Chapter 10 - Gene Isolation and Manipulation

• Design of experiments is used to address yet-to-be-solved genetic problems.
• Techniques for isolating and manipulating fragments of DNA, amplifying DNA, sequencing DNA, and introducing DNA into an organism are examples of such techniques.
• CRISPR-Cas9 is a recently developed technique for the precise engineering of genomes.
• Genome engineering techniques have made reverse genetic studies possible, which aim to understand the function of a gene by analyzing the phenotypic consequences of altering the gene sequence or its expression.
• Genes are the central focus of genetics, and isolating a gene of interest from the genome for study can be a daunting task due to the size of the genome.
• Forward genetic approaches, such as mutagenesis and crosses or pedigree analysis, are used to search for mutants that exhibit an altered phenotype and determine whether that phenotype is determined by a single gene.
• Mapping by recombination helps locate the gene at the DNA level.
• The first step in studying gene function is to isolate its DNA and reproduce it in quantities suitable for study.
• The tools of the genetic engineer are molecules isolated from cells, many of which were the product of scientific discovery.
• Restriction enzymes are a cornerstone of genetic engineering and a common tool found in the genetic engineer’s toolbox.
• DNA polymerase, discovered by Arthur Kornberg, can be fashioned into two powerful tools for DNA isolation and analysis.
• DNA technologies are the collective techniques for obtaining, amplifying, and manipulating specific DNA fragments.
• Mice have two insulin genes, Ins1 and Ins2, on chromosomes 19 and 7, respectively.
• The open reading frame of the human Ins mRNA is 81 percent identical in nucleotide sequence to the mouse Ins1 mRNA and 83 percent to Ins2, and the human Ins protein is 78 percent identical in amino acid sequence to the mouseIns1 protein and 82 percent to Ins2.
• Southern blot; Probe: D N A or R N A fragment
• Polymerase chain reaction, P C R; Probe: D N A primers
• Fluorescence in situ hybridization, F I S H; Probe: D N A or R N A fragment
• Northern blot; Probe: D N A or R N A fragment
• Reverse transcription P C R, R T P C R; Probe: D N A primers
• In situ hybridization; Probe: D N A or R N A fragment
• Restriction enzyme Eco RI is used to make a staggered double-strand cut at a single site in a circular vector such as a bacterial plasmid, converting the circular DNA into a single linear molecule with half of an Eco RI site at each end.
• Plasmids can be engineered to contain a multiple cloning site (MCS) or polylinker that contains restriction enzyme recognition sites that do not occur elsewhere in the plasmid.
• Cleavage at any one of these sites linearizes the plasmid rather than cutting it into multiple pieces.
• Eco RI digestion of a linear piece of DNA at two sites produces a DNA fragment with half of an Eco RI site at each end.
• Mixing the linearized vector with the linear insert allows the “sticky” ends of the vector and insert to hybridize and form a recombinant molecule.
• DNA ligase finishes the job by creating phosphodiester linkages at the junctions between vector and insert sequences.
• If a single restriction enzyme is used for cloning, or if two restriction enzymes are used that both create blunt ends, the insert can hybridize in two orientations relative to the vector.
• Cloning with two different restriction enzymes limits hybridization of the insert and vector to one orientation.
• To form a recombinant DNA molecule, restriction enzymes are used to cut a vector and an insert.
• Because of sequence complementarity at the ends of the vector and insert, the vector and insert anneal.
• DNA ligase then permanently links the vector and insert together.
• Depending on the restriction enzymes used, the insert can hybridize with the vector in two orientations or only one orientation.
• The expected size of a protein on a Western blot can be estimated based on the average molecular weight of an amino acid, 110 Daltons (Da).
• Only one insulin protein band should be detected in mouse β cells because the Ins1 and Ins2 proteins are the same size and, thus, should migrate to the same position upon gel electrophoresis.
• Northern, Western, and Southern blot analyses for insulin can be performed in different human and mouse tissues.
• Sometimes, the starting material for Southern blot analysis is chromosome-sized DNA molecules of genomic DNA.
• Restriction enzymes are endonucleases that cleave phosphodiester bonds between nucleotides at specific DNA sequences, called restriction sites, that are usually 4 to 8 base pairs long.
• Restriction sites are palindromic, meaning that both strands have the same nucleotide sequence but in antiparallel orientation.
• Restriction enzyme names are based on the organism in which they were discovered.
• Approximately 3000 restriction enzymes have been identified that recognize over 230 restriction sites.