nuclear transplantation - can be used to clone animals...

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CLONING OF PLANTS AND ANIMALS

Copyright © 2009 Pearson Education, Inc.

Nuclear transplantation - can be used to clone animals

  Nuclear transplantation

–  Replacing the nucleus of an egg cell or zygote with a nucleus from an adult somatic cell

–  Early embryo (blastocyst) can be used in

–  Reproductive cloning

–  Implant embryo in surrogate mother for development

–  New animal is genetically identical to nuclear donor

–  Therapeutic cloning (as seen in Clone)

–  Remove embryonic stem cells and grow in culture for medical treatments

–  Induce stem cells to differentiate


Remove nucleus from egg cell

Implant blastocyst in surrogate mother

Add somatic cell from adult donor

Donor cell

Remove embryonic stem cells from blastocyst and grow in culture

Reproductive cloning

Nucleus from donor cell

Grow in culture to produce an early embryo (blastocyst)

Therapeutic cloning

Clone of donor is born

Induce stem cells to form specialized cells

Therapeutic cloning can produce stem cells with great medical potential

  Stem cells can be induced to give rise to differentiated cells

–  Embryonic stem cells can differentiate into a variety of types

–  Adult stem cells can give rise to many but not all types of cells

  Therapeutic cloning can supply cells to treat human diseases

  Research continues into ways to use and produce stem cells


Blood cells

Different types of differentiated cells

Nerve cells

Adult stem cells in bone

marrow

Different culture conditions

Cultured embryonic stem cells

Heart muscle cells

12.1 Genes can be cloned in recombinant plasmids

  Biotechnology is the manipulation of organisms or their components to make useful products.

  For thousands of years, humans have

  used microbes to make wine and cheese and

  selectively bred stock, dogs, and other animals.

  DNA technology is the set of modern techniques used to study and manipulate genetic material.

© 2012 Pearson Education, Inc.

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12.1 Genes can be cloned in recombinant plasmids

  Genetic engineering involves manipulating genes for practical purposes

–  Gene cloning leads to the production of multiple identical copies of a gene-carrying piece of DNA

–  Recombinant DNA is formed by joining DNA sequences from two different sources

–  One source contains the gene that will be cloned

–  Another source is a gene carrier, called a vector

–  *Remember Plasmids (small, circular DNA molecules independent of the bacterial chromosome) are often used as vectors


Examples of gene use

Recombinant DNA plasmid

E. coli bacterium Plasmid

Bacterial chromosome

Gene of interest DNA

Gene of interest

Cell with DNA containing gene of interest

Recombinant bacterium

Clone of cells

Genes may be inserted into other organisms

Genes or proteins are isolated from the cloned bacterium

Harvested proteins may be used directly

Examples of protein use

Gene of interest

Isolate plasmid

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Isolate DNA

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Cut plasmid with enzyme

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Cut cell’s DNA with same enzyme

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Combine targeted fragment and plasmid DNA

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Add DNA ligase, which closes the circle with covalent bonds

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Put plasmid into bacterium by transformation

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Allow bacterium to reproduce

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E. coli bacterium Plasmid

Bacterial chromosome

Gene of interest DNA

Cell with DNA containing gene of interest

Gene of interest

Isolate plasmid

Isolate DNA

Cut plasmid with enzyme

Cut cell’s DNA with same enzyme

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2

3

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Gene of interest

Combine targeted fragment and plasmid DNA

Add DNA ligase, which closes the circle with covalent bonds

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Gene of interest

Recombinant bacterium

Clone of cells

Genes or proteins are isolated from the cloned bacterium

Harvested proteins may be used directly

Examples of protein use

Put plasmid into bacterium by transformation

Allow bacterium to reproduce

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Genes may be inserted into other organisms

Examples of gene use

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12.2 Enzymes are used to “cut and paste” DNA

  Restriction enzymes (aka ENDONUCLEASES) cut DNA at specific sequences

–  Each enzyme binds to DNA at a different restriction site

–  Many restriction enzymes make staggered cuts that produce restriction fragments with single-stranded ends called “sticky ends”

–  Fragments with complementary sticky ends can associate with each other, forming recombinant DNA

  DNA ligase joins DNA fragments together


Restriction enzyme recognition sequence

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2

DNA

Restriction enzyme cuts the DNA into fragments

Sticky end

3 Addition of a DNA fragment from another source

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Two (or more) fragments stick together by base-pairing

DNA ligase pastes the strands

Recombinant DNA molecule 5

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GENETICALLY MODIFIED ORGANISMS


12.6 Recombinant cells and organisms can mass-produce gene products

  Cells and organisms containing cloned genes are used to manufacture large quantities of gene products

  Examples

  E. coli

–  Can produce and secrete proteins (such as replacement insulin)

–  Yeast: S. cerevisiae

–  Can produce and secrete complex eukaryotic proteins

–  Many vaccines produced in this way

12.7 CONNECTION: DNA technology has changed the pharmaceutical industry and medicine

  Advantages of recombinant DNA products

–  Identity to human protein

–  Purity

–  Quantity


12.8 CONNECTION: Genetically modified organisms are transforming agriculture

  Genetically modified (GM) organisms contain one or more genes introduced by artificial means

  Transgenic organisms contain at least one gene from another species

  GM plants –  Resistance to herbicides –  Resistance to pests –  Improved nutritional profile

  GM animals –  Improved qualities –  Production of proteins or therapeutics


Agrobacterium tumefaciens

DNA containing gene for desired trait

Ti plasmid Insertion of gene

into plasmid

Recombinant Ti plasmid

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Restriction site

Plant cell

Introduction into plant cells

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DNA carrying new gene

Regeneration of plant

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Plant with new trait

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12.9 Genetically modified organisms raise concerns about human and environmental health

  Scientists use safety measures to guard against production and release of new pathogens

  Concerns related to GM organisms

–  Can introduce allergens into the food supply

–  FDA requires evidence of safety before approval

–  Exporters must identify GM organisms in food shipments

–  May spread genes to closely related organisms

–  Hybrids with native plants may be prevented by modifying GM plants

  Regulatory agencies address the safe use of biotechnology


12.10 CONNECTION: Gene therapy may someday help treat a variety of diseases

  Gene therapy aims to treat a disease by supplying a functional allele

  One possible procedure

–  Clone the functional allele and insert it in a retroviral vector

–  Use the virus to deliver the gene to an affected cell type from the patient, such as a bone marrow cell

–  Viral DNA and the functional allele will insert into the patient’s chromosome

–  Return the cells to the patient for growth and division


DNA PROFILING


12.11 The analysis of genetic markers can produce a DNA profile

  DNA profiling (“fingerprinting” is the analysis of DNA fragments to determine whether they come from a particular individual

–  Compares genetic markers from noncoding regions that show variation between individuals

–  Involves amplification (copying) of markers for analysis

–  Sizes of amplified fragments are compared


Crime scene DNA isolated 1

Suspect 1 Suspect 2

DNA of selected markers amplified

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Amplified DNA compared

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12.12 The PCR method is used to amplify DNA sequences

  Polymerase chain reaction (PCR) is a method of amplifying a specific segment of a DNA molecule

  Repeated cycle of steps for PCR –  Sample is heated to separate DNA strands –  Sample is cooled and primer binds to specific target

sequence –  Target sequence is copied with form of DNA

polymerase


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Cycle 1 yields 2 molecules

2 1 3

Genomic DNA



3′ 5′ 3′ 5′ 3′ 5′

Target sequence

Heat to separate DNA strands

Cool to allow primers to form hydrogen bonds with ends of target sequences

3′ 5′

3′ 5′

3′ 5′

3′ 5′ 3′ 5′

Primer New DNA

5′

DNA polymerase adds nucleotides to the 3′ end of each primer

5′

  Advantages of PCR

–  Can amplify DNA from a small sample

–  Results are obtained rapidly

–  Reaction is highly sensitive, copying only the target sequence


12.12 The PCR method is used to amplify DNA sequences

12.13 Gel electrophoresis sorts DNA molecules by size

  Gel electrophoresis separates DNA molecules based on size

–  Know the step and underlying concepts of this process from labs!


12.15 CONNECTION: DNA profiling has provided evidence in many forensic investigations

  Forensics –  Evidence to show guilt or innocence

  Establishing family relationships –  Paternity analysis

  Identification of human remains

–  After tragedies such as the September 11, 2001, attack on the World Trade Center

  Species identification –  Evidence for sale of products from endangered species


GENOMICS


12.21 EVOLUTION CONNECTION: Genomes hold clues to the evolutionary divergence of humans and chimps

  Comparisons of human and chimp genomes

–  Differ by 1.2% in single-base substitutions

–  Differ by 2.7% in insertions and deletions of larger DNA sequences

–  Human genome shows greater incidence of duplications

–  Genes showing rapid evolution in humans –  Genes for defense against malaria and tuberculosis

–  Gene regulating brain size

–  FOXP2 gene involved with speech and vocalization


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