Bio 100 DNA Technology 1 Chapter 12 - DNA Technology Among bacteria, there are 3 mechanisms for transferring genes from one cell to another cell: transformation, transduction, and conjugation 1. Transformation It involves the taking up of DNA from the fluid surrounding the cell 2. Transduction Among bacteriophages - a fragment of DNA belonging to a phage's former host cell gets into the new bacteria when the virus infects the new bacteria 3. Conjugation The union of 2 bacteria and the transfer of DNA between the cells Plasmids The ability of bacteria cells to carry out conjugation is usually due to a specific segment of DNA called the F factor The F factor carries genes for making sex pili and other things needed for conjugation It also contains a site where DNA replication can start An F factor can exist as a plasmid, a small, circular DNA molecule separate from the much larger bacterial chromosome in the bacterial cell A plasmid can replicate itself within the cell, and some, including the F factor plasmid, can serve as carriers (also called vectors) that move genes from one cell to another cell R Plasmids E. coli and other bacteria have several different kinds of plasmids One class of plasmids called R plasmids pose serious problems for human medicine Transferable R plasmids carry genes for conjugation and also genes for enzymes that destroy antibiotics such as penicillin and tetracycline Bacteria carrying R plasmids are resistant to antibiotics
Bio 100 DNA Technology 2 Plasmids and Biotechnology How to custom build a bacterial cell: 1. A plasmid is isolated from a bacterium such as E. coili 2. At the same time, DNA carrying a particular gene of interest is removed from another cell 3. A piece of DNA containing the gene is inserted into the plasmid producing recombinant DNA 4. The bacteria takes up the plasmid by transformation 5. This genetically engineered, recombinant bacterium is then cloned in large numbers to make multiple copies of the gene Biotechnology - the use of living organisms to perform practical tasks Restriction Enzymes Extracting a gene from a cell and inserting it into another requires precise cutting and pasting The cutting tools of recombinant DNA technology are bacterial enzymes called restriction enzymes Most restriction enzymes recognize short nucleotide sequences in DNA molecules and cut at specific points within these so-called recognition sequences There are several hundred restriction enzymes and about one hundred different recognition sequences known
Bio 100 DNA Technology 3 Cloning Making recombinant DNA in large enough amounts to be useful requires several steps 1. The biologist isolates 2 kinds of DNA: The bacterial plasmid that serves as the vector - the carrier that move genes from one cell to another cell Eukaryotic DNA containing the protein V gene 2. The researcher treats both the plasmid and the human DNA with the same restriction enzyme The enzyme cuts the plasmid DNA at one specific restriction site; it also cuts the human DNA 3. The human DNA is mixed with the cut plasmid 4. The enzyme DNA ligase joins the 2 DNA molecules by covalent bonds 5. The recombinant plasmid is added to a bacterium 6. The production of multiple copies of the gene - gene cloning Biologists refer to the result of cloning DNA taken directly from living cells as a genomic library The Role of Reverse Transcriptase in Cloning Not all DNA that is cloned comes directly from cells Some eukarytoic genes are too large to clone easily because they contain long noncoding regions (introns) 1. The cell makes a RNA transcript of the intron-containing gene 2. Cellular enzymes then remove the introns and splice the exons together producing mrna 3. The researcher isolates the mrna molecules from the cell and uses them as templates for DNA synthesis This synthesis of DNA on an RNA template is the reverse of transcription; catalyzed by an enzyme called reverse transcriptase
Bio 100 DNA Technology 4 4. After a single strand of DNA is synthesized the RNA and DNA strand separate 5. The second DNA strand is made using the first as a template (DNA of gene without introns) The DNA from this procedure is called complementary DNA (cdna) Polymerase Chain Reaction Biologists are often interested in copying a specific segment within a bunch of DNA This is done through a technique called polymerase chain reaction or PCR - essentially DNA is isolated and segments of DNA are amplified Barbara McClintock and the Discovery of Jumping Genes In the 1940s while studying the inheritance pattern in corn plants, McClintock made a rather startling discovery - certain genetic elements can move from one location to another in a chromosome or even from one chromosome to another She found that these mobile elements, often called transposons or "jumping genes" may land in the middle of other genes and disrupt them Strong evidence now suggests that all cells have segments of DNA, (transposable elements or transposons) that are able to move from one site to another McClintock found that a transposon inserted into the middle of a gene can make the gene non-functional These mobile genetic units can act as natural mutagens that generate genentic diversity DNA Fingerprinting DNA fingerprinting is a technique that allows you to determine genetic variation among closely related individuals based on the specific kinds of bands that are produced on gels The technique works based on the fact that there are duplicated noncoding regions (or repetitive regions) of the DNA referred to as mini- and microsatellite sequences This DNA is similar among closely related organisms that is, there is a core sequence of nucleotides shared among closely related individuals However, each individual also has a rather unique sequence - these are highly variable regions and they experience random mutations through time giving
Bio 100 DNA Technology 5 The Process of DNA Fingerprinting a. DNA is isolated from cells and cleaved at specific sites with an endonuclease b. The sample containing DNA fragments from each individual is placed in an electrophoretic gel where the fragments are separated by size and charge, producing a streak of fragments of different sizes in each lane of the gel c. The DNA fragments are then denatured into single-stranded segments and transferred to a nitrocellulose membrane by a Southern blot transfer d. The membrane is then washed with a solution containing single-stranded, radioactively labeled probes for the minisatellite DNA. The probe hybridizes with homologous fragments on the filter e. A piece of X-ray film is placed over the membrane and exposed. Each labeled hybrid fragment exposes the film and upon development shows up as a band. The pattern of bands constitutes an individual s unique DNA fingerprint Genetic Engineering and Agriculture Genetic engineers often use a plasmid vector to introduce new genes into plant cells Often the plasmid they use is from the soil bacterium, Agrobacterium tumefaciens When plants are infected with this bacterium in nature the develop tumors These are induced by the bacterium's Ti plasmid (Ti = tumor inducing) But researchers have found ways to eliminate the plasmid's cancer causing properties while keeping its ability to transfer DNA to plant cells 1. A gene of interest is inserted into a segment of the plasmid - T DNA 2. The recombinant plasmid is put into a plant cell, where the T DNA carrying the new gene integrates into a plant chromosome 3. The recombinant cell is cultured and grows into a new plant An organism that contains genes from another species is called a transgenic organism