Microarrays: Use in Mutation Detection


Microarrays are used in assay formats that allow parallel analyses of tens, hundreds or thousands of biomolecules in very small reaction volumes. One of the many application areas of the microarray format is to genotype or detect diseaseā€causing or diseaseā€predisposing mutations in the human genome for diagnostics, carrier identification and pharmacogenetic profiling.

Keywords: genotyping; microarrays; minisequencing; single nucleotide polymorphism; disease mutation; pharmacognetics; fluorescence detection

Figure 1.

Strategies for single nucleotide polymorphism (SNP) genotyping by primer extension using microarrays. Genotyping of SNP1 with the nucleotide variation A/G, SNP2 with G/C and SNP3 with C/T is illustrated for a sample with the genotypes AA, GC and CC. (a) Minisequencing, arrayed primer extension. One primer for each SNP to be genotyped is immobilized covalently on the surface of a microscope slide. Multiplex polymerase chain reaction (PCR) products spanning the SNP sites, a mixture of fluorescently labeled terminating nucleotide analogs, and a deoxyribonucleic acid (DNA) polymerase are added to the arrays. The primer extension reactions are allowed to proceed on the array surface, and the microscope slides are scanned. The position of the primers on the microarray surface defines which SNP is analyzed, and the fluorescent nucleotide(s) by which a primer becomes extended defines the genotype of the SNP. (b) Allele‐specific primer extension. Two allele‐specific primers, with the 3′ base complementary to the two possible nucleotides of each SNP, are immobilized on the array. The multiplex PCR products spanning the SNPs are transcribed into multiple ribonucleic acid (RNA) copies by an RNA polymerase. The RNA molecules serve as templates for a primer extension reaction catalyzed by a reverse transcriptase, in which multiple fluorescent deoxynucleotides become incorporated into each product. For homozygous genotypes, a signal is generated from one of the allele‐specific primers, and for heterozygous genotypes, a signal is generated from both primers. (c) Primer extension using tag arrays. Cyclic single nucleotide primer extension reactions are performed in solution in the presence of fluorescently labeled dideoxynucleotides, using primers carrying an extra tag sequence in their 5′ end. Generic arrays of oligonucleotides that are complementary to the tags of the primers are used to capture the products of the cyclic minisequencing reactions. (Reproduced from Syvänen, )

Figure 2.

Genotyping by minisequencing using the array of arrays format. (a) Schematic view of a microscope slide divided into 80 subarrays at the same spacing as the wells in a 384‐well microtiter plate. For individual minisequencing reactions, separate reaction chambers are formed by using a silicon rubber grid placed on the slide, to allow simultaneous genotyping of up to 200 single nucleotide polymorphisms (SNPs) in 80 samples, generating over 10000 genotypes per microscope slide. (b) Fluorescence image of 16 subarrays, in which polymerase chain reaction (PCR) products amplified from deoxyribonucleic acid (DNA) from four individuals have been genotyped for 74 SNPs in duplicate in four separate minisequencing reactions for each individual. The primers immobilized in the subarrays have been extended with dideoxynucleoside triphosphates (ddNTPs) labeled with the fluorophore 6‐carboxytetramethylrhodamide (TAMRA) using a DNA polymerase. The horizontal rows of four subarrays correspond to one individual, and the vertical columns are the reactions for detecting the nucleotides A, C, G and T respectively. (c) Enlarged fluorescent image of one individual reaction well, where a minisequencing reaction to detect the T allele has been performed. The rainbow‐color scale corresponds to different signal intensities measured in the array scanner, with blue as low and white as a saturated signal. (Figure provided by Ulrika Liljedahl, Department of Medical Sciences, Uppsala University, Sweden.)



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Further Reading

Evans WE and Relling MV (1999) Pharmacogenomics: translating functional genomics into rational therapeutics. Science 286: 487–491.

Hamosh A, Scott AF, Amberger J, et al. (2002) Online Mendelian Inheritance in Man (OMIM), a knowledgebase of human genes and genetic disorders. Nucleic Acids Research 30: 52–55.

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Web Links

http://www.genet.sickkids.on.ca/cftr/ Cystic Fibrosis Mutation Data Base. Contains a collection of mutations in the CFTR gene

OMIM database. Online Mendelian Inheritance in Man. The OMIM database is a catalog of human genes and genetic disorders. It contains textual information, pictures and reference information on most monogenic disorders http://www.ncbi.nlm.nih.gov/omim

Cystic fibrosis transmembrane conductance regulator, ATP‐binding cassette (CFTR); LocusID: 1080. LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=1080

Cystic fibrosis transmembrane conductance regulator, ATP‐binding cassette (CFTR); MIM number: 602421. OMIM: http://www.ncbi.nlm.nih.gov/htbin‐post/Omim/dispmim?602421

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Syvänen, Ann‐Christine(Jan 2006) Microarrays: Use in Mutation Detection. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0005954]