TOOLS OF GENETIC ENGINEERING By: Russel Jean Gallo

Recombinant DNA Technology and Procedures Molecular cloning permits the replication of a specific DNA sequence in a living microorganism. Although a very large number of host organisms and molecular cloning vectors are in use, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector. E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment. Modern bacterial cloning vectors (e.g. pUC19) use the blue-white screening system to distinguish colonies (clones) of transgenic cells from those that contain the parental vector. polymerase chain reaction  A technique in molecular biology for creating multiple copies of DNA from a sample; used in genetic fingerprinting etc. molecular cloning  a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. restriction enzyme  An endonuclease that catalyzes double-strand cleavage of DNA containing a specific sequence.

Selection A selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells. Recombinant DNA is introduced into the organism from which the replication sequences were obtained, then the foreign DNA will be replicated along with the host cell's DNA in the transgenic organism. Artificial genetic selection is the process in which cells that have not taken up DNA are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to surviveFigure 1. When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin. PCR  polymerase chain reaction molecular cloning  a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms.

Restriction Enzymes Cloning a gene involves cutting DNA using restriction enzymes and uniting it with a cut plasmid, then rejoining the strands with ligase. Restriction enzymes are present in bacteria to cut up foreign DNA when it enters the cell, such as another organism or phage. A restriction enzyme will identify a very specific nucleotide sequence called a restriction site and cut the bands of DNA within this site. Methyl groups protect the DNA of the bacteria from its own restriction enzymes. DNA cut by restriction enzymes yields restriction fragments. Most restriction enzymes used will recognize four to eight nucleotide sequences. Treatment of DNA copies with restriction enzymes always results in the same restriction fragment pattern because the DNA sequence is identical among copies. A sticky end of DNA will occur if a short extension of bases is left on one of the DNA strands. This staggered cut will allow the extension to pair with complementary sticky ends from other DNA fragments. DNA ligase is an enzyme that "seals" DNA back up. This enzyme re-forms covalent bonds in the sugar-phosphate backbone. The complementary binding between sticky ends will not remain unless made permanent by DNA ligase. restriction enzyme  An endonuclease that catalyzes double-strand cleavage of DNA containing a specific sequence ligase  Any of a group of enzymes that catalyze the binding of two molecules; synthetase

Mutation Mutations are accidental changes in a genomic sequence of DNA; this includes the DNA sequence of a cell's genome or the DNA or RNA sequence. Mutations are caused by radiation, viruses, transposons, and mutagenic chemicals. They are also caused by errors that occur during meiosis or DNA replication. Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique often used in biomolecular engineering in which a mutation is created at a defined site in a DNA molecule. The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation. It is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. mutation  Any heritable change of the base-pair sequence of genetic material. site-directed mutagenesis  Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique often used in biomolecular engineering in which a mutation is created at a defined site in a DNA molecule.

Southern Blots Agarose gel electrophoresis is a commonly used method for separating nucleic acid molecules by length using an electric field. Gel electrophoresis is a very common technique to separate DNA, RNA or proteins based on their size or charge. To perform electrophoresis, a gel, often made of agarose, is used. These gels form polymers and will allow the molecules to be separated. DNA has a negative charge so when electricity is applied, the DNA molecules move to the anode end of gel. Because the agarose forms a polymer structure, the longer the DNA fragment, the slower it moves through the gel. This phenomenon will separate the different DNA molecules into bands on the gel. Restriction fragment analysis is the unique band pattern that DNA will have after being treated with a particular restriction enzyme. Different DNA can be compared with restriction fragment analysis. Differences among DNA sequences in a population is called a polymorphism. When the difference occurs within a restriction site, it is called a restriction fragment length polymorphism (RFLP), which will have a different restriction fragment pattern based on the sequence of the individual. Using a restriction enzyme on genomic DNA yields too many bands to identify single genes. To overcome this, methods such as Southern blotting can be used. Southern blotting is a technique that allows specific genes in a band to be detected by combining gel electrophoresis and nucleic hybridation. electrophoresis  A method for the separation and analysis of large molecules, such as proteins, by migrating a colloidal solution of them through a gel; gel electrophoresis RFLP  Restriction fragment length polymorphism; a section of DNA whose length varies among individuals and which is delimited by a base which does not occur within it

Essentials of Molecular Cloning In molecular cloning, DNA is obtained from an organism of interest, combined with vector DNA, and replicated in a host organism. Plasmids are commonly used in cloning. A plasmid is small, circular DNA found in E. coli and other bacteria (also yeast). The plasmids replicate separately from the chromosomal DNA. To initiate cloning, the DNA from a foreign source (with gene of interest) is inserted into the plasmid. Because DNA from two different sources are present, it is called recombinant DNA. A recombinant bacteria forms when the recombinant plasmid is inserted into a bacteria. Now, every time the bacteria divides, it passes on the foreign DNA to its offspring. This can quickly result in a large population of bacteria that contain the foreign gene. Gene cloning is a process that results in multiple copies of a gene of interest. It is important because it can be used to amplify the gene for protein production. The copies can also be retrieved from the plasmid and used for research or inserted into another organism to produce a new trait. plasmid  A circle of double-stranded DNA that is separate from the chromosomes, and which is found in bacteria and protozoa. molecular cloning  A biological method that creates many identical DNA molecules and directs their replication within a host organism.

Plasmids as Cloning Vectors Plasmids can be used as cloning vectors, allowing the insertion of exogenous DNA into a bacterial target. All engineered vectors have an origin of replication, a multi-cloning site, and a selectable marker. Expression vectors (expression constructs) express the transgene in the target cell, and they have a promoter sequence that drives expression of the transgene. Transcription is needed for a plasmid to function, without the proper sequences to transcribe parts of a plasmid it will not be expressed or even maintained in host cells. Vectors can have many additional sequences that can be used for downstream applications—purification of proteins encoded by the plasmid and expressing proteins targeted to be exported or to a certain compartment of the cell. transcription  The synthesis of RNA under the direction of DNA. Kozak sequence  a sequence which occurs on eukaryotic mRNA and has the consensus (gcc)gccRccAUGG. The Kozak consensus sequence plays a major role in the initiation of the translation process. The sequence was named after the person who brought it to prominence, Marilyn Kozak. polyadenylation  The formation of a polyadenylate, especially that of a nucleic acid