Concepts Summary

• Restriction endonucleases are enzymes that make double-stranded cuts in DNA at specific base sequences.
• DNA fragments can be separated with the use of gel electrophoresis and visualized by staining the gel with a dye that is specific for nucleic acids or by labeling the fragments with a radioactive or chemical tag.
• In gene cloning, a gene or a DNA fragment is placed into a bacterial cell, where it will be multiplied as the cell divides.
• Plasmids, small circular pieces of DNA, are often used as vectors to ensure that a cloned gene is stable and replicated within the recipient cells. Expression vectors contain sequences necessary for foreign DNA to be transcribed and translated.
• The polymerase chain reaction is a method for amplifying DNA enzymatically without cloning. A solution containing DNA is heated, so that the two DNA strands separate, and then quickly cooled, allowing primers to attach to the template DNA. The solution is then heated again, and DNA polymerase synthesizes new strands from the primers. Each time the cycle is repeated, the amount of DNA doubles.
• Genes can be isolated by creating a DNA library—a set of bacterial colonies or viral plaques that each contain a different cloned fragment of DNA. A genomic library contains the entire genome of an organism; a cDNA library contains DNA fragments complementary to all the different mRNAs in a cell.
• In situ hybridization can be used to determine the chromosomal location of a gene and the distribution of the mRNA produced by a gene.
• Positional cloning uses linkage relations to determine the location of genes without any knowledge of their products.
• The Sanger (dideoxy) method of DNA sequencing uses special substrates for DNA synthesis (dideoxynucleoside triphosphates, ddNTPs) that terminate synthesis after they are incorporated into the newly made DNA. Next-generation and third generation sequencing methods sequence many DNA fragments simultaneously, providing a much faster and less-expensive determination of a DNA sequence.
• Short tandem repeats (STRs) and microsatellites are used to identify people by their DNA sequences (DNA fingerprinting).
• Forward genetics begins with a phenotype and conducts analyses to locate the responsible genes. Reverse genetics starts with a DNA sequence and conducts analyses to determine its phenotypic effect.
• Site-directed mutagenesis can be used to produce mutations at specific sites in DNA, allowing genes to be tailored for a particular purpose.
• Transgenic animals, produced by injecting DNA into fertilized eggs, contain foreign DNA that is integrated into a chromosome. Knockout mice are mice in which a normal gene is disabled.
• RNA interference is used to silence the expression of specific genes.
• Techniques of molecular genetics are being used to create products of commercial importance, to develop diagnostic tests, and to treat diseases.
• In gene therapy, diseases are being treated by altering the genes of human cells.