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