12: Techniques of Molecular Genetics
Genetics is the study of the inheritance and variation of biological traits. We have previously noted that it is possible to conduct genetic research without directly studying DNA. Indeed some of the greatest geneticists had no special knowledge of DNA at all, but relied instead on analysis of phenotypes, inheritance patterns, and their ratios in carefully designed crosses.
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- 12.1: Prelude to Molecular Genetics
- Today, classical genetics is often complemented by molecular biology, to give molecular genetics, which involves the study of DNA and other macromolecules that have been isolated from an organism. Usually, molecular genetics experiments involve some combination of techniques to isolate and analyze the DNA or RNA transcribed from a particular gene.
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- 12.2: Isolating Genomic DNA
- DNA purification strategies rely on the chemical properties of DNA that distinguish it from other molecules in the cell, namely that it is a very long, negatively charged molecule. To extract purified DNA from a tissue sample, cells are broken open by grinding or lysing in a solution that contains chemicals that protect the DNA while disrupting other components of the cell (Figure 8.2). These chemicals may include detergents, which dissolve lipid membranes and denature proteins.
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- 12.3: Isolating or Detecting a Specific Sequence by PCR
- The Polymerase Chain Reaction (PCR) is a method of DNA replication that is performed in a test tube (i.e. in vitro). Here “polymerase” refers to a DNA polymerase enzyme extracted and purified from bacteria, and “chain reaction” refers to the ability of this technique produce millions of copies of a DNA molecule, by using each newly replicated double helix as a template to synthesize two new DNA double helices. PCR is therefore a very efficient method of amplifying DNA.
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- 12.4: Cutting and Pasting DNA- Restriction Digests and DNA Ligation
- Many bacteria have enzymes that recognize specific DNA sequences and then cut the double stranded DNA helix at this sequence. These enzymes are called site-specific restriction endonucleases, or more simply “restriction enzymes”, and they naturally function as part of bacterial defenses against viruses and other sources of foreign DNA. To cut DNA at known locations, researchers use restriction enzymes from various bacterial species, and which can be purchased from various commercial sources.
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- 12.5: Cloning DNA - Plasmid Vectors
- Many bacteria contain extra-chromosomal DNA elements called plasmids. These are usually small (a few 1000 bp), circular, double stranded molecules that replicate independently of the chromosome and can be present in high copy numbers within a cell. In the wild, plasmids can be transferred between individuals during bacterial mating and are sometimes even transferred between different species. Plasmids often carry genes for pathogenicity and drug-resistance.
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- 12.7: DNA Analysis- Blotting and Hybridization
- Bands of DNA in an electrophoretic gel form only if most of the DNA molecules are of the same size, such as following a PCR reaction, or restriction digestion of a plasmid. In other situations, such as after restriction digestion of chromosomal (genomic) DNA, there will be a large number of variable size fragments in the digest and it will appear as a continuous smear of DNA, rather than distinct bands.
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- 12.8: Transgenic organisms
- Transgenic organisms contain foreign DNA that has been introduced using biotechnology. Foreign DNA (the transgene) is defined here as DNA from another species, or else recombinant DNA from the same species that has been manipulated in the laboratory then reintroduced. The terms transgenic organism and genetically modified organism (GMO) are generally synonymous.
Contributors
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Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.