Genetic Engineering

Genetic Engineering: Enzymes for Dicing and Splicing Nucleic Acids Restriction endonucleases enzymes capable of recognizing foreign DNA and breaking the phosphodiester bonds between adjacent nucleotides on both strands of DNA protects bacteria against incompatible DNA of bacteriophages allows biotechnologists to cleave DNA at desired sites necessary for recombinant DNA technology recognize and clip at palindromes

Restriction Endonucleases Make staggered symmetrical cuts that leave short tails called “sticky ends” cut four to five bases on the 3’ strand and on the 5’ strand, leaving overhangs on each end adhesive tails will base-pair with complementary tails on other DNA fragments or plasmids

Restriction Endonucleases restriction fragments: pieces of DNA produced by restriction endonucleases restriction fragment length polymorphisms (RFLPs): differences in the cutting patterns of specific restriction endonucleases

Analysis of DNA Gel electrophoresis produces a readable pattern of DNA fragments samples are placed in compartments in a soft agar gel and subjected to an electrical current phosphate groups have a negative charge, which causes DNA to move toward the positive pole in the gel larger fragments migrate more slowly; smaller fragments migrate more quickly position of fragments are determined by staining the gel creates a genetic fingerprint

Revealing the Patterns with Electrophoresis Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Electrophoresis Restriction endonucleases selectively cleave sites of DNA Known DNA size markers Samples 1 2 3 4 5 Wells Restriction fragments DNA for sample 3 Larger Smaller 1 2 3 4 5 Samples (–) Wells Size markers (b) (+) DNA migrates toward positive electrode. (a) © Kathy Park Talaro

Polymerase Chain Reaction: A Molecular Xerox Machine for DNA Rapidly increases the amount of DNA in a sample without the need for making cultures or carrying out complex purification techniques Sensitive enough to detect cancer from a single cell or diagnose an infection from a single gene copy Rapid enough to replicate target DNA from a few copies to billions of copies in a few hours

Polymerase Chain Reaction Primers: DNA strands 15 – 30 bases long that serve as landmarks where DNA amplification should begin DNA polymerase from thermophilic bacteria “Taq” polymerase isolated from Thermus aquaticus remain active at elevated temperatures used in PCR Thermal cycler: automatically performs the cyclic temperature changes required for PCR

Recombinant DNA Technology Remove genetic material from one organism and combine it with that of a different organism Bacteria can be genetically engineered to mass produce substances such as hormones, enzymes, and vaccines difficult to synthesize by usual industrial methods

Recombinant DNA Technology Genetic clones/cloning involves removal of a selected gene from an animal, plant, or microorganism and propagated in a host microorganism gene must be inserted into a vector (usually a plasmid or a virus) vector inserts the gene into the cloning host cloning host is usually a bacterium or yeast which can translate the gene into the desired protein

Cloning Vectors Plasmids small, well characterized, easy to manipulate can be transferred into appropriate cells through transformation Bacteriophages: have the natural ability to inject DNA into bacterial hosts through transduction Vectors typically contain a gene that confers drug resistance to their cloning host cells can be grown on drug containing media only those cells that harbor a plasmid will be selected for growth

Cloning vectors Carry a significant piece of the donor DNA (gene of interest) Readily accepted DNA by the cloning host Contain an origin of replication Contain a selective antibiotic resistant gene Ex. Plasmids, phages

Producing Recombinant DNA is easy Start with a cloning vector (special plasmid) and DNA with your gene of choice. Cut the cloning vector and your desired gene out of the parent chromosome with specific enzymes.

Producing Recombinant DNA is easy Mix the vector and the gene together with a ligase enzyme which “seals” the DNA together. Use various techniques to insert the vector +gene into a new cell.

Producing Recombinant DNA is easy Grow cell on selective or differential media to find out which cells possess the recombinant plasmid.

Modified bacteria Pseudomonas syringae Pseudomonas fluorescens Natural bacteria that grow on plants but promote frost crystals Alteration of the normal frost gene now prevents frost crystals from forming on plants (applied with crop duster to compete with the natural bacteria in the field) Pseudomonas fluorescens This bacteria was engineered to contain an insecticide gene. The bacteria is sprayed on fields with crop dusting planes. The bacteria grow on the plants and when the insects start to eat the plant they will also eat some bacteria with the insecticide. The ingestion of insecticide kills the insects.

Transgenic plants using bacteria Agrobacterium tumefaciens Ti plasmid contains gene of interest, and is integrated into plant chromosome Ex. tobacco, garden pea, rice

Schematic of Agrobacterium tumefaciens transferring and integrating the Ti plasmid into the plant chromosome. Fig. 10.11 Bioengineering of plants

Transgenic animals Pharmaceutical production Knockout mouse Tailor-made genetic defects Cystic fibrosis Gaucher’s disease Alzheimer’s disease Sickle-cell anemia Allow us to research cures to these diseases

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Genotypic Diagnostics: DNA Analysis Using Genetic Probes Hybridization used to identify bacterial species by analyzing the sequences of nitrogenous bases in DNA probes: small fragments of single-stranded DNA or RNA complementary to the specific DNA sequence of a particular microbe unknown test DNA is extracted from cells and bound to blotter paper probes are added to the blotter paper and visible signs that probes have been hybridized to the test DNA

Complementary base pairing DNA Analysis Using Genetic Probes Probes can be labeled with chemically luminescent materials that can be measured by “light meters,” which avoids the use of radioactivity Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ATGGTC No complementary base pairing Addition of probes Blotter paper containing DNA from unknown organism No color development on the paper CTGGTA Complementary base pairing TACCAG ATGGTC Color development on the paper

Nucleic Acid Sequencing and rRNA Analysis One of the most viable indicators of evolutionary relatedness and affiliation is the comparison of 16s rRNA sequences 16s rRNA is part of the 30s subunit of the bacterial ribosome 16s rRNA is highly conserved across species and evolutionary time perfectly suited for bacterial identification and diagnosis of infection

rRNA Analysis Fluorescent in situ hybridization (FISH) rapidly identifies 16s RNA sequences without first culturing the organism relies on dyes to emit visible light in response to UV radiation turnaround time for identifying suspect pathogens present in blood cultures has been reduced from 24 hours to 90 minutes

Peptide Nucleic Acid FISH Testing for S. aureus Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. S. aureus PNA probe Nucleic acid Peptide Fluorescent molecule 90 minutes Gram-positive cocci in clusters S. aureus on a slide Positive blood culture tube rRNA in ribosome Image provided by AdvanDx, Inc.