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Biotechnology
Pre-AP BiologyCh.12
Ms. Haut
DNA technology has many useful applications
– The Human Genome Project– The production of vaccines, cancer drugs, and
pesticides– Engineered
bacteria that can clean up toxic wastes
Copyright © 2003 Pearson Education, Inc. publishing Benjamin Cummings
•DNA and Crime Scene Investigations– Many violent crimes go unsolved
• For lack of enough evidence
– If biological fluids are left at a crime scene• DNA can be isolated from them
– DNA fingerprinting is a set of laboratory procedures• That determines with near certainty whether two
samples of DNA are from the same individual• That has provided a powerful tool for crime scene
investigators
Investigator at oneof the crime scenes(above), Narborough,England (left)
BACTERIAL PLASMIDS AND GENE CLONING
•Plasmids are used to customize bacteria: An overview
– Gene cloning is one application of DNA technology
• Methods for studying and manipulating genetic material
The Bacterial Chromosome
• One double-stranded, circular molecule of DNA
• Located in nucleoid region, so transcription and translation can occur simultaneously
• Many also contain extrachromosomal DNA in plasmids
Binary Fission
Plasmids
• Short, circular DNA molecules outside the chromosome
• Carry genes that are beneficial but not essential
• Replicate independently of chromosome
en.wikipedia.org/?title=Plasmid
R Plasmids
• Contain genes that confer antibiotic resistance
• Medical consequences: resistant strains of pathogens due to overuse of antibiotics
http://www.slic2.wsu.edu:82/hurlbert/micro101/images/AntibioticSelection.gif
Bacteria as Tools
• Bacterial Transformation—– Uptake of DNA from the fluid surrounding the cell– Causes genetic recombination
Transformation
• Biotech companies use this technique to artificially introduce foreign genes into bacterial genomes (human insulin, human growth hormone)
– Researchers can insert desired genes into plasmids, creating recombinant DNA
• And insert those plasmids into bacteria (transformation)
Figure 12.1
– If the recombinant bacteria multiply into a clone
• The foreign genes are also copied
Isolate DNAfrom two sources
1E. coli
Cut both DNAs with the same
restrictionenzyme
2
Plasmid
Human cell
DNA
Gene VSticky ends
Mix the DNAs; they joinby base-pairing
3
Add DNA ligaseto bond the DNA covalently
4
Recombinant DNAplasmid Gene V
Put plasmid into bacteriumby transformation
5
Clone the bacterium6
Bacterial clone carrying manycopies of the human gene
Restriction Enzymes
•Used to “cut and paste” DNA
•The tools used to make recombinant DNA are
– Restriction enzymes, which cut DNA at specific sequences
– DNA ligase, which “pastes” DNA fragments together
Genes can be cloned in recombinant plasmids: A closer look
– Bacteria take the recombinant plasmids from their surroundings
– And reproduce, thereby cloning the plasmids and the genes they carry
Cloned genes can be stored in genomic libraries
•Genomic libraries, sets of DNA fragments containing all of an organism’s genes
– Can be constructed and stored in cloned bacterial plasmids or phages
Figure 12.4
Recombinantplasmid
Genome cut up withrestriction enzyme
Recombinantphage DNA
or
Bacterialclone
Phageclone
Phage libraryPlasmid library
Recombinantplasmid
Genome cut up withrestriction enzyme
Recombinantphage DNA
or
Bacterialclone
Phageclone
Phage libraryPlasmid library
Recombinantplasmid
Genome cut up withrestriction enzyme
Recombinantphage DNA
or
Bacterialclone
Phageclone
Phage libraryPlasmid library
Nucleic acid probes
•A short, single-stranded molecule of radioactively labeled or fluorescently labeled DNA or RNA
– Can tag a desired gene in a library
Radioactiveprobe (DNA)
Single-strandedDNA
Mix with single-stranded DNA fromvarious bacterial(or phage) clones
Base pairingindicates thegene of interest
A T C C G A
A T G C G C T T A T C G
A G C
C T
T A
T G
C A
T
A T C C
G A
A G G T A G G C T A A
Radioactiveprobe (DNA)
Single-strandedDNA
Mix with single-stranded DNA fromvarious bacterial(or phage) clones
Base pairingindicates thegene of interest
A T C C G A
A T G C G C T T A T C G
A G C
C T
T A
T G
C A
T
A T C C
G A
A G G T A G G C T A A
Master plate
Filter
Solutioncontainingprobe
Filter liftedand flipped over
Radioactivesingle-strandedDNA
ProbeDNA
Gene ofinterest
Single-strandedDNA from cell
Film
Hybridizationon filter
Master plate
Coloniescontaininggene ofinterest
A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter.
The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter.
The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography).
After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest.
Master plate
Filter
Solutioncontainingprobe
Filter liftedand flipped over
Radioactivesingle-strandedDNA
ProbeDNA
Gene ofinterest
Single-strandedDNA from cell
Film
Hybridizationon filter
Master plate
Coloniescontaininggene ofinterest
A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter.
The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter.
The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography).
After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest.
CONNECTION
•Recombinant cells and organisms can mass-produce gene products
– Applications of gene cloning include
• The mass production of gene products for medical and other uses
Table 12.6
• New genetic varieties of animals and plants are being produced– A plant with a new trait can be created using the Ti
plasmid
Genetically modified organisms are transforming agriculture
• Biotech companies can artificially induce transformation of bacteria
• “Golden rice” has been genetically modified to contain beta-carotene– This rice could help prevent vitamin A
deficiency
Figure 12.18B
Drought resistant corn
Flavr Savr Tomato (CalGene)
•Transgenic organisms– Are those that have had genes from
other organisms inserted into their genomes
– Different organisms, including bacteria, yeast, and mammals
• Can be used for this purpose
Figure 12.6
These sheep carry a gene for a human blood protein that is a potential treatment for cystic fibrosis
•DNA technology is changing the pharmaceutical industry
– DNA technology• Is widely used to produce medicines and to
diagnose diseases
CONNECTION
• Hormones, cancer-fighting drugs, and new vaccines are being produced using DNA technology– This lab equipment
is used to produce a vaccine against hepatitis B
DNA technology is changing the pharmaceutical industry and medicine
Figure 12.17
•Therapeutic hormones– In 1982, humulin, human insulin produced by
bacteria• Became the first recombinant drug approved
by the Food and Drug Administration
Figure 12.7A
•Diagnosis and Treatment of Disease – DNA technology
• Is being used increasingly in disease diagnosis
•Vaccines– DNA technology
• Is also helping medical researchers develop vaccines
Figure 12.7B