DNA Technology• In laboratory experiments

– Genes can be transcribed and translated after being transplanted from one species to another

• Called “Recombinant DNA” technology• Can be produced via “Genetic Engineering” (laboratory manipulation)

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Overview: Understanding and Manipulating Genomes

• One of the greatest achievements of modern science has been the sequencing of the human genome, which was largely completed by 2003

• DNA sequencing accomplishments– Have all depended on advances in DNA

technology, starting with the invention of methods for making recombinant DNA

– DNA sequencing animation

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How can we modify a person’s genome?

• Gene therapy - insertion of genetic material into human cells to treat a disorder

– Ex vivo therapy – cells are removed for a person altered and then returned to the patient

– In vivo therapy – a gene is directly inserted into an individual through a vector (e.g. viruses) or directly injected to replace mutated genes or to restore normal controls over gene activity

• Gene therapy has been most successful in treating cancer, to date.

Genomics

Ex vivo gene therapy

Genomics

defective gene

retrovirus

normal gene

normal gene

viral recombinant RNA

viral recombinant DNA

1. Remove bone marrow stem cells.

2. Use retroviruses to bring the normal gene into the bone marrow stem cells.

4. Return genetically engineered cells to patient.

3. Viral recombinant DNA carries normal gene into genome.

viral recombinantRNA

reverse transcription

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

DNA Cloning

• Concept 20.1: DNA cloning permits production of multiple copies of a specific gene or other DNA segment

• To work directly with specific genes– Scientists have developed methods for

preparing well-defined, gene-sized pieces of DNA in multiple identical copies, a process called gene cloning

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24.3 DNA Technology

• The Cloning of a Gene

– Cloning: Production of many identical copies of

an organism through some

asexual means.

– Gene Cloning: The production of many identical copies of a single gene

– Two Ways to Clone a Gene:– Recombinant DNA

– Polymerase Chain Reaction

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Using Restriction Enzymes to Make Recombinant DNA

• Bacterial restriction enzymes– Cut DNA molecules at

a limited number of specific DNA sequences, called restriction sites

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Restriction Enzymes and Sticky Ends

Step through animation of cut/splice using EcoRI

Narrated animation8

Cloning of a Human Gene / Recombinant DNA

– Restriction enzymes breaks open a plasmid vector at specific sequence of bases “sticky ends”

– Foreign DNA that is to be inserted is also cleaved with same restriction enzyme so ends match

– Foreign DNA is inserted into plasmid DNA and “sticky ends” pair up

– DNA ligase seals them together

– Narrated animation of “Cloning a Gene” 9

24.3 DNA Technology• Polymerase Chain Reaction

– Amplifies a targeted DNA sequence– Requires DNA polymerase, a set of

primers, and a supply of nucleotides• Primers are single stranded DNA

sequences that start replication process

– Amount of DNA doubles with each replication cycle

– Process is now automated– Narrated animation– Step by step animation

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24.3 DNA Technology• DNA Fingerprinting

– Permits identification of individuals and their relatives– Based on, polymorphisms differences between

sequences in nucleotides between individuals– RFLPs : restriction fragment length polymorphisms– Narrated animation– Detection of the number of repeating segments (called

repeats) are present at specific locations in DNA• Different numbers in different people• PCR amplifies only particular portions of the DNA• Procedure is performed at several locations to identify

repeats

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DNA Fingerprints

DNA fragments (after digest with restriction enzymes) can be separated through gel ELECTROPHORESIS

See How:Animation http://highered.mcgraw-hill.com/sites/007337797x/student_view0/chapter14/animation_quiz_-_dna_fingerprinting.html

Step-by-step electrophoresis

Another walk-through explanation

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Forensic Evidence• DNA “fingerprints”

obtained by analysis of tissue or body fluids found at crime scenes– Can provide definitive

evidence that a suspect is guilty or, more specifically, not guilty

– Is a specific pattern of bands of RFLP markers on a gel

Defendant’sblood (D)

Blood fromdefendant’sclothes

Victim’sblood (V)

D Jeans shirt V

4 g 8 g

Figure 20.1713

DNA fingerprinting

Can also be used in establishing paternity

Figure: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint.

http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Paternity_testing

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24.3 DNA Technology

• Biotechnology

– Biotechnology uses natural biological systems to create a product or to achieve a goal desired by humans.

– “Model Organisms” favored for genetics research

15

Environmental Cleanup• Genetic engineering can be used to modify

the metabolism of microorganisms– So that they can be used to extract minerals

from the environment or degrade various types of potentially toxic waste materials

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24.3 DNA Technology

• Transgenic Bacteria

– Medical Uses: Production of Insulin, Human Growth

Hormone, Hepatitis B Vaccine

– Agricultural Uses: Bacteria that protects plants from freezing, bacteria that protect plant roots from insects

– Environmental: Bacteria that degrade oil (clean up after oil spills), bacteria that remove sulfur from coal

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24.3 DNA Technology

• Transgenic (GM) Plants– Plants have been engineered to secrete a toxin that

kills insects (ex: Bt corn)

– Plants have been engineered to be resistant to

herbicides (ex: Roundup Ready)

– Animation: Gene Transfer in Plants Using a Ti Plasmid

http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter1

7/genes_into_plants_using_the_ti-plasmid.html

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Agricultural Applications• DNA technology

– Is being used to improve agricultural productivity and food quality

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In 2008:92% of the soybeans and 80% of the corn planted in the United States had been genetically engineered.

Genetic Engineering in Plants• Agricultural scientists

– Have already endowed a number of crop plants with genes for desirable traits

Bt corn (right) 20

Biotechnology products: Transgenic plants

DNA technology

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

b. Salt-intolerant Salt-toleranta. Desirable traits

Disease-protected Wheat, corn, potatoes

Herbicide-resistant

Salt-tolerant

Drought-tolerant

Cold-tolerant

Improved yield

Modified wood pulp

Wheat, rice, sugar beets, canola

Cereals, rice, sugarcane

Cereals, rice, sugarcane

Cereals, rice, sugarcane

Cereals, rice, corn, cotton

Fatty acid/oil content

Protein/starch content

Amino acid content

Corn, soybeans

Cereals, potatoes, soybeans, rice, corn

Corn, soybeans

Transgenic Crops of the Future

Improved Agricultural Traits

Improved Food Quality Traits

Trees

Health focus: Ecological concern about GMO crops

• Resistance increasing in the target pest

• Exchange of genetic material between the transgenic plant and a related species

• Concern about the impact of BT crops on nontarget species (ex: pollinators)

DNA technology

GM Animals and “Pharm” Animals

• Transgenic animals– Contain genes from other organisms– Sometimes called “chimeras”– Fig 1. transgenic mouse lines expressing GFP known as “green

mice.”

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24.3 DNA Technology

• Transgenic

Animals

– Fish, cows, pigs,

rabbits and sheep

have been

engineered to

produce human

growth hormone in

order to increase

size of the animals

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– Transgenic organisms have a foreign gene inserted into their DNA– Have been engineered to be pharmaceutical “factories”

Figure 20.18

“Pharm” Animals

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Human breast milk from a cow (2011)

Pharmaceutical Products

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• “Knockout” mice

A number of mice models have been developed: either possessing an inactivated tumor suppressor gene (p53), an activated oncogene (Tg.AC), over-expression of a (human) oncogene (rasH2) or being deficient in nucleotide excision repair (Xpa, de Vries et al., 1995).

These mice models have several advantages: • the number of animals needed for one study is 120 instead of 400-500• the duration of the study is 6-9 instead of 24 months leading to less

distress of the animals• the transgenic mouse model is considered more discriminating hence

improving the accuracy and reliability of human carcinogen identification.

http://www.nca-nl.org/English/Newsletters/Nb13/nl13txt.html

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Safety and Ethical Questions Raised by DNA Technology

• The potential benefits of genetic engineering– Must be carefully weighed against the potential

hazards of creating products or developing procedures that are harmful to humans or the environment

• Today, much public concern about possible hazards

– Centers on genetically modified (GMOs) organisms used as food (allergic reactions, etc)

– Gene “escape” 28