Nucleic Acids: Hybridisation

Abstract

Nucleic acid hybridisation is the pairing of complementary deoxyribonucleic acid (DNA) strands to produce DNA–DNA hybrids or the pairing of complementary DNA–RNA strands to produce DNA–RNA hybrids. Nucleic acid hybridisation is the basis of many research and diagnostic applications with all relying on simple base pairing specificity of nucleic acids to generate a signal through a labelled probe. This fundamental principle has led to DNA/RNA detection and quantification on solid phase blots, DNA/RNA cytogenetic localisation on cells, detection and purification of specific DNA and comparative gene expression analysis. Most recently, principles of nucleic acid hybridisation have been combined with next generation sequencing technology to create powerful new platforms for analysis which will increase in utility as we enter this new age of genomics and personalised medicine. The concepts and applications of nucleic acid hybridisation will be discussed in this review.

Key Concepts:

  • Nucleic acid hybridisation using base paring complementary allows detection of genes, mutations and RNA permitting accurate diagnosis of disease in the clinic and providing researchers with many powerful tools to analyse and interpret their work.

  • Traditional hybridisation technologies have enabled detection of target DNA or RNA sequences on a solid‐base membrane or on chromosomes.

  • Development of radioactive and nonradioactive probe labelling systems allowed for significantly improved sensitivity and specificity of detection of the target nucleic acids.

  • Nucleic acid hybridisation in quantitative analysis permits detection of mutations such as deletion, insertion and copy number variation for disease diagnosis.

  • Incorporation of next‐generation sequencing with nucleic acid hybridisation has opened a new genomic era and has enabled high‐throughput sophisticated analysis for personalised medicine by discovering novel genes and single nucleotide polymorphisms (SNP).

Keywords: nucleic acids; DNA/RNA; hybridisation; probe; microarray; complementary; next generation sequencing; comparative gene analysis

Figure 1.

A schematic diagram of Southern hybridisation analysing DNA fragments that share homology with a nucleic acid probe.

Figure 2.

Autogradioagraphs of a Southern blot and a spot blot. (a) Example of a Southern screen for the gene targeting the chicken DT40 CAP‐D3 locus. A probe external to the targeting vector is used to screen genomic DNA digested with the restriction enzyme EcoRI. Wildtype genomic DNA digested with EcoRI gives a 8.3 kb band when probed with a radiolabled external probe (1st lane). When a targeting event occurs an extra EcoRI site is inserted at the locus producing a lower 6.3 kb band (lanes 2–4). CAP‐D3 in the chicken genome exists as a trisomy, hence the first targeting event (2nd lane) gives a 2:1 band intensity consistent with the CAP‐D3 gene containing 3 alleles. Hence 3 rounds of gene targeting are required to disrupt all wildtype alleles (4th lane). The CAP‐D3 trisomy was confirmed using FISH (see Figure ). (b) DNA probes of Y pericentric domain of mouse genome, as well as controls, on a nylon filter were hybridised with the purified centromeric DNA probes. Each row represents hybridisation of different probes. The higher intensity of the top row suggests enrichment of the centromeric sequence compared to the bottom 3 rows which include a nonspecific control.

Figure 3.

An example of a FISH image captured by a digital camera. Hybridisation of metaphase (bottom) and interphase (top) cells (DAPI; blue) from chicken DT 40 cells with a CAP‐D3 BAC probe (green) and stained for DNA (blue) with DAPI. The presence of 3 signals in both metphase and interphase shows CAP‐D3 in chicken contains 3 copies as suggested from the Southern blot analysis in Figure a.

Figure 4.

A schematic diagram showing how microarray technology is used to measure gene expression of two different samples. Templates of genes to be tested are produced usually by PCR and the total reaction mixture is coated onto glass slides using a computer controlled high‐speed robot. Each gene is represented by a single dot. Messenger RNA from the two samples to be compared are labelled by different coloured fluorophores by reverse transcription, mixed and hybridised to the DNA microarray. Each cDNA hybridises to its individual target in the array and the relative abundance of the test and the reference transcripts of each gene is assessed by the ratio of red to green signals measured by scanning confocal laser microscope and specialised software.

Figure 5.

Chromosome 15 microdeletion detected using SNP microarray. (a) Deviation of Log2 ratio (LogR, red line) towards −1.0 and loss of heterozygous (0.5) B‐allele frequency SNP calls identifies a 5 Mb microdeletion within chromosome region 15q11.2q13. (b) Metaphase FISH analysis using SNRPN probe (red) in combination with PML control probe (green) confirms the microdeletion on one chromosome 15 (arrowhead) and DNA stained by DAPI (blue). (c) Methylation‐specific PCR (MS‐PCR) assay shows absence of 174 bp maternally imprinted (methylated) allele and presence of 100 bp paternally expressed allele only (at left). Microdeletion of the maternal chromosome 15 is associated with Angelman syndrome.

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Kim, Ji Hun, Kalitsis, Paul, Pertile, Mark D, Magliano, Dianna, Wong, Lee, Choo, Andy, and Hudson, Damien F(Aug 2012) Nucleic Acids: Hybridisation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003148.pub2]