12.1 Microbes and the Tools of Genetic Engineering
- Biotechology is the science of utilizing living systems to benefit humankind. In recent years, the ability to directly alter an organism’s genome through genetic engineering has been made possible due to advances in recombinant DNA technology, which allows researchers to create recombinant DNA molecules with new combinations of genetic material.
- Molecular cloning involves methods used to construct recombinant DNA and facilitate their replication in host organisms. These methods include the use of restriction enzymes (to cut both foreign DNA and plasmid vectors), ligation (to paste fragments of DNA together), and the introduction of recombinant DNA into a host organism (often bacteria).
- Blue-white screening allows selection of bacterial transformants that contain recombinant plasmids using the phenotype of a reporter gene that is disabled by insertion of the DNA fragment.
- Genomic libraries can be made by cloning genomic fragments from one organism into plasmid vectors or into bacteriophage.
- cDNA libraries can be generated to represent the mRNA molecules expressed in a cell at a given point.
- Transfection of eukaryotic hosts can be achieved through various methods using electroporation, gene guns, microinjection, shuttle vectors, and viral vectors.
12.2 Visualizing and Characterizing DNA, RNA, and Protein
- Finding a gene of interest within a sample requires the use of a single-stranded DNA probe labeled with a molecular beacon (typically radioactivity or fluorescence) that can hybridize with a complementary single-stranded nucleic acid in the sample.
- Agarose gel electrophoresis allows for the separation of DNA molecules based on size.
- Restriction fragment length polymorphism (RFLP) analysis allows for the visualization by agarose gel electrophoresis of distinct variants of a DNA sequence caused by differences in restriction sites.
- Southern blot analysis allows researchers to find a particular DNA sequence within a sample whereas northern blot analysis allows researchers to detect a particular mRNA sequence expressed in a sample.
- Microarray technology is a nucleic acid hybridization technique that allows for the examination of many thousands of genes at once to find differences in genes or gene expression patterns between two samples of genomic DNA or cDNA,
- Polyacrylamide gel electrophoresis (PAGE) allows for the separation of proteins by size, especially if native protein charges are masked through pretreatment with SDS.
- Polymerase chain reaction allows for the rapid amplification of a specific DNA sequence. Variations of PCR can be used to detect mRNA expression (reverse transcriptase PCR) or to quantify a particular sequence in the original sample (real-time PCR).
- Although the development of Sanger DNA sequencing was revolutionary, advances in next generation sequencing allow for the rapid and inexpensive sequencing of the genomes of many organisms, accelerating the volume of new sequence data.
12.3 Whole Genome Methods and Pharmaceutical Applications of Genetic Engineering
- The science of genomics allows researchers to study organisms on a holistic level and has many applications of medical relevance.
- Transcriptomics and proteomics allow researchers to compare gene expression patterns between different cells and shows great promise in better understanding global responses to various conditions.
- The various –omics technologies complement each other and together provide a more complete picture of an organism’s or microbial community’s (metagenomics) state.
- The analysis required for large data sets produced through genomics, transcriptomics, and proteomics has led to the emergence of bioinformatics.
- Reporter genes encoding easily observable characteristics are commonly used to track gene expression patterns of genes of unknown function.
- The use of recombinant DNA technology has revolutionized the pharmaceutical industry, allowing for the rapid production of high-quality recombinant DNA pharmaceuticals used to treat a wide variety of human conditions.
- RNA interference technology has great promise as a method of treating viral infections by silencing the expression of specific genes
12.4 Gene Therapy
- While gene therapy shows great promise for the treatment of genetic diseases, there are also significant risks involved.
- There is considerable federal and local regulation of the development of gene therapies by pharmaceutical companies for use in humans.
- Before gene therapy use can increase dramatically, there are many ethical issues that need to be addressed by the medical and research communities, politicians, and society at large.