Identification of Active Genes (Edexcel International A Level Biology)

• Microarrays are laboratory tools used to identify active genes 
  • Active genes are genes that are expressed; mRNA is transcribed from them and the resulting mRNA strand is translated into a polypeptide
• Microarrays can be used to identify active genes in thousands of gene samples at a time
• Microarrays are used for
  • Medical diagnosis and treatment, e.g. identification of harmful mutations
  • Biotechnology, e.g. identifying genes for the process of producing recombinant DNA
  • Forensic analysis, e.g. in criminal investigations
• A microarray consists of a small piece of glass, plastic or silicon that has DNA probes attached to many spots, called gene spots, in a grid pattern
  • DNA probes are short, single stranded lengths of DNA linked to an easily identifiable label such as a fluorescence protein or a radioactive tag; these single stranded probes bind to any complementary sequences present in a DNA sample, indicating their presence by fluorescing or under an x-ray
  • There can be 10 000 or more spots per cm²
• When producing a microarray, scientists compare experimental samples of genetic material with a known reference sample, e.g. a genetic sample taken from an individual known to have a particular disease mutation

• When a microarray is used to analyse genomes
  • Samples of genetic material, known as chips, are collected from
    • A reference source, e.g. an individual known to have a particular genetic mutation; this provides a control sample for comparison
    • An unknown source, e.g. a patient to be diagnosed
      • Note that many unknown samples can be simultaneously compared to a single reference sample
  • Collecting mRNA rather than DNA at this stage enables active genes to be identified; only active genes will be undergoing the process of transcription into mRNA
  • Enzymes called reverse transcriptase enzymes are used to convert the mRNA back into DNA; the DNA produced in this way is known as complementary DNA, or cDNA
  • The cDNA samples are labelled, e.g. using fluorescence labels
    • The reference samples are labelled with a different label to the unknown experimental samples
      • Usually the reference samples fluoresce green while the unknown samples fluoresce red
  • Once the reference and unknown samples are mixed together, they are then allowed to hybridise with the probes on the microarray
    • After a set period of time any DNA that did not hybridise with the probes is washed off
  • The microarray is then examined using ultraviolet light which causes the tags to fluoresce, or scanned; colours are detected by the computer and the information is analysed
  • The fluorescence colour detected indicates where hybridisation has occurred, as the DNA fragment is complementary to the probe
    • If reference (green) and unknown (red) samples both hybridise in equal proportions then the overall colour detected will be yellow; this shows that the gene in question is being expressed in equal quantities in the reference individual and the individual from whom the unknown sample was taken
    • If reference samples hybridise more than the unknown samples then the overall colour detected will be green; this shows that the gene is being expressed more in the reference individual than in the individual providing the unknown sample
    • If unknown samples hybridise more than the reference samples then the overall colour detected will be red; this shows that the gene is being expressed more in the individual providing the unknown sample than in the reference individual
    • Note that a lack of fluorescence indicates that the gene in question is not being expressed at all

Microarrays can be used to identify active genes

• Microarrays can be used to test for expression of genes that increase the risk of certain cancers
  • E.g. High levels of expression of genes that code for receptors that bind to the hormone oestrogen can be a factor in the progression of some cancers; if doctors know that these genes are being expressed at high levels then drugs that block oestrogen receptors are likely to be an effective treatment