Biotechnolog y and DNA Technology

Transcription

1 PowerPoint Lecture Presentations prepared by Bradley W. Christian, McLennan Community College C H A P T E R 9 Biotechnolog y and DNA Technology

2 Introduction to Biotechnology Biotechnology: the use of microorganisms, cells, or cell components to make a product Foods, antibiotics, vitamins, enzymes Recombinant DNA (rdna) technology: the insertion or modification of genes to produce desired proteins

3 An Overview of Recombinant DNA Procedures Vector: self-replicating DNA molecule used to transport foreign DNA into a cell Clone: population of genetically identical cells arising from one cell; each carries the vector

4 Figure 9.1 A Typical Genetic Modification Procedure.

5 Tools of Biotechnology Selection: selecting for a naturally occurring microbe that produces a desired product Mutation: Mutagens cause mutations that might result in a microbe with a desirable trait Site-directed mutagenesis: a targeted and specific change in a gene

6 Restriction Enzymes Cut specific sequences of DNA Destroy bacteriophage DNA in bacterial cells Methylated cytosines in bacteria protect their own DNA from digestion Create blunt ends or staggered cuts known as sticky ends

7 Recombinant DNA Technology PLAY Animation: Recombinant DNA Technology

8 Table 9.1 Selected Restriction Enzymes Used in rdna Technology

9 Figure 9.2 A restriction enzyme's role in making rdna. Recognition sites A restriction enzyme cuts (red arrows) double-stranded DNA at its particular recognition sites, shown in blue. DNA Cut Cut Cut Cut These cuts produce a DNA fragment with two sticky ends. DNA from another source, perhaps a plasmid, cut with the same restriction enzyme Sticky end When two such fragments of DNA cut by the same restriction enzyme come together, they can join by base pairing. The joined fragments will usually form either a linear molecule or a circular one, as shown here for a plasmid. Other combinations of fragments can also occur. The enzyme DNA ligase is used to unite the backbones of the two DNA fragments, producing a molecule of rdna. rdna

10 Vectors Carry new DNA to desired cells Must be able to self-replicate Plasmids and viruses can be used as vectors Shuttle vectors exist in several different species and can move cloned sequences among various organisms

11 Figure 9.3 A plasmid used for cloning. amp R puc19 lacz HindIII BamHI EcoRI ori

12 Polymerase Chain Reaction Process of increasing small quantities (amplifying) of DNA for analysis Used for diagnostic tests for genetic diseases and detecting pathogens Reverse-transcription PCR uses mrna as template

13 PCR: Overview PLAY Animation: PCR: Overview

14 PCR: Components PLAY Animation: PCR: Components

15 Figure 9.4 The polymerase chain reaction.

16 Inserting Foreign DNA into Cells DNA can be inserted into a cell by: Transformation: Cells take up DNA from the surrounding environment Electroporation: Electrical current forms pores in cell membranes Protoplast fusion: Removing cell walls from two bacteria allows them to fuse

17 Figure 9.5 Protoplast fusion. Chromosome Plasma membrane Cell wall Bacterial cells Bacterial cell walls are enzymatically digested, producing protoplasts. Protoplasts In solution, protoplasts are treated with polyethylene glycol. Protoplasts fuse. Segments of the two chromosomes recombine. Recombinant cell Recombinant cell grows new cell wall.

18 Inserting Foreign DNA into Cells DNA can be inserted into a cell by: Gene gun Microinjection

19 Figure 9.7 The microinjection of foreign DNA into an egg.

20 Genomic Libraries Collections of clones containing different DNA fragments An organism's DNA is digested and spliced into plasmid or phage vectors and introduced into bacteria At least one clone exists for every gene in the organism

21 Figure 9.8 Genomic libraries. Genome to be stored in library is cut up with restriction enzyme Host cell Recombinant plasmid OR Recombinant phage DNA Phage cloning vector Plasmid Library Phage Library

22 Genomic Libraries Complementary DNA (cdna) is made from mrna by reverse transcriptase Used for obtaining eukaryotic genes because eukaryotic DNA has introns that do not code for protein mrna has the introns removed, coding only for the protein product

23 Figure 9.9 Making complementary DNA (cdna) for a eukaryotic gene. Exon Intron Exon Intron Exon Nucleus DNA RNA transcript A gene composed of exons and introns is transcribed to RNA by RNA polymerase. Processing enzymes in the nucleus remove the intron-derived RNA and splice together the exon-derived RNA into mrna. mrna mrna is isolated from the cell, and reverse transcriptase is added. Cytoplasm DNA strand being synthesized First strand of DNA is synthesized. The mrna is digested by reverse transcriptase. cdna of gene without introns DNA polymerase is added to synthesize second strand of DNA. Test tube

24 Synthetic DNA Builds genes using a DNA synthesis machine

25 Selecting a Clone Blue-white screening Uses plasmid vector containing ampicillin resistance gene (amp R ) and β-galactosidase gene (lacz) Bacteria is grown in media containing ampicillin and X-gal, a substrate for β-galactosidase

26 Figure 9.11 Blue-white screening, one method of selecting recombinant bacteria. β-galactosidase gene (lacz) Ampicillin-resistance gene (amp R ) Plasmid DNA and foreign DNA are both cut with the same restriction enzyme. The plasmid has the genes for lactose hydrolysis (the lacz gene encodes the enzyme β-galactosidase) and ampicillin resistance. Plasmid Foreign DNA Restriction sites Restriction site Foreign DNA will insert into the lacz gene. The bacterium receiving the plasmid vector will not produce the enzyme β-galactosidase if foreign DNA has been inserted into the plasmid. Recombinant plasmid The recombinant plasmid is introduced into a bacterium, which becomes ampicillin resistant. Bacterium All treated bacteria are spread on a nutrient agar plate containing ampicillin and a β-galactosidase substrate and incubated. The β-galactosidase substrate is called X-gal. Only bacteria that picked up the plasmid will grow in the presence of ampicillin. Bacteria that hydrolyze X-gal produce galactose and an indigo compound. The indigo turns the colonies blue. Bacteria that cannot hydrolyze X-gal produce white colonies. Colonies with foreign DNA

27 Selecting a Clone Colony hybridization Use DNA probes: short segments of single-stranded DNA complementary to the desired gene

28 Figure 9.12 Colony hybridization: using a DNA probe to identify a cloned gene of interest. Master plate with colonies of bacteria containing cloned segments of foreign genes Colonies containing genes of interest Replica plate Compare filter with replica of master plate to identify colonies containing gene of interest. Nitrocellulose filter Make replica of master plate on nitrocellulose filter. Wash filter to remove unbound probe. Strands of bacterial DNA Treat filter with detergent (SDS) to lyse bacteria. Treat filter with sodium hydroxide (NaOH) to separate DNA into single strands. Bound DNA probe Fluorescence labeled probes Gene of interest Singlestranded DNA Probe will hybridize with desired gene from bacterial cells. Add labeled probes.

29 Making a Gene Product E. coli Advantages: easily grown and its genomics are known Disadvantages: produces endotoxins and does not secrete its protein products

30 Making a Gene Product Saccharomyces cerevisiae Easily grown and has a larger genome than bacteria Expresses eukaryotic genes easily Plant cells and whole plants Express eukaryotic genes easily Plants are easily grown, large-scale, and low-cost Mammalian cells Express eukaryotic genes easily Can make products for medical use Harder to grow

31 Therapeutic Applications Human enzymes and other proteins such as insulin Subunit vaccines: made from pathogen proteins in genetically modified yeasts Nonpathogenic viruses carrying genes for pathogen's antigens as DNA vaccines Gene therapy to replace defective or missing genes

32 Table 9.2 Some Pharmaceutical Products of rdna (1 of 2)

33 Table 9.2 Some Pharmaceutical Products of rdna (2 of 2)

34 Therapeutic Applications Gene silencing Small interfering RNAs (sirnas) bind to mrna, which is then destroyed by RNA-induced silencing complex (RISC) RNA interference (RNAi) inserts DNA encoding sirna into a plasmid and transferred into a cell

35 Figure 9.14 Gene silencing could provide treatments for a wide range of diseases.

36 Genome Projects Shotgun sequencing sequences small pieces of genomes which are assembled by a computer Metagenomics is the study of genetic material directly from environmental samples The Human Genome Project sequenced the entire human genome The Human Proteome Project will map proteins expressed in human cells

37 Figure 9.15 Shotgun sequencing. Isolate DNA. Sequence DNA fragments. Assemble sequences. Fragment DNA with restriction enzymes. Clone DNA in a bacterial artificial chromosome (BAC). Edit sequences; fill in gaps. Construct a gene library Random sequencing Closure phase

38 Scientific Applications Bioinformatics: understanding gene function via computer-assisted analysis Proteomics: determining proteins expressed in a cell Reverse genetics: discovering gene function from a genetic sequence

39 Scientific Applications Southern blotting: DNA probes detect specific DNA in fragments (RFLPs) separated by gel electrophoresis

40 Figure 9.16 Southern blotting. Restriction enzyme Gene of interest DNA containing the gene of interest is extracted from human cells and cut into fragments by restriction enzymes. Fragments are called restriction fragment length polymorphisms, or RFLPs (pronounced "rif-lips"). Gel Human Larger DNA fragments Smaller The fragments are separated according to size by gel electrophoresis. Each band contains many copies of a particular DNA fragment. The bands are invisible but can be made visible by staining. Paper towels Salt solution Sponge Nitrocellulose filter Gel Nitrocellulose filter The DNA bands are transferred to a nitrocellulose filter by blotting. The solution passes through the gel and filter to the paper towels by capillary action. Gel DNA transferred to filter This produces a nitrocellulose filter with DNA fragments positioned exactly as on the gel. Labeled probes Sealable plastic bag The filter is exposed to a labeled probe for a specific gene. The probe will base-pair (hybridize) with a short sequence present on the gene. The fragment containing the gene of interest is identified by a band on the filter.

41 Forensic Microbiology DNA fingerprinting is used to identify pathogens PCR microarrays and DNA chips can screen samples for multiple pathogens Differs from medicine because it requires: Proper collection of evidence Establishing a chain of custody

42 Figure 9.17 DNA fingerprints used to track an infectious disease. E. coli isolates from patients whose infections were not juice related E. coli isolates from patients who drank contaminated juice Apple juice isolates

43 Nanotechnology Bacteria can make molecule-sized particles Nanospheres used in drug targeting and delivery

44 Agricultural Applications Ti plasmid: occurs in Agrobacterium tumefaciens Integrates into the plant genome and causes a tumorlike growth Can be used to introduce rdna into a plant

45 Figure 9.19 Crown gall disease on a rose plant. Crown gall

46 Figure 9.20 Using the Ti plasmid as a vector for genetic modification in plants. Agrobacterium tumefaciens bacterium Inserted T-DNA carrying foreign gene Restriction cleavage site T-DNA Ti plasmid The plasmid is removed from the bacterium, and the T-DNA is cut by a restriction enzyme. The plasmid is reinserted into a bacterium. Recombinant Ti plasmid The bacterium is used to insert the T-DNA carrying the foreign gene into the chromosome of a plant cell. The plant cells are grown in culture. Foreign DNA is cut by the same enzyme. The foreign DNA is inserted into the T-DNA of the plasmid. A plant is generated from a cell clone. All of its cells carry the foreign gene and may express it as a new trait.

47 Agricultural Applications Bt toxin Herbicide resistance Suppression of genes Antisense DNA Nutrition Human proteins

48 Table 9.3 Some Agriculturally Important Products of rdna Technology

49 Safety Issues and Ethics of Using DNA Technology Need to avoid accidental release into the environment Genetically modified crops must be safe for consumption and for the environment Who will have access to an individual's genetic information?