I. Gene Cloning & Recombinant DNA Biotechnology: Figure 1: Restriction Enzyme Activity Restriction Enzyme: Most restriction enzymes recognize a single short base sequence, or Restriction Site. Restriction sites are usually palindromic sequences that read the same on each strand in the 5 3 direction. Restriction enzymes bind to DNA at these sites & make a break in each strand, resulting in Restriction Fragments. The ends of each fragment have an overhanging piece of single stranded DNA, called a sticky end because it is able to base pair with any other fragment having a complementary sticky end. Recombinant DNA:
Gene Cloning Restriction fragments containing a gene of interest (GOI) can be incorporated into bacterial Plasmids (small, circular, self-replicating pieces of DNA). Figure 2: Bacterial Plasmids Plasmids within Living Cells Sample Plasmid Plasmids act as convenient vectors ( vehicles ) to introduce the GOI into a living system such as bacterial cells that can express the gene to obtain an economically valuable protein product (e.g. human insulin). Figure 3: Gene Cloning Phase 1: Isolation of GOI & Introduction into Plasmid Both plasmid DNA & restriction fragments must be treated with the SAME restriction enzyme so that they have complimentary sticky ends. The restriction fragment is then incorporated into the plasmid & the nicks in the resulting recombinant DNA are sealed with ligase.
Figure 3.1: Gene Cloning Phase 2: Introduction of rplasmid into Host System The recombinant plasmid is introduced into bacterial cell by Transformation. As the transformed bacterial cells divide, the plasmid & gene of interest are replicated (cloned) & passed onto the resulting daughter cells. Thus, each new cell has inherited the gene of interest & has the ability to express the selected trait/gene product. Gene Cloning: II. DNA Fingerprinting Short Tandem Repeats (STR) Individuals are highly variable in the number of repetitive sequences within junk DNA. Short Tandem Repeats are short sequences that repeat in tandem (in sequence), such as CTA. The number of such repeats varies between homologous chromosomes & from person to person. In the example below, each person has a different number of the STR CTA within the same chromosome: When such differences in STR copy numbers are taken into account at multiple loci throughout the genome, they can be used to distinguish a person from all others. In other words, a DNA Fingerprint for an individual can be produced. For identification purposes, the FBI examines the copy number of a given STR at 12 different loci, as well as a gender marker (e.g. presence of Y chromosome). The probability that 2 people have the same STR copy at all 12 loci is astronomically small!
In order to create a DNA fingerprint, each STR must be amplified millions of times in order to potentially visualize within a gel. The technique used to increase the copy number of select regions of the genome is called Polymerase Chain Reaction (PCR): Figure 4: PCR Polymerase Chain Reaction (PCR): a) DNA chains are separated by heating. During the cooling cycle, the templates associate with primers & taq polymerase to form new strands. The process is repeated with each heating & cooling cycle in which the amount of DNA is doubled. Thus after only 20 cycles, this exponential cycle yields 2 20 copies of the target sequence. Figure 5: Gel Electrophoresis Gel Electrophoresis: a) STR s amplified by PCR are of different lengths that can be separated by passing them through a gel. Agarose gel is prepared containing wells for the digested samples to be placed. An electric field is applied & the negatively charged DNA fragments move toward the positive terminal, their rate depending on the electrical field strength &
size of the fragments. As the DNA moves through the tiny passages in the gel, the larger fragments encounter more resistance & do not travel as great a distance. b) The separated fragments may be stained with ethidium bromide. The larger the fragment, the more intense the staining. When exposed to UV light, the fragments fluoresce to produce discrete bands. When compared to a known sample, the banding pattern can be used for identification purposes. Figure 5.1: DNA Fingerprint via Gel Electrophoresis *The bands with the greatest STR copy numbers are found closest to the wells while those having the least STR copy numbers the farthest. The banding pattern produced reflects differences in the STR copy numbers at each loci & constitutes a unique DNA fingerprint.
III. Genetically Modified Organisms Figure 6: Transgenic Organisms: General Overview Transgenic Organism: Figure 6.1: Transgenic Organisms: Atlantic Salmon Transgenic Atlantic salmon exhibit rapid growth, reaching market size in 16-18 months instead of 3 years. Additionally, the salmon requires 20% less food compared to non-transgenic salmon.
Figure 6.2: Transgenic Organisms: Bt Crops Bt Crops: engineered with a gene from the bacterium Bacillus thrugingiensis that codes for a toxin. When pests eat ANY part of the Bt crop plant, the toxin degrades their gut lining & kills them. Bt crops (corn, potato, cotton, soybeans) target specific pests & reduce the need for pesticides. Figure 7: Reproductive Cloning: Overview
Figure 7.1: Reproductive Cloning: Reactivating Embryonic Genes Reproductive Cloning: Figure 8: Therapeutic Cloning: Embryonic Stem Cell Types Stem Cells: