Introduction Plasmids Bacteriophage Lambda Vectors Cosmids Yeast Artificial Chromosomes (YACs) Bacterial Artificial Chromosomes (BACs) Library Screening and Gene Sequencing Course Topics Course Home Page

Plasmids Characteristics of plasmids extrachromosomal circular DNA molecules which are not part of the bacterial genome size range: 1-200 kb carry functions advantageous to the host such as: produce enzymes which degrade antibiotics or heavy metals produce restriction and modifying enzymes Replication is coupled to host replication in a: stringent manner - one (or two) plasmids made during each round of bacterial replication relaxed manner - 10-200 copies of the plasmid made during each round of bacterial replication; (this can be increased to 1000-2000 plasmids by stopping host protein synthesis and replication with the antibiotic chloramphenicol) Foreign DNA is inserted into a plasmid (or any cloning vector) by ligating the DNA into a complementary site in the plasmid. These sites are generated by digesting the DNA and vector with the same restriction enzyme. (The site for the restriction enzyme that is chosen should only be represented once in the plasmid. Thus, when the plasmid is digested, a single, linear molecule would be generated.) The foreign DNA is then inserted into the plasmid by the action of the enzyme DNA ligase. The next step is to insert the ligated DNA into a bacterial cell for propagation. This is done by a technique called transformation. Bacterial cells are treated with either Ca2Cl or Rb2Cl. This treatment produces pores in the bacterial cell wall and membrane through which the plasmid enters. Although there is no size limitation to the ligation reaction, transformation efficiency is dictated by the size of the plasmid. Effect of plasmid size on transformation efficiency Molecule size (kb) % Maximum probability 2.0 57 3.2 100 4.3 86 12.5 43 20.0 36 39.0 14 54.0 6 The goal of the ligation reaction is to insert the foreign DNA into the vector. An unwanted ligation product also occurs - religated vector. One way to minimize this event is to treat the vector with phosphatase. This removes the terminal phosphate group from the restriction site of the vector and theoretically prevents religation of the two ends. In reality though, this treatment is never 100%, so a low level of religation does occur. Thus after transformation you will have two types of cells: those which contain the original plasmid and those that contain a plasmid containing foreign DNA. Plasmids are designed to distinguish the two types of transformation products. pBR322, the first widely used vector, utilizes differential antibiotic screening to distinguish the two types of transformation products. Let's say that we clone into the BamHI site of the vector. The insert DNA will then split the gene responsible for tetracycline resistance. But at the same time, the gene for ampicillin resistance is left intact. Transformed cells are first grown on bacterial plates containing ampicillin. This will kill all the cells that do not contain a plasmid. But we still cannot say which cells contain foreign DNA. Those cells that grew on ampicillin are then replica plated on plates with ampicillin and tetracycline. Those cells which grow in the presence of the ampicillin, but die under tetracycline selection contain plasmids which have foreign DNA inserts. pBR322 was a breakthrough for molecular biology, but the double screening procedure was time consuming and could be subject to error. In 1981, a new series of plasmids were developed that permitted the identification of the foreign DNA containing cells in a single screening step. These are called the pUC plasmids. As with pBR322, ampicillin resistance is used as one selectable marker. The second marker is based on insertional inactivation of the E. coli lacZ gene. The wild type gene can hydrolyze a specific dye [X-Gal (5-bromo-4-indoyl-B-D- galactopyranoside)] to a blue color, and the bacterial colony is stained blue. A multiple cloning site has been inserted into this gene. This site will accept fragments ending in a number of different restriction enzymes. Upon insertion of DNA into this site, the activity of the gene is eliminated and the colony appears white in color. Thus transformed colonies containing plasmids with inserts can be distinguished from those with plasmids without inserts based on the color of the colony and the ability of the colony to grow on an ampicillin containing media. cDNA Cloning (Cloning Eukaryotic mRNA) cDNA cloning is a method of obtaining a DNA copy of the mRNAs that are expressed at a specific stage in the development of the plant. In this manner, you can enrich the library that you will screen for those sequences in which you are interested. The reagent required for this type of cloning approach is mRNA. These mRNAs have a poly A+ tail. This tail permits the isolation of poly A+ mRNA by using either oligo-dT or oligo-U columns. Total RNA is run through one of these columns under conditions which favor the binding of the tail to the matrix on the column. After the column is extensively washed, the conditions are changed and the bound mRNA is isolated. This is the starting reagent for cDNA cloning. Steps in cDNA Cloning Bind oligo-dT to the poly +A tail of the mRNA. Add reverse transcriptase and make a DNA copy of mRNA (cDNA). Erase the mRNA with alkali and high temperature. (First strand synthesis) Add a C-tail to the 3' end of the cDNA with terminal transferase. Add oligo-dG to the tailed-cDNA and make the second strand with reverse transcriptase or the Klenow fragment of DNA Polymerase I. Add dC's to the 3' end of the double-stranded cDNA with terminal transferase Add C-tailed, ds-cDNA to G-tailed, PstI cut pBR322 and anneal. (DNA ligase is not required for this step.) Transform E. coli cells. Select Tetr/Amps cells. Most, but not all inserts can be removed by PstI digestions. Copyright © 1998. Phillip McClean