Chap 10

Return to Essential Concepts

  • Recombinant DNA technology has revolutionized the study of the cell, making it possible for researchers to pick out any gene at will from the thousands of genes in a cell and to determine the exact molecular structure of the gene.
  • A crucial element in this technology is the ability to cut a large DNA molecule into a specific and reproducible set of DNA fragments by using restriction nucleases, each of which cuts the DNA double helix only at a particular nucleotide sequence.
  • DNA fragments can be separated from one another on the basis of size by gel electrophoresis.
  • Nucleic acid hybridization can defect any given DNA or RNA sequence in a mixture of nucleic acid fragments. This technique relies on the fact that a single strand of DNA or RNA will form a double helix only with another nucleic acid strand of the complementary nucleotide sequence.
  • Single-stranded DNAs of known sequence and labeled with fluorescent dyes or radioisotopes are used as probes in hybridization reactions.
  • Short DNA strands of any sequence can be made by chemical synthesis in the laboratory.
  • DNA cloning techniques enable a DNA sequence to be selected from millions of other sequences and produced in unlimited amounts in pure form.
  • DNA fragments can be joined together in vitro by using DNA ligase to form recombinant DNA molecules that are not found in nature.
  • DNA fragments can be maintained and amplified by inserting them into a DNA molecule capable of replication, such as a plasmid. This recombinant DNA molecule is then introduced into a rapidly dividing host cell, usually a bacterium, so that the DNA is replicated at each cell division.
  • A collection of cloned fragments of chromosomal DNA representing the complete genome of an organism is known as a genomic library. The library is often maintained as millions of clones of bacteria, each clone carrying a different DNA fragment.
  • cDNA libraries contain cloned DNA copies of the total mRNA of a particular cell type or tissue. Unlike genomic DNA clones, cloned cDNAs contain predominantly protein-coding sequences; they lack introns, regulatory DNA sequences and promoters. They are thus most suitable for use when the cloned gene is to be expressed to male a protein.
  • The PCR is a powerful form of DNA amplification that is carried out in vitro using a purified DNA polymerase. PCR requires prior knowledge of the sequence to be amplified, because two synthetic oligonucleotide primers must be synthesized that bracket the portion of DNA to be replicated.
  • Historically, genes are cloned using hybridization techniques to identify the plasmid carrying the desired sequence from a DNA library. Today, most genes are cloned using PCR to greatly amplify them and thereby obtain a specific sequence from a sample of DNA or mRNA.
  • Techniques are now available for rapidly determining the nucleotide sequence of any piece of DNA.
  • The complete nucleotide sequences of the genome of hundreds of different organisms have been determined. These include bacteria, archaea, yeasts, insects, fish, plants and mammals.
  • Bacteria, yeasts, and mammalian cells can be engineered to synthesize large quantities of any protein from any organism, thus making it possible to study proteins that are otherwise rare or difficult to isolate.
  • Using recombinant DNA techniques, a protein can be joined to a molecular tag, such as the green fluorescent protein (GFP), which allows the tracking of its movement inside the cell. In the case of GFP, the protein can be monitored over time in living organisms.
  • In situ nucleic acid hybridization can be used to detect the precise location of genes in chromosomes, or RNAs in cells and tissues.
  • By presenting a platform for performing a large number of simultaneous hybridization reactions, DNA microarrays can be used to monitor the expression of tens of thousands of genes at once.
  • Cloned genes can be permanently inserted into the genome of a cell or an organism by using recombinant DNA technology. Clones DNA can be altered in vitro to create mutant genes that can then be reinserted into a cell or an organism to study gene function.
  • A straightforward strategy for studying the function of a gene is to delete it from the organism's genome and then to study the effect of this knockout on the behavior or appearance of the organism.
  • The expression of particular genes can be inhibited in cells or organisms by the technique of RNA interference (RNAi), which prevents an mRNA from being translated into protein.
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