16. Recombinant DNA and Biotechnology

Recombinant DNA and Biotechnology16 Recombinant DNA and Biotechnology 16.1 How Are Large DNA Molecules Analyzed?16.2 What Is Recombinant DNA?16.3 How Are New Genes Inserted into Cells?16.4 What Are the Sources of DNA Used in Cloning?16.5 What Other Tools Are Used to Manipulate DNA?16.6 What Is Biotechnology?16.1 How Are Large DNA Molecules Analyzed?Naturally occurring enzymes that cleave and repair DNA are used in the laboratory to manipulate and recombine DNA.16.1 How Are Large DNA Molecules Analyzed?Restriction enzymes (restriction endonucleases) cut double-stranded DNA into smaller pieces.Bacteria use these as defense against DNA from bacteriophage.DNA is cut between the 3′ hydroxyl group of one nucleotide and the 5′ phosphate group of the next—restriction digestion.Figure 16.1 Bacteria Fight Invading Viruses with Restriction Enzymes16.1 How Are Large DNA Molecules Analyzed?There are many restriction enzymes that cut DNA at specific base sequences—the recognition sequence, or restriction site.16.1 How Are Large DNA Molecules Analyzed?Restriction enzymes do not cut bacteria’s own DNA because the recognition sequences are modified.Methylases add methyl groups after replication; makes sequence unrecognizable by restriction enzyme.16.1 How Are Large DNA Molecules Analyzed?Bacterial restriction enzymes can be isolated from cells.DNA from any organism will be cut wherever the recognition site occurs.EcoRI (from E. coli) cuts DNA at this sequence:16.1 How Are Large DNA Molecules Analyzed?The sequence is palindromic—it reads the same in both directions from the 5′ end.EcoRI occurs about once every four genes in prokaryotes. DNA can be chopped into small pieces containing a few genes.16.1 How Are Large DNA Molecules Analyzed?The EcoRI sequence does not occur anywhere in the genome of the phage T7. Thus it can survive in its host, E. coli.16.1 How Are Large DNA Molecules Analyzed?After DNA is cut, fragments of different sizes can be separated by gel electrophoresis.Mixture of fragments is place on a well in a porous gel. An electric field is applied across the gel. Negatively charged DNA fragments move towards positive end.Smaller fragments move faster than larger ones.Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 2)Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 3)16.1 How Are Large DNA Molecules Analyzed?Electrophoresis provides information on:Size of fragments. Fragments of known size provide comparison.Presence of specific sequences. These can be determined using probes. DNA is denatured while in the gel, then transferred to a nylon filter to make a “blot.”Figure 16.3 Analyzing DNA Fragments by Southern Blotting16.1 How Are Large DNA Molecules Analyzed?DNA fingerprinting uses restriction analysis and electrophoresis to identify individuals.Works best with genes that are polymorphic—have multiple alleles.16.1 How Are Large DNA Molecules Analyzed?Two types of polymorphisms:Single nucleotide polymorphisms (SNPs): inherited variation involving a single baseShort tandem repeats (STRs): moderately repetitive sequences side by side16.1 How Are Large DNA Molecules Analyzed?STRs are recognizable if they lie between two restriction sites.Several different STRs can be used to determine the unique pattern for an individual.Figure 16.4 DNA Fingerprinting with Short Tandem Repeats16.1 How Are Large DNA Molecules Analyzed?DNA fingerprinting requires at least 1 μg of DNA (amount in about 100,000 human cells).This is not always available, so amplification by PCR is used.16.1 How Are Large DNA Molecules Analyzed?DNA fingerprinting is used in forensics.It is more often used to prove innocence than guilt.Only a small portion of the genome is examined; there is the possibility that two people could have the same sequence.16.1 How Are Large DNA Molecules Analyzed?DNA fingerprinting has been used to analyze historical events.The skeletal remains of Russian Tsar Nicholas II and his family were identified from DNA in bone fragments.DNA also showed relationships with living descendents of the Tsar.Figure 16.5 DNA Fingerprinting the Russian Royal Family16.1 How Are Large DNA Molecules Analyzed?DNA technology can be used to identify species.A proposal to identify all known species and look for unknowns has been put forth by the Consortium for the Barcode of Life (CBOL):Use a short sequence from a gene (cytochrome oxidase) as a “barcode” for each species.Figure 16.6 A DNA Barcode16.1 How Are Large DNA Molecules Analyzed?The barcode project could contribute to:Evolution researchSpecies diversity issuesIdentification of new speciesIdentification of undesirable microbes or bioterrorism agents16.2 What Is Recombinant DNA?DNA fragments can be rejoined by DNA ligase.Any two DNA sequences can be spliced.First done in 1973 with two E. coli plasmids; Recombinant DNA was bornFigure 16.7 Making Recombinant DNA (Part 1)Figure 16.7 Making Recombinant DNA (Part 2)16.2 What Is Recombinant DNA?Some restriction enzymes cut both DNA strands exactly opposite each other.Others (such as EcoRI) make a staggered cut. Results in single-stranded “tails” at the ends of fragments.Tails are called sticky ends—can bind by base pairing to other sticky ends.Figure 16.8 Cutting and Splicing DNA16.2 What Is Recombinant DNA?Sticky ends of fragments that were cut by the same restriction enzyme are all the same—thus fragments from different species can be joined.When temperature is lowered, the fragments anneal—join by hydrogen bonding. Must be permanently spliced by DNA ligase.16.3 How Are New Genes Inserted into Cells?Recombinant DNA technology can be used to clone, or make exact copies of genes.The gene can be used to make a protein—but it must first be inserted, or transfected, into host cells.The altered host cell is called transgenic.16.3 How Are New Genes Inserted into Cells?To determine which of the host cells contain the new sequence, the recombinant DNA is often tagged with reporter genes.Reporter genes have easily observed phenotypes or genetic markers.16.3 How Are New Genes Inserted into Cells?The first host cells used were bacteria, especially E. coli.Yeasts (Saccharomyces) are commonly used as eukaryotic hosts. Plant cells are also used—they have totipotency, the ability of any differentiated cell to develop into a new plant.16.3 How Are New Genes Inserted into Cells?The new DNA must also replicate as the host cell divides. It must become a segment with an origin of replication—a replicon or replication unit.16.3 How Are New Genes Inserted into Cells?New DNA can become part of a replicon in two ways:Inserted near an origin of replication in host chromosome.It can be part of a carrier sequence or vector that already has an origin of replication.16.3 How Are New Genes Inserted into Cells?A vector should have four characteristics:Ability to replicate independently of the host cellA recognition sequence for a restriction enzymeA reporter geneSmall size in comparison with host’s chromosomes16.3 How Are New Genes Inserted into Cells?Plasmids have all these characteristics.Plasmids are small, many have only one restriction site.Genes for antibiotic resistance can be used as reporter genes.And they have an origin of replication and can replicate independently.Figure 16.9 Vectors for Carrying DNA into Cells (A)16.3 How Are New Genes Inserted into Cells?Plasmids can be used for genes of 10,000 bp or less. Most eukaryote genes are larger than this.Viruses can be used as vectors—e.g., bacteriophage. The genes that cause host cell to lyse can be cut out and replaced with other DNA.16.3 How Are New Genes Inserted into Cells?Bacterial plasmids don’t work for yeasts because the origins of replication use different sequences.A yeast artificial chromosome (YAC) has been created: contains yeast origin of replication, plus yeast centromere and telomere sequences.Also contains artificial restriction sites and reporter genesFigure 16.9 Vectors for Carrying DNA into Cells (B)16.3 How Are New Genes Inserted into Cells?A plasmid from the soil bacterium Agrobacterium tumefaciens is used as a vector for plant cells.Plasmid Ti (tumor inducing) causes crown gall. Plasmid has a region called T DNA, which inserts copies of itself into chromosomes of infected plants.16.3 How Are New Genes Inserted into Cells?T DNA has several restriction sites, where new DNA can be inserted. With altered T DNA, plasmid no longer causes tumors, but can still insert itself into host chromosomes.Figure 16.9 Vectors for Carrying DNA into Cells (C)16.3 How Are New Genes Inserted into Cells?Usually only a small proportion of host cells take up the vector, and they may not have the appropriate sequence.Host cells with the desired sequence must be identifiable.16.3 How Are New Genes Inserted into Cells?One method:E. coli is host; pBR322 plasmid is the vector.Plasmid has genes for resistance to ampicillin and tetracycline.Plasmid has only one restriction site for enzyme BamHI, within the gene for tetracycline resistance.16.3 How Are New Genes Inserted into Cells?If new DNA is inserted at that restriction site, it inactivates the gene for tetracycline resistance.Plasmid then has gene for ampicillin resistance, but not for tetracycline. This can be used to select for host cells with new DNA.Figure 16.10 Marking Recombinant DNA by Inactivating a Gene16.3 How Are New Genes Inserted into Cells?Other reporter genes:Artificial vectors with restriction sites within the lac operon. If new DNA is inserted there, vector no longer carries its original function into the host cell.Green fluorescent protein, which normally occurs in the jellyfish Aequopora victoriana.16.4 What Are the Sources of DNA Used in Cloning?DNA fragments used for cloning come from three sources:Gene librariesReverse transcription from mRNAArtificial synthesis or mutation of DNA16.4 What Are the Sources of DNA Used in Cloning?Human chromosomes contain an average of 80 million bp each.The DNA is cut into fragments by restriction enzymes, the fragments are “stored” as a gene library.Each fragment is inserted into a vector, which goes into a host cell.Figure 16.11 Constructing a Gene Library16.4 What Are the Sources of DNA Used in Cloning?If phage λ is used as a vector, about 50,000 volumes are required to store the library.One petri plate can hold 80,000 phage colonies, or plaques.DNA in the plaques is screened using specific probes.16.4 What Are the Sources of DNA Used in Cloning?Smaller DNA libraries can be made from complementary DNA (cDNA).mRNA is extracted from a tissue and the poly A tails allowed to hybridize with oligo dT—a string of thymine bases.Oligo dT serves as a primer for reverse transcriptase to synthesize a complementary DNA strand.Figure 16.12 Synthesizing Complementary DNA16.4 What Are the Sources of DNA Used in Cloning?cDNA libraries are made from particular tissues at particular times and represent a snapshot of the mRNA present at that time.Used to compare gene expression in different tissues at different stages of development.cDNA is also used to clone eukaryotic genes.16.4 What Are the Sources of DNA Used in Cloning?DNA can be synthesized if the amino acid sequence of a protein is known.This process is now automated, and labs can make custom DNA sequences overnight.Flanking sequences for transcription initiation, termination, and regulation and start and stop codons are also added.16.4 What Are the Sources of DNA Used in Cloning?Synthetic DNA can be used to create specific mutations in order to study the consequences of the mutation.Called mutagenesis techniques.These techniques have revealed many cause-and-effect relationships, e.g., determining signal sequences.16.5 What Other Tools Are Used to Manipulate DNA?Three additional ways of manipulating DNA:Knockout experimentsGene silencingDNA chips16.5 What Other Tools Are Used to Manipulate DNA?A knockout experiment involves homologous replication to replace a gene with an inactive gene, and determine results in a living organism.The normal allele of a gene is inserted into a plasmid; restriction enzymes are used to insert a reporter gene in the middle of the normal gene.16.5 What Other Tools Are Used to Manipulate DNA?The gene is thus inactivated.The plasmid is then transfected into a stem cell of a mouse embryo.Stem cell: undifferentiated cell that divides and differentiates to form different tissues.16.5 What Other Tools Are Used to Manipulate DNA?Much of the normal gene is still present, so homologous recognition takes place between the normal allele and the inactive allele on the plasmid.Recombination can occur, and inactive allele is swapped for the normal allele.The transfected stem cell is then inserted into an early mouse embryo.Figure 16.13 Making a Knockout Mouse (Part 1)Figure 16.13 Making a Knockout Mouse (Part 2)16.5 What Other Tools Are Used to Manipulate DNA?Translation of mRNA can be blocked by complementary micro RNAs—antisense RNA.Antisense RNA can be synthesized, and added to cells to prevent translation—the effects of the missing protein can then be determined.16.5 What Other Tools Are Used to Manipulate DNA?Interference RNA (RNAi) is a rare natural mechanism that blocks translation. Short, double stranded RNA is unwound and binds to complementary mRNA by a protein complex, which also catalyzes the breakdown of the mRNA.Small interfering RNA (siRNA) can be synthesized in the laboratory. Figure 16.14 Using Antisense RNA and RNAi to Block Translation of mRNA16.5 What Other Tools Are Used to Manipulate DNA?Antisense RNA and RNAi are also used to study cause-and-effect relationships.Example: Antisense RNA is used to block translation of proteins essential for growth of cancer cells—the cells revert to normal phenotype.16.5 What Other Tools Are Used to Manipulate DNA?DNA chip technology provides a large array of sequences for hybridization experiments.A series of DNA sequences are attached to a glass slide in a precise order.The slide has microscopic wells which each contain thousands of copies of sequences up to 20 nucleotides long.Figure 16.15 DNA on a Chip16.5 What Other Tools Are Used to Manipulate DNA?To analyze mRNA, it is incubated with reverse transcriptase to make cDNA.The cDNA is amplified using PCR.Technique is called RT-PCR.Amplified cDNA is tagged with a fluorescent dye and used as a probe of the DNA on the chip.16.5 What Other Tools Are Used to Manipulate DNA?DNA chip technology has been developed to identify gene expression patterns in women with a propensity for breast cancer tumors to recur—a gene expression signature.16.6 What Is Biotechnology?Biotechnology is the use of living cells to produce materials useful to people.Examples: use of yeasts to brew beer and wine, use of bacteria to produce cheese, yogurt, etc.Use of microbes to produce antibiotics such as penicillin, alcohol, and other products.16.6 What Is Biotechnology?Gene cloning is now used to produce proteins in large amounts.Almost any gene can be inserted into bacteria or yeasts, and the resulting cells induced to make large quantities of the product.Requires specialized vectors.16.6 What Is Biotechnology?Expression vectors are synthesized that include sequences needed for expression of the transgene in the host cell.Figure 16.16 An Expression Vector Allows a Transgene to Be Expressed in a Host Cell16.6 What Is Biotechnology?Expression vectors can be modified by:Inducible promoters; enhancers can also be added so that protein synthesis takes place at high rates.Tissue-specific promotersSignal sequences—e.g., a signal to secrete the product to the extracellular medium.Table 16.116.6 What Is Biotechnology?Example of a medical application:After wounds heal, blood clots are dissolved by plasmin. Plasmin is stored as an inactive form called plasminogen.Conversion of plasminogen is activated by tissue plasminogen activator (TPA).TPA can be used to treat strokes and heart attacks, but large quantities are needed—can be made using recombinant DNA technology.Figure 16.17 Tissue Plasminogen Activator: From Protein to Gene to Drug (Part 1)Figure 16.17 Tissue Plasminogen Activator: From Protein to Gene to Drug (Part 2)16.6 What Is Biotechnology?Pharming: production of medically useful proteins in milk.Transgenes for a protein are inserted into the egg of a domestic animal, next to the promoter for lactoglobulin—a protein in milk. The transgenic animal then produces large quantities of the protein in its milk.Figure 16.18 Pharming16.6 What Is Biotechnology?Through cultivation and selective breeding, humans have been altering the traits of plants and animals for thousands of years.Recombinant DNA technology has several advantages:Specific genes can be targeted.Any gene can be introduced into any other organism.New organisms are generated quickly.Table 16.216.6 What Is Biotechnology?Crop plants have been modified to produce their own insecticides:The bacterium Bacillus thuringiensis produces a protein that kills insect larvae.Dried preparation of B. thuringiensis are sold as a safe alternative to synthetic insecticides. The toxin is easily biodegradable.16.6 What Is Biotechnology?Genes for the toxin have been isolated, cloned, and modified, and inserted into plant cells using the Ti plasmid vector.Transgenic corn, cotton, soybeans, tomatoes, and other crops are being grown. Pesticide use is reduced.16.6 What Is Biotechnology?Some transgenic crops are resistant to herbicides.Glyphosate (Roundup) is widely used to kill weeds.Expression vectors have been used to make plants that synthesize so much of the target enzyme of glyphosate that they are unaffected by the herbicide.16.6 What Is Biotechnology?The gene has been inserted into corn, soybeans, and cotton.About half of U.S. crops of these plants contain this gene.16.6 What Is Biotechnology?Crops with improved nutritional characteristics:Rice does not have β-carotene, but does have a precursor molecule.Genes for enzymes that synthesize β-carotene from the precursor are taken from daffodils and inserted into rice by the Ti plasmid.16.6 What Is Biotechnology?The transgenic rice is yellow, and can supply β-carotene to improve the diets of many people.β-carotene is converted to vitamin A in the body.Figure 16.19 Transgenic Rice Is Rich in β-Carotene16.6 What Is Biotechnology?Recombinant DNA is also used to adapt a crop plant to an environment.Example: plants that are salt-tolerantGenes from a protein that moves sodium ions into the central vacuole were isolated from Arabidopsis and inserted into tomato plants.Figure 16.20 Salt-Tolerant Tomato Plants16.6 What Is Biotechnology?Concerns over biotechnology:Genetic manipulation is an unnatural interference in nature.Genetically altered foods are unsafe to eat.Genetically altered crop plants are dangerous to the environment.16.6 What Is Biotechnology?Advocates of biotechnology point out that all crop plants have been manipulated by humans.Advocates say that since only single genes for plant function are inserted into crop plants, they are still safe for human consumption.Genes that affect human nutrition may raise more concerns.16.6 What Is Biotechnology?Concern over environmental effects centers on escape of transgenes into wild populations:For example, if the gene for herbicide resistance made its way into the weed plants.Beneficial insects can also be killed from eating plants with B. thuringiensis genes.