Recombinant DNA Technology

Recombinant DNA Technology
8 Recombinant DNA Technology

The Role of Recombinant DNA Technology in Biotechnology
Biotechnology — the use of microorganisms to make practical products Recombinant deoxyribonucleic acid (DNA) technology Intentionally modifying genomes of organisms for practical purposes Three goals: Eliminate undesirable phenotypic traits Combine beneficial traits of two or more organisms Create organisms that synthesize products humans need

Figure 8.1 Overview of recombinant DNA technology.
Bacterial cell DNA containing gene of interest Bacterial chromosome Plasmid Isolate plasmid. Gene of interest Enzymatically cleave DNA into fragments. Isolate fragment with the gene of interest. Insert gene into plasmid. Insert plasmid and gene into bacterium. Culture bacteria. Harvest copies of gene to insert into plants or animals. Harvest proteins coded by gene. Eliminate undesirable phenotypic traits. Create beneficial combination of traits. Produce vaccines, antibiotics, hormones, or enzymes.

The Tools of Recombinant DNA Technology
Mutagens Physical and chemical agents that produce mutations Scientists utilize mutagens to: create changes in microbes' genomes to change phenotypes. select for and culture cells with beneficial characteristics. Mutated genes alone can be isolated

The Use of Reverse Transcriptase to Synthesize complementary DNA (cDNA) Isolated from retroviruses Uses ribonucleic acid (RNA) template to transcribe molecule of cDNA Easier to isolate mitochondrial RNA (mRNA) molecule for desired protein first cDNA generated from mRNA of eukaryotes has introns removed Allows cloning in prokaryotic cells

Synthetic Nucleic Acids Molecules of DNA and RNA produced in cell-free solutions Uses of synthetic nucleic acids: Elucidating the genetic code Creating genes for specific proteins Synthesizing DNA and RNA probes to locate specific sequences of nucleotides Synthesizing antisense nucleic acid molecules Synthesizing polymerase chain reaction (PCR) primers

Restriction Enzymes Bacterial enzymes that cut DNA molecules only at restriction sites Restriction site usually sequences palindromes Categorized into two groups based on type of cut: Cuts with sticky ends Cuts with blunt ends

Figure 8.2 Actions of representative restriction enzymes.
Restriction site (palindrome) 5′ 3′ 5′ 3′ 5′ 3′ Restriction enzyme Restriction enzyme 1 Restriction enzyme 2 5′ 3′ 5′ 3′ Blunt ends Sticky ends Production of blunt ends Production of sticky ends Ligase 5′ 3′ 5′ 3′ Recombinant DNA molecules Restriction fragments from two different organisms cut by the same restriction enzyme Recombinants using blunt ends Ligase 5′ 3′ 5′ 3′ Recombinant DNA molecules Recombinants using sticky ends

Recombinant DNA Technology
PLAY Recombinant DNA Technology

Vectors Nucleic acid molecules that deliver a gene into a cell Useful properties: Small enough to manipulate in a lab Survive inside cells Contain recognizable genetic marker Ensure genetic expression of gene Include viral genomes, transposons, and plasmids

Figure 8.3 An example of the process for producing a recombinant vector.
mRNA for human growth hormone (HGH) Antibiotic- resistance gene Restriction site Reverse transcription cDNA for HGH Plasmid (vector) Restriction enzyme Restriction enzyme Sticky ends Gene for human growth hormone Ligase Recombinant plasmid Introduce recombinant plasmid into bacteria. Bacterial chromosome Recombinant plasmid Inoculate bacteria on media containing antibiotic. Bacteria containing the plasmid with HGH gene survive because they also have resistance gene.

Gene Libraries A collection of bacterial or phage clones Each clone in library often contains one gene of an organism's genome Library may contain all genes of a single chromosome Library may contain set of cDNA complementary to mRNA

Figure 8.4 Production of a gene library.
Genome Isolate genome of organism. Generate fragments using restriction enzymes. 1 2 3 4 5 6 7 8 9 10 11 Insert each fragment into a vector. 1 2 3 4 5 6 7 8 9 10 11 Introduce vectors into cells. 1 2 3 4 5 6 7 8 9 10 11 Culture recombinant cells; descendants are clones. 1 2 3 4 5 6 7 8 9 10 11

Techniques of Recombinant DNA Technology
Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR) Large number of identical molecules of DNA produced in vitro Critical to amplify DNA in variety of situations Epidemiologists used PCR to determine that two separate Ebola outbreaks occurred in Africa in 2014

Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR) Repetitive process consisting of three steps: Denaturation Priming Extension Can be automated using a thermocycler

PCR: Overview PLAY PCR: Overview

PCR: Components PLAY PCR: Components

Figure 8.5a Use of the polymerase chain reaction (PCR) to replicate DNA.
Original DNA molecule 3′ 3′ 5′ 5′ Heat to 94°C Denaturation DNA primer Deoxyribonucleotide triphosphates Priming DNA polymerase Repeat Cool to 65°C DNA polymerase 3′ 5′ 5′ Extension DNA primer 5′ 5′ 3′ 72°C

First cycle Second cycle Third cycle Fourth cycle 2 DNA molecules
Figure 8.5b Use of the polymerase chain reaction (PCR) to replicate DNA. First cycle Second cycle Third cycle Fourth cycle 2 DNA molecules 4 DNA molecules 8 DNA molecules 16 DNA molecules

PCR: The Process PLAY PCR: The Process

Selecting a Clone of Recombinant Cells Must find clone containing DNA of interest Probes are used

Separating DNA Molecules: Gel Electrophoresis and the Southern Blot Gel electrophoresis: Separates molecules based on electrical charge, size, and shape Allows scientists to isolate DNA of interest Negatively charged DNA drawn toward positive electrode Agarose makes up gel; acts as molecular sieve Smaller fragments migrate faster and farther than larger ones Determine size by comparing distance migrated to standards

Figure 8.6 Gel electrophoresis.
Wells (–) Electrophoresis chamber filled with buffer solution E D C (50) Agarose gel B (40) (+) A (35) (15) (10) a (5) Movement of DNA DNA b Lane of DNA fragments of known sizes (kilobase pairs) Wire

Separating DNA Molecules: Gel Electrophoresis and the Southern Blot Southern blot DNA transferred from gel to nitrocellulose membrane Probes used to localize DNA sequence of interest Northern blot — similar technique used to detect RNA

DNA Microarrays Consist of molecules of immobilized single-stranded DNA Fluorescently labeled DNA washed over array will adhere only at locations where there are complementary DNA sequences Variety of scientific uses of DNA microarrays: Monitoring gene expression Diagnosis of infection Identification of organisms in an environmental sample

Figure 8.7 DNA microarray. cDNA sequences from reverse
transcription, a microbe, or a gene library Multiple copies of each single-stranded sequence are attached to substrate in precisely defined locations. DNA microarray Fluorescently labeled DNA

Inserting DNA into Cells Goal of DNA technology is insertion of DNA into cell Natural methods: Transformation Transduction Conjugation Artificial methods: Electroporation Protoplast fusion Injection — gene gun and microinjection

Figure 8.8a-b Artificial methods of inserting DNA into cells.
Pores in wall and membrane Chromosome Cell synthesizes new wall Electrical ﬁeld applied Competent cell Recombinant cell DNA from another source Electroporation Cell walls Cell synthesizes new wall Enzymes remove cell walls Polyethylene glycol Recombinant cell New cell Protoplasts Fused protoplasts Protoplast fusion

Figure 8.8c-d Artificial methods of inserting DNA into cells.
Suction tube to hold target cell in place Micropipette containing DNA Target cell's nucleus Target cell Blank .22- caliber shell Nylon projectile Vent Plate to stop nylon projectile DNA-coated beads Target cell Nylon projectile Gene gun Microinjection

Applications of Recombinant DNA Technology
Genetic Mapping Locating genes on a nucleic acid molecule Provides useful facts concerning metabolism, growth characteristics, and relatedness to others

Genetic Mapping Locating genes Until 1970, genes identified by labor-intensive methods Simpler and universal methods now available Restriction fragmentation Fluorescent in situ hybridization (FISH)

Figure 8.9 Fluorescent in situ hybridization (FISH).

Genetic Mapping Genomics and Nucleotide Sequencing Genomics sequencing and analysis of the nucleotide bases of genomes Elucidation of the genomes of pathogens is a priority Used to relate DNA sequence to protein function Now use next-generation sequencing

Figure 8.10 Next-generation DNA sequencing.
Attach identical ssDNA fragments at known locations on a slide. Template strand Pass all four types of modified nucleotides over the slide. Each type of nucleotide is modified to fluoresce a color and has a molecular group that causes synthesis to cease. Multiple copies of a different template strand Multiple copies of ssDNA DNA polymerase ssDNA Fluorescent dye and chemical group that stops DNA synthesis. The laser stimulates the dyes and another image is captured. Laser shines across the slide, causing each fluorescent dye to flash its color. The sequencer then removes the fluorescent dye and the stop group. Synthesis restarts, but stops as soon as another modified nucleotide is added. The process repeats until a complementary DNA strand has been synthesized for each ssDNA on the slide. A light detector records the event. The sequence of colors recorded by the light detector at each location corresponds to the sequence of nucleotides in the ssDNA molecules at that site.

Genetic Mapping Functional Genomics Use of genomics to determine what genes' products do Common methods include the use of gene knockouts or the overexpression of a gene of interest

Table 8.2 Tools and Techniques of Recombinant DNA Technology

Microbial Communities Studies Most microorganisms have never been grown in a laboratory Scientists know them only by their DNA fingerprints Allowed identification of over 500 species of bacteria from human mouths Next-generation sequencing allows for the determination of all of the members of a microbiome

Pharmaceutical and Therapeutic Applications Protein synthesis Creation of synthetic proteins by bacteria and yeast cells Vaccines Production of safer vaccines Subunit vaccines Introduce genes of pathogens into common fruits and vegetables Injecting humans with plasmid-carrying gene from pathogen Humans synthesize pathogen's proteins

Pharmaceutical and Therapeutic Applications Genetic screening DNA microarrays used to screen individuals for inherited disease caused by mutations Can also identify viral DNA in blood or tissues Gene therapy Missing or defective genes replaced with normal copies Difficult to get a functioning gene into enough cells to affect the disease

Pharmaceutical and Therapeutic Applications Medical diagnosis Patient specimens can be examined for presence of gene sequences unique to certain pathogens Xenotransplants Animal cells, tissues, or organs introduced into human body Biomedical Animal Models Animals are used in biomedical research to study diseases and develop new diagnostic and therapeutic procedures

Agricultural Applications Production of transgenic organisms Recombinant plants and animals altered by addition of genes from other organisms Also called genetically modified organisms (GMOs)

Agricultural Applications Herbicide tolerance Gene from Agrobacterium tumefaciens conveys resistance to glyphosate (Roundup) Farmers can kill weeds without killing crops Salt tolerance Scientists have inserted a gene for salt tolerance into tomato and canola plants Transgenic plants survive, produce fruit, and remove salt from soil

Agricultural Applications Freeze resistance Crops sprayed with genetically modified bacteria can tolerate mild freezes Pest resistance Bacillus thuringiensis (Bt) toxin Naturally occurring toxin only harmful to insects Organic farmers use to reduce insect damage to crops Gene for Bt toxin inserted into various crop plants Genes for Phytophthora resistance inserted into potato crops

Figure 8.11 Genetically modified papaya plants.

Agricultural Applications Improvements in nutritional value and yield Enzyme that breaks down pectin suppressed in some tomatoes Allows tomatoes to ripen on vine and increases shelf life Bovine growth hormone (BGH) allows cattle to gain weight more rapidly Have meat with lower fat content and produce 10% more milk Gene for β-carotene (vitamin A precursor) inserted into rice Scientists considering transplanting genes coding for entire metabolic pathways

The Ethics and Safety of Recombinant DNA Technology
Long-term effects of transgenic manipulations are unknown Unforeseen problems arise from every new technology and procedure Natural genetic transfer could deliver genes from transgenic plants and animals into other organisms Transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic

Studies have not shown any risks to human health or environment Standards imposed on labs involved in recombinant DNA technology Can create biological weapons using same technology

Ethical issues Routine screenings? Who should pay? Genetic privacy rights? Profits from genetically altered organisms? Required genetic screening? Forced correction of "genetic abnormalities"?

Micro Matters In the Micro Matters video in Chapter 8, Jose and his classmates examine the origin of a salmonellosis outbreak. Pathogenic salmonella strains contain virulence plasmids and pathogenicity islands. DNA is transmitted between donor and recipient bacteria via horizontal gene transfer. DNA fingerprinting is used to compare the genomes of different strains of bacteria. Gel electrophoresis is a tool used to examine DNA.

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