Biotechnology

Chapter 10

10.1 Impacts/Issues Golden Rice or Frankenfood? 

Genes from one species may be inserted into an individual of another species ± or a gene may be modified and reinserted into an individual of the same species

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 (³sticky ends´) 3. DNA fragments from different sources are mixed together; matching sticky ends base-pair 4. DNA ligase joins fragments, forming recombinant DNA

Making Recombinant DNA

restriction enzyme (cut)

mix

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.

Fig. 10-2, p. 181

restriction enzyme (cut)

mix

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.

Stepped Art Fig. 10-2, p. 181

Commercial Plasmid Cloning Vector

Kpn l Sph l Pst l Bam Hl Eco RI Sal l

pDrive Cloning Vector 3.85 kb

Acc l Xho l Xba l Bst XI Sac l Not l
Fig. 10-3, p. 181

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

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.
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 Fig. 10-4, p. 182

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

Two Rounds of PCR

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

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

3 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.

5 Taq polymerase begins DNA synthesis at the primers, and complementary strands of DNA form on the single-stranded templates.
Fig. 10-5, p. 183

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

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

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

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

Stepped Art Fig. 10-5, p. 183

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

DNA Fingerprinting: A Forensic Case

Size Reference Control DNA Size Reference Victim Suspect 1 Suspect 2 Female Cells Semen Size Reference Boyfriend Control DNA Control DNA Size Reference

Evidence from Crime Scene

Fig. 10-6, p. 184

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

Using the Ti plasmid to Make a Transgenic Plant

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.
Fig. 10-8, p. 187

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

Fig. 10-11a, p. 193

Fig. 10-11b, p. 193

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