Chapter 15 Recombinant DNA and Genetic Engineering In this chapter you will learn How restriction enzyme work and why they are essential to DNA technology. About various procedures such as cloning and isolating DNA, and detecting recombinant DNA molecules. Restriction Enzymes Function as Nature s Pinking Shears Nomenclature The first letter of the genus followed by the first two letters of the species name of their bacterial source. EcoR I from Escherichia coli Hind III from Hemophilus influenzue Taq I from Thermus aquaticus 1
Restriction enzymes make breaks in symmetric sequences Restriction enzyme recognizes only one short sequence, usually 4 to 6 base-pairs long. The sequences recognized by many restriction enzymes are palindromes Isolation and Characterization of DNA Fragments The breaks are usually not directly opposite one another Sticky, cohesive, or complementary end Make two singlestrand breaks two 3 -OH and two 5'- P groups 5 -G-A-A-T-T-C-3 3 -C-T-T-A-A-G-5 5 -G-3 -OH 3 -C-T-T-A-A-5 -p + p-5 -A-A-T-T-C-3 OH-3 -G-5 2
Table 15-1 Some restriction endonucleases and their cleavage sites These cohesive, compatible ends can be ligated Figure 15-1 Restriction fragments that have overhanging cohesive ends following digestion with some restriction enzymes can circularize 3
Map of Enzyme Cutting Sites is a Useful Tool Restriction maps indicate the cutting sites for specific restriction enzymes Restriction maps provide a convenient method for comparing DNA fragments. Figure 15-2 Procedure for preparing a restriction enzyme map for a DNA fragment 4
Large Maps Require Gene Libraries and Chromosome Walks In order to map a large segment or an entire genome, it is essential to isolate and put in linear order experimentally manageable (i.e., smaller) mapped segments. To achieve this potentially overwhelming task, chromosome walks are taken on large (10-20 Kb) DNA fragments (Figure 15-4). The collection of these fragments, containing pieces of DNA from the entire genome of the organism, is called a genomic library. Figure 15-4 Chromosome walking and jumping The problem is to localize Y with respect to X 5
PCR (Polymerase Chain Reaction) Yields Large Quantities of a Specific Sequence In 1983 a new technique, the polymerase chain reaction (PCR), was devised. It has made the detection and cloning of rare DNA sequences possible. Repeat Denature: heat, 95 C Anneal: 45 ~ 55 C Polymerization: extension, 65 or 72 C Figure 15-5 polymerase chain reaction 6
Figure 15-5 polymerase chain reaction The process of generating recombinant DNA: Production of desired DNA fragments: p383-386 Insertion of these fragments into a suitable vector such as a plasmid or phage: p392-397 Introduction of that vector into an appropriate host (usually a strain of E. coli) Identification, selection, & characterization of recombinant clones: p399-401 7
Genetic Interlopers Vectors function as vehicles for transferring genes To be useful, a vector must have three properties 1. Able to enter a bacterial host. 2. An origin of DNA replication that allows it to replicate in the host 3. Cells thus transformed must be selectable 8
Two types of vectors are commonly used in cloning Bacterial plasmids (often pbr322) small size, ease of handling most commonly used cloning vectors Antibiotic resistance is one of the most important features for selection Phages (l and M13) like hypodermic needles; inject DNA into the bacterial host where it replicates Many plasmids have been genetically engineered to serve as specialized vectors Polylinkers (multiple cloning sites) One or more specific restriction sites Shuttle vectors Received a eukaryotic transcriptional promoter can be expressed in both bacteria and eukaryotes. Still other vectors have unique promoters useful for sequencing DNA or synthesizing RNA in vitro 9
Table 15-2 A variety of cloning vectors are available Type of vector plasmids Lambda phage Cosmids Yeast plasmids Plant plasmids Advantage Easy to use and store. Recombinant plasmids readily selected with antibiotics. Useful for cloning large (15-20 Kb fragments). Combined plasmid/phage vector permits cloning of even larger (45 Kb) DNA fragments. Permit direct studies on eukaryotic gene regulation Bacterial (Agrobacterium) infection of plant transfers Ti plasmid into host plant cells Insertion of a DNA Fragment Into a Vector To select a vector with two restriction sites that are compatible with the restriction sites on the 5' and 3' termini of the fragment of foreign DNA The fragment of foreign DNA can then be inserted into the vector by directional cloning After a fragment of DNA is cloned into a plasmid, inserted into a bacterium, & grown to large amounts in a selecting (antibiotic-containing) medium, a method must be available to recover the fragment from its hybrid plasmid 10
Figure 15-6 Cloning foreign DNA Ends of DNA fragments Table 15-3 Parameters of the ligation reaction Cloning requirements Features Blunt ends (ex: Bal I) DNA and ligase present in high concentrations Restriction sites at junctions of DNAs may be lost; number of wildtype plasmids without inserts may be high; multiple tandem copies of the foreign DNA may be inserted 11
Table 15-3 Parameters of the ligation reaction Ends of DNA fragments Identical overhanging cohesive sticky ends (EcoR I) Cloning requirements Phosphatase treatment of linearized plasmid DNA reduces religation of wild type plasmids Features Restriction sites at junctions of ligated ends are constituted; foreign DNA inserted bidirestionally; multiple tandem copies of the foreign DNA may be inserted G Ends of DNA fragments Different cohesive ends (directional cloning) Table 15-3 Parameters of the ligation reaction Cloning requirements Purification of lenearized plasmid increases cloning efficiency Features Restriction sites at junctions of ligated DNA are usually reconstituted; recircularization of wildtype plasmid is low; insertion of foreign DNA is unidirectional 12
Figure 15-7 Directional cloning into a plasmid vector (puc19) Figure 15-7 Directional cloning into a plasmid vector (puc19) 13
After cloning, several types of molecules result 1. A re-ligated vector that has not acquired any fragments of foreign DNA 2. A vector with one or more foreign DNA fragments 3. A molecule without a vector, consisting only of join fragments or the organism s DNA. Do not contain an origin of replication, and therefore cannot be replicated in bacteria Detection?? Detection of Recombinant DNA Molecules Four methods are used to identify bacterial colonies that contain recombinant plasmids: 1. Screening by colony hybridization 2. Insertional inactivation of a plasmid gene (often for antibiotic resistance) 3. Analysis of plasmid DNA by restriction enzymes 4. a-complementation 14
Figure 15-8 Detection of transformed cells by colony hybridization with a radioactive probe Bacteria colonies are replica-plated onto a solid support Figure 15-9 Detection of transformed cells by insertional inactivation 15
a-complementation (figure 15-7) Vectors (puc19) carry a segment of regulatory sequences & coding information for first 146 amino acids of lacz gene (b-galasidase) Host E. coli cells that code for carboxyterminal portion of b-galasidase Neither the host-encoded nor the plasmidencoded fragment is active, but together they associate to form an active enzyme Site-Specific Mutagenesis Using Bacteriophage M-13 Vector 16
Site-Specific Mutagenesis Using Bacteriophage M-13 Vector The Recombinant DNA Concept DNA is DNA, regardless of the organism from which it is isolated Hence, genes from diverse species can be recombined to construct novel genes, or in certain instances, even new organisms It is used in research of all types: basic, medical agricultural, commercial, & industrial To filling some of most fundamental needs of humankind 17
Application of Genetic Engineering 1. Facilitating production of useful proteins 2. Creating bacteria capable of synthesizing economically important molecules 3. Supplying DNA & RNA sequences as research tools 4. Altering genotype of organisms (both animals & plants) 5. Potentially correcting genetic defects in animals (gene therapy) Figure 15-10 Direct injection of foreign genes into animal eggs & embryos A newly fertilized mouse egg Displaying both egg & sperm nuclei With a micropipette 18