Genetic markers uses enzymes that produces fluorescent substances. There used to be antibiotic resistance gene markers, but there was the chance that they would spread the antibiotic resistance to other new strains of bacteria.
Genetic markers
Green fluorescent protein (GFP) from jellyfish:
- gene inserted into plasmid --> taken up by bacteria
- shine UV light --> identify genetically modified bacteria
β-glucuronidase (GUS) from E.coli:
- transform into incubated with colourless/non-fluorescent substrate
---> transform into coloured/fluorescent products
---> detect activity of inserted genes
Microarray
- identify genes present in an organism's genome
- find out which genes are expressed within cells
---> microarrays contain thousands of gene probes
1. Genome analysis: compare genes present in two different species
- DNA collected from each species, cut to fragments and denatured, labelled with fluorescent tags
- DNA samples are mixed together and hybridised with DNA probes on the microarray --> inspected with UV light, causing the tags to fluoresce
- colour: DNA has hybridised with probe
- no colour: DNA not hybridised, gene not present
2. Gene expression - detecting mRNA
- to identify genes that are being transcribed to mRNA
- mRNA collected --> reverse transcriptase --> cDNA
- cDNA labelled with fluorescent tags, denatured, hybridised with probes on microarray
- spots that fluoresce microarrays show transcribed genes
* intensity of light emitted from spots = level of activity of genes
19.1 Principles of genetic technology
Genetic engineering involves the manipulation of naturally occurring processes and enzymes.
Genome sequencing gives information about the location of genes and provides evidence for the evolutionary links between organisms.
a) define the term recombinant DNA
b) explain that genetic engineering involves the extraction of genes from one organism, or the synthesis of genes, in order to place them in another organism (of the same or another species) such that the receiving organism expresses the gene product
c) describe the principles of the polymerase chain reaction (PCR) to clone and amplify DNA (the role of Taq polymerase should be emphasised)
d) describe and explain how gel electrophoresis is used to analyse proteins and nucleic acids, and to distinguish between the alleles of a gene (limited to the separation of polypeptides and the separation of DNA fragments cut with restriction endonucleases)
e) describe the properties of plasmids that allow them to be used in gene cloning
f) explain why promoters and other control sequences may have to be transferred as well as the desired gene
g) explain the use of genes for fluorescent or easily stained substances as markers in gene technology
h) explain the roles of restriction endonucleases, reverse transcriptase and ligases in genetic engineering
i) explain, in outline, how microarrays are used in the analysis of genomes and in detecting mRNA in studies of gene expression
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