of copies of a particular oligonucleotide probe, and this also means that the detection of low levels of mRNA is sensitive and accurate.

    Oligonucleotide arrays used for gene expression have an advantage over those used for genotyping: they can be used to analyse longer fragments.

    A further benefit with gene expression arrays is that although knowing what each array spot is can be advantageous, it is not a necessity. It is quite common to use probe cells that sequences that represent genes with unknown sequence or function.

    Commercially Produced Gene Expression Arrays
    As well as Affymetrix's GeneChip, the following examples are used for analysis of gene expression:

      Atlas arrays, by Clontech, are used in the detection of expression of specific genes. They have been produced for a range of genes including regulators of the cell cycle, cytokines and transcription factors
      DisplayARRAY membranes, by Display Systems Biotech, have been designed to search for novel homologous genes or study the expression patterns of specific genes
      Panorama gene arrays, by Genosys Biotechnologies are specific for use with Eschericia coli. They contain DNA that is representative of the whole genome of this bacteria, and therefore probing these arrays provides a method for quantifying the expression levels from all 4,290 E.coli genes, and under any growth condition. These are the first of their kind, but it is expected that similar arrays will be shortly available for a range of organisms.

    See (Sinclair , 1999)

  3. Research into Diseases

    4. Tumours and Cancers
    As mentioned in     Applications, microarray technology may provide many benefits in the study of tumours, and also many different types of cancer.

      One particular example of this is the use of the GeneChip with 96,000 oligonucleotide probes in the detection of all the possible heterozygous mutations in the BRCA1 breast and ovarian cancer gene.

    Mutations Relevant to Disease
    Microarray technology also has a vast number of possibilities for the detection of mutations with particular relevance to humans. These include:

      the use of GeneChips to investigate the HIV virus and the genes which make up the human mitochondrial genome,
      mutations in the cystic fibrosis transmembrane conductance regulator, and
      studies into beta-thalassaemia mutations in the beta globin gene in blood.

  4. Mapping Genomic Libraries

    5. GeneChips have been used for mapping genomic libraries by determining the order of the overlapping clones.

Specific Examples

In S.cerevisiae, oligonucleotide arrays have also been used to study the whole genome.

In experiments carried out by Wodicka et al. four GeneChips were used with 260,000 25-mer oligonucleotide probes - 65,000 probe sites on each chip - which covered every open reading frame of the yeast genome. The yeast were grown on rich or minimal media, and the gene expression under each condition was compared. It was found that as much as 90% of the genes in the genome were expressed under both culture conditions.

It has been shown by this experiment that this technique can be used for the resequencing of complex samples. This includes those with ORFs up to 1kb in length, and is due to the abundance of data generated by experiments such as these, particularly the information provided by the number of genes that are characterised.

Oligonucleotide arrays have been used in a number of studies on bacterial genomes.

An example of this is measuring the levels of expression of the genes of the bacterium.
In these situations bacterial mRNA can be expressed without the usual purification of the total mRNA of the genome, which is very beneficial to this technique: reproducible purification of bacterial mRNA is very difficult because of its low concentration in total RNA.

de Saizieu et al. designed an oligonucleotide probe array - again using Affymetrix's GeneChip - that contained probes representing 100 Streptococcus pneumoniae genes and 106 Haemophilus influenzae genes.

    Their experiments revealed that hybridisation of the bacterial RNA to the array permits quantification of the RNA transcript levels simultaneously. It was possible to analyse the entire genomes of the bacteria on a single oligonucleotide probe array.
    Their experiments also showed that again the DNA microarray results agreed with those obtained by Northern blot analysis.


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