Chapter 8: Recombinant DNA Technology

Chapter 8: Recombinant DNA Technology Linnea Fletcher Ph.D. BIOL 2316 Principal Points  Cloning is the making of many copies of a segment of DNA  Cloning vectors range from plasmids to artificial chromosomes to viruses  Genomic libraries are collections of clones that contain at least one copy of every DNA sequence in an organism’s genome  cDNA made from mRNA represent eukaryotic genes without introns  Identification of clones can be done using cDNA probes, antibodies, complementation of mutations, and sequencing  The PCR is a procedure for amplifying a specific segment of DNA DNA fragments separated by gel electrophoresis and visualized under UV light. (EtBr intercalates between the bases; allowing for visualization of the DNA)  What are the 3 general steps used to clone DNA?  Isolate DNA from an organism  Cut the organismal DNA and the vector with restriction enzymes making recombinant DNA  Introduce the recombinant DNA into a host  Restriction Enzymes  Recognize a specific site (generally a pallidromic sequence)  Produce overhangs or straight cuts  Naturally found in bacteria, they protect against viruses and foreign DNA  More than 400 enzymes have been isolated  Named for the organism they from which they are isolated  The first letter is that of the genus and the 2 nd and 3 rd are from the species Figure 8.1 Restriction site in DNA, showing symmetry of the sequence around the center point. The sequence is a palindrome, reading the same from left to right (5’-to-3’) on the top strand (GAATTC, here) as it does from right to left (5’-to-3’) on the bottom strand. Shown is the restriction site for EcoRI. How are restriction enzymes named? Figure 8.2 Examples of how restriction enzymes cleave DNA. (a) SmaI results in blunt ends. (b) BamHI results in 5’ overhanging (“sticky”) ends. (c) PstI results in 3’ overhanging (“sticky”) ends. Figure 8.3 Cleavage of DNA by the restriction enzyme EcoRI. EcoRI makes staggered, symmetrical cuts in DNA, leaving “sticky” ends. A DNA fragment with a sticky end produced by EcoRI digestion can bind by complementary base pairing (anneal) to any other DNA fragment with a sticky end produced by EcoRI cleavage. The gaps can then be sealed by DNA ligase.  What types of nucleic acids are used as cloning vectors?  What are some elements that are necessary for a plasmid cloning vector and in what ways is the pUC19 vector especially useful for cloning DNA?  Why must a cloning plasmid be recircularized before transforming a host bacterium?  Describe how insertional mutagenesis is used to distinguish nonrecombinant plasmid from recombinant plasmid when screening transformed hosts.  What are some elements that are necessary for a shuttle plasmid cloning vector?  What are some elements that are necessary for an expression cloning vector, a YAC, and a BAC?  Compare the size of DNA insert that can be carried by a plasmid, a YAC, and a BAC. Must have: 1) Ori 2) A dominant selectable Marker 3) Cleavage sites for cloning 4) (high copy no.) Figure 8.4 The plasmid cloning vector pUC19. This plasmid has an origin of replication (ori), an ampR selectable marker, and a polylinker located within part of the β-galactosidase gene lacZ+. [...]... for eukaryotes Figure 8.8 The synthesis of double-stranded complementary DNA (cDNA) from a polyadenylated mRNA, using reverse transcriptase, RNase H, DNA polymerase I, and DNA ligase Cloning of cDNA using BamHI linkers Screening     What screening method can be used to identify a recombinant clone with a target gene in a cDNA library cloned into an expression vector? (Western Blot) What role does... Genosys makes custom oligos) Radioactive Labeling of DNA Using DNA probes to screen plasmid genomic libraries for specific DNA sequences PowerPoint® Layered Art Figure 8.11 Using DNA probes to screen plasmid genomic libraries for specific DNA sequences PowerPoint® Layered Art Figure 8.11 Using DNA probes to screen plasmid genomic libraries for specific DNA sequences PowerPoint® Layered Art PowerPoint®...Figure 8.5 Insertion of a piece of DNA into the plasmid cloning vector pUC19 to produce a recombinant DNA molecule The vector pUC19 contains several unique restriction enzyme sites localized in a polylinker that are convenient for constructing recombinant DNA molecules The insertion of a DNA fragment into the polylinker disrupts part of the β-galactosidase (lacZ+)... some advantages of constructing a cDNA library? How is mRNA isolated for constructing a cDNA library and what special enzymes are required to prepare cDNA? What is adapter and linker DNA, and what advantages do they offer? Figure 8.7 Use of partial digestion with a restriction enzyme to produce DNA fragments of appropriate size for constructing a genomic library cDNA Libraries are best for eukaryotes... Review the next slide—know how to do this! Screening for specific cDNA plasmids in a cDNA Library by using an antibody probe The antibody binds to a specific Site on a protein that is made via The inserted foreign DNA This Is a Western Blot since it uses antibody To detect a protein          Southern Blot: Detection of A Specific DNA Sequence Using a Probe Once some sequence information is available... Describe the two apposing forces that allows DNA fragments to be separated by size in gel electrophoresis How are DNA fragments detected in an agarose gel? How are the sizes of DNA fragments determined in an agarose gel? Figure 8.14 An agarose gel electrophoresis apparatus (right) and power supply (left) Restriction Mapping      How is a restriction map of a recombinant plasmid determined, and what... that allows autonomous replication in yeast (ARS), and restriction sites for cloning      What types of DNA inserts are found in genomic libraries, chromosomal libraries, and a cDNA library? Describe the steps in constructing a genomic library What strategies can be used to ensure that the DNA inserts for a genomic library are especially large? How can you avoid “gaps” in a genomic library? How... in a genomic library? What is replica plating, and how does it make library screens more efficient? What are some requirements for probing DNA with a complementary DNA fragment and how are these requirements met in screening a library with a probe? Describe how DNA probes can be labeled for detection of annealing to a complementary sequence Which method(s) have the greatest sensitivity of detection?... applications of northern blot analysis? DNA Sequencing      Describe the rationale behind the Sanger dideoxy sequencing technique What are the necessary components of sequencing reactions? How is the radiogram interpreted in sequencing reactions? In what ways are the sequencing reactions different in an automated DNA procedure? What types of information does DNA sequencing reveal? ... cloned fragment, the orientation of the clones can be distinguished by the restriction fragment sizes Figure 8.16 Southern blot procedure for analyzing cellular DNA for the presence of sequences complementary to a labeled probe, such as a cDNA molecule made from an isolated mRNA molecule The hybrids, shown as three bands in this theoretical example, are visualized by autoradiography or chemiluminescence