Biotechnology

Chapter 10

Golden Rice

Rice plants with added genes make and store beta carotene

Video: Golden rice or Frankenfood?

GMOs and Transgenic Organisms

Transgenic• An organism that has been genetically modified

with genes from a different species

Genetically modified organisms (GMOs)• Organism whose genome has been modified by

genetic engineering

10.2 Finding Needles in Haystacks

Gene research was limited until enzymes produced by bacteria to cut viral DNA were discovered

Restriction enzyme• Enzyme that cuts DNA at specific base sequences • Used in DNA cloning to cut DNA into pieces that

are inserted into cloning vectors

DNA Cloning

DNA cloning mass-produces DNA fragments for research

DNA cloning• Set of procedures that uses living cells to make

many identical copies of a DNA fragment

Clone• A genetically identical copy of DNA, a cell, or an

organism

Cloning Vectors

Cloning vector• A DNA molecule that can accept foreign DNA,

resulting in a hybrid molecule that can be transferred to a host cell, and get replicated in it

Plasmid• A small, circular DNA molecule in bacteria,

replicated independently of the chromosomes• A cloning vector

Recombinant DNA

Recombinant DNA molecules are introduced into host cells such as bacteria, which copy the DNA as they divide

Recombinant DNA• Contains genetic material from more than one

organism

Making Recombinant DNA

1. A restriction enzyme recognizes specific base sequences in DNA from two different sources

2. Restriction enzymes cut DNA into fragments with single-stranded tails (“sticky ends”)

3. DNA fragments from different sources are mixed together; matching sticky ends base-pair

4. DNA ligase joins fragments, forming recombinant DNA

Fig. 10-2, p. 181

restriction enzyme (cut)

DNA ligase (paste)

1 A restriction enzyme recognizes a specific base sequence in DNA (green boxes) from two sources.

2 The enzyme cuts DNA from both sources into fragments that have sticky ends.

3 The DNA fragments from the two sources are mixed together. The matching sticky ends base-pair with each other.

4 DNA ligase joins the fragments of DNA where they overlap. Molecules of recombinant DNA are the result.

mix

Making Recombinant DNA

Fig. 10-3, p. 181

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Cloning Vector 3.85 kb

Plasmid

The use of mRNA for the Identification of DNA

• mRNA sequence comes from specific regions of DNA (Genes)

• mRNA sequence is used to make proteins and defines the physical/behavioral characteristics of the organism

• Therefore we use mRNA to identify active regions of DNA

• Use mRNA sequence and base pairing rules to identify DNA original sequence

cDNA Cloning

RNA cannot be cloned directly; reverse transcriptase is used to copy single-stranded RNA into cDNA for cloning

Reverse transcriptase• Viral enzyme that uses mRNA as a template to

make a strand of DNA

cDNA• DNA synthesized from an RNA template by the

enzyme reverse transcriptase

Making cDNA

Fig. 10-4, p. 182

mRNA

A The enzyme reverse transcriptase transcribes mRNA into DNA.

mRNA

cDNA

B DNA polymerase replicates the DNA strand.

cDNA

cDNA

Eco RI recognition site

C The result is a double-stranded molecule of DNA that can be cut and pasted into a cloning vector. Stepped Art

Libraries

A library is a collection of cells that host different fragments of DNA, often representing an organism’s entire genome

Researchers make DNA libraries to isolate one gene from the many other genes in a genome

Genome• An organism’s complete set of genetic material

Nucleic Acid Hybridization

Probes are used to identify one clone that hosts a DNA fragment of interest among many other clones in a DNA library

Probe• Short fragment of DNA labeled with a tracer• Hybridizes with a specific nucleotide sequence

Nucleic acid hybridization• Base-pairing between DNA or RNA from different

sources

PCR

PCR quickly mass-produces copies of a particular DNA fragment for study

Polymerase chain reaction (PCR)• Uses primers and heat-resistant DNA polymerase

to rapidly generate many copies of a DNA fragment

Primer• Short, single-strand of DNA designed to hybridize

with a DNA fragment

Steps in PCR

1. Starting material is mixed with DNA polymerase, nucleotides and primers

2. Mixture is heated and cooled in cycles• At high temperature, DNA unwinds• At low temperature, primers base-pair with

template DNA

3. Taq polymerase synthesizes complementary DNA strands on templates

2 When the mixture is heated, the double-stranded DNA separates into single strands. When it is cooled, some of the primers base-pair with the template DNA.

4 The mixture is heated again, and the double-stranded DNA separates into single strands. When it is cooled, some of the primers base-pair with the template DNA.

Fig. 10-5, p. 183

1 DNA template (blue) is mixed with primers (red), nucleotides, and heat-tolerant Taq DNA polymerase.

Stepped Art

3 Taq polymerase begins DNA synthesis at the primers, and complementary strands of DNA form on the single-stranded templates.

2 When the mixture is heated, the double-stranded DNA separates into single strands. When it is cooled, some of the primers base-pair with the template DNA.

5 Taq polymerase begins DNA synthesis at the primers, and complementary strands of DNA form on the single-stranded templates.

4 The mixture is heated again, and the double-stranded DNA separates into single strands. When it is cooled, some of the primers base-pair with the template DNA.

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Animation: Polymerase chain reaction (PCR)

Animation: Formation of recombinant DNA

Animation: Use of a radioactive probe

Animation: Base-pairing of DNA fragments

Animation: How to make cDNA

Animation: Restriction enzymes

Animation: F2 ratios interaction

10.3 Studying DNA

Short tandem repeats are multiple copies of a short DNA sequence that follow one another along a chromosome

The number and distribution of short tandem repeats, unique in each individual, is revealed by electrophoresis as a DNA fingerprint

DNA Fingerprinting

DNA fingerprinting is used in forensics, court evidence, and other applications

DNA fingerprint• An individual’s unique array of short tandem

repeats

Electrophoresis• Used to separate DNA fragments by size

Fig. 10-6, p. 184

Evidence from Crime Scene

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The Human Genome Project

Automated DNA sequencing and PCR enabled scientists to sequence the more than 3 billion bases of the human genome

Sequencing• Method of determining the order of nucleotides in

DNA

Sequencing a Fragment of DNA

The order of colors is the order of DNA bases (A, T, G, C)

Genomics

Analysis of the human genome sequence is yielding new information about human genes and how they work

Genomics• The study of genomes (structural genomics,

comparative genomics)

Some Sequenced Genomes

Animation: Automated DNA sequencing

Animation: DNA fingerprinting

Video: ABC News: DNA mystery: Human chimeras

Video: ABC News: Family ties: Paternity testing

3D Animation: Gene sequencing

10.4 Genetic Engineering

Recombinant DNA technology and genome analysis are the basis of genetic engineering

Genetic engineering is the directed alteration of an individual’s genome, resulting in a genetically modified organism (GMO)

Genetic engineering• Process by which deliberate changes are

introduced into an individual’s genome

Genetically Modified Microorganisms

A transgenic organism carries a gene from a different species

Transgenic organisms are used in research, medicine, and industry

Transgenic bacteria and yeast produce medically valuable proteins

Designer Plants

Transgenic crop plants help farmers produce food more efficiently

Plants with modified or foreign genes are now common in farm crops

Fig. 10-8, p. 187

1 An A. tumefaciens bacterium has been engineered to contain a Ti plasmid. The plasmid carries a foreign gene.

2 The bacterium infects a plant cell and transfers the Ti plasmid into it. The plasmid DNA becomes integrated into one of the cell’s chromosomes.

3 The plant cell divides, and its descendants form an embryo.

4 The embryo develops into a transgenic plant.

5 The transgenic plant expresses the foreign gene. This tobacco plant is expressing a gene from a firefly.

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Animation: Gene transfer using a Ti plasmid

Genetically Modified Crops

Bt gene confers insect resistance to corn

Biotech Barnyards

Transgenic animals produce human proteins

Animals that would be impossible to produce by traditional breeding methods are being created by genetic engineering

Transgenic animals are used in research, medicine, and industry

Transgenic Animals

Knockout Cells and Organ Factories

Transgenic animals may one day provide a source of organs and tissues for transplantation into humans

Xenotransplantation• Transplant of an organ from one species to

another

Animation: Transferring genes into plants

Video: ABC News: Cloned pooch

Video: ABC News: Mule clones

Video: ABC News: Glow-in-the-dark pigs

Video: ABC News: Cloned food approved

10.5 Genetically Modified Humans

Genes can be transferred into a person’s cells to correct a genetic defect or treat a disease

However, the outcome of altering a person’s genome remains unpredictable

Gene therapy• Transfer of a normal or modified gene into an

individual with the goal of treating a genetic defect or disorder

Unpredictable Outcomes

There are more than 15,000 serious genetic disorders – gene therapy is the only real cure

In some cases, gene therapy works – in other cases it leads to death• Inserting a virus-injected gene into a

chromosome can disrupt normal function and cause cancer

• Severe allergic reaction to the viral vector can cause death

One Successful Case of Gene Therapy

Rhys Evans, born with a severe immune disorder (SCID-X1) received a normal gene and no longer lives in isolation

Getting Perfect

Eugenics• Idea of deliberately improving the genetic

qualities of the human race

The potential benefits of genetically modifying humans must be weighed against the potential risks, including social implications

10.6 Impacts/Issues Revisited

Golden rice with beta carotene was ready for distribution in 2005 but is still not available for human consumption – the biosafety experiments required are too expensive for the public humanitarian agency that developed it

Digging Into Data: Enhanced Spatial Learning in Mice With Autism Mutation