Chapters 7, 8, And 13 - Biotechnology

8/11/2019 Chapters 7, 8, And 13 - Biotechnology

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Chapters 7, 8, and 13: Biotechnology

Genes can be cloned in recombinant plasmids

Genetic engineering involves manipulating genes for practical purposes

Gene cloningleads to the production of multiple identical copies of a gene-carrying piece of DNA

Recombinant DNAis formed by joining DNA sequences from two different sources

One source contains the gene that will becloned

Another source is a gene carrier, called a vector

Vector: vehicle to move gene we want toclone

Plasmids (small, circular DNA moleculesindependent of the bacterial chromosome)

are often used as vectors

Transgenic: DNA from 2 or more species splicedtogether

Bt Corn: produces its own pesticide

Toxin gene that comes from bacteria

Steps in cloning a gene

1. Plasmid DNA is isolated

2. DNA containing the gene of interest is isolated

3. Plasmid DNA is treated with restriction enzyme that cuts in one place, opening the circle

4. DNA with the target gene is treated with the same enzyme and many fragments are produced

5. Plasmid and target DNA are mixed and associate with each other

6. Recombinant DNA molecules are produced when DNA ligasejoins plasmid and target segmentstogether

7. The recombinant DNA is taken up by a bacterial cell

8. The bacterial cell reproduces to form a clone of cells


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Enzymes are used to cut and paste DNA

Restriction enzymescut DNA at specific sequences (steps 3-4)

Each enzyme binds to DNA at a different restriction site (DNA sequence where they bind andcut)

Many restriction enzymes make staggered cuts that produce restriction fragmentswith single-stranded ends called sticky ends(see graphic)

Fragments with complementary sticky ends can associate with each other, forming recombinantDNA

DNA ligase joins DNA fragments together


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Cloned genes can be stored in genomic libraries

A genomic libraryis a collection of all of the clonedDNA fragments from a target genome for ease offuture research

Genomic libraries can be constructed with differenttypes of vectors

Plasmid library: genomic DNA is carried byplasmids

Phage library: genomic DNA is incorporated intobacteriophage DNA

Nucleic acid probes identify clones carrying specific genes

Screening a gene libraryoften the most tedious part of the process!

Figuring out which bacterial clone has our gene of interest

Genetically Modified Organisms


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Recombinant cells and organisms can mass-produce gene products

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

Capabilities of the host cell are matched to the characteristics of the desired product

Prokaryotic host: E. coli

Can produce eukaryotic proteins that do not require post-translational modification

Has many advantages in gene transfer, cell growth, and quantity of protein production

Can be engineered to secrete proteins

Capabilities of the host cell are matched to the characteristics of the desired product

Eukaryotic hosts

Yeast: S. cerevisiae

Can produce and secrete complex eukaryotic proteins

Mammalian cells in culture

Can attach sugars to form glycoproteinsPharm animals

Will secrete gene product in milk

DNA technology has changed the pharmaceutical industry and medicine

Products of DNA technology

Therapeutic hormones

Insulin to treat diabetes

Human growth hormone to treat dwarfism

Diagnosis and treatment of disease

Testing for inherited diseases


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Detecting infectious agents such as HIV

Vaccines

Stimulate an immune response by injecting

Protein from the surface of an infectious agent

A harmless version of the infectious agent

A harmless version of the smallpox virus containing genes from other infectious agents

Advantages of recombinant DNA products:

Identity to human protein Purity Quantity

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

Golden Rice: transgenic; contains genes form rice, bacteria, and daffodil

Resulting rice makes beta carotene, which body uses to make vitamin A

GM animals

Improved qualities

Production of proteins or therapeutics

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


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Hybrids with native plants may be prevented by modifying GM plants

Regulatory agencies address the safe use of biotechnology

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 marrowcell

Viral DNA and the functional allele will insert into the patients chromosome

Return the cells to the patient for growth and division

SCID (severe combined immune deficiency) was the first disease treatedby gene therapy

First trial in 1990 was inconclusive

Second trial in 2000 led to the development of leukemia in somepatients due to the site of gene insertion

Challenges

Safe delivery to the area of the body affected by the disease

Achieving a long-lasting therapeutic effect

Addressing ethical questions

DNA Profiling

The analysis of genetic markers can produce a DNA profile

DNA profiling is the analysis of DNA fragments to determine whether they come from a particularindividual

Compares genetic markers from noncoding regions that show variation between individualsInvolves amplification (copying) of markers for analysis

Sizes of amplified fragments are compared


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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

Relies upon a pair ofprimers

Short DNA molecules that bind to sequences at each end of the sequence to be copied

Used as a starting point for DNA replication

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 heat-stable DNA polymeraseAdvantages of PCR

Can amplify DNA from a small sample

Results are obtained rapidly

Reaction is highly sensitive, copying only the target sequence

Gel electrophoresis separates DNA molecules based on size

DNA sample is placed at one end of a porous gel

Current is applied and DNA molecules move from the negative electrode toward the positive electrode

Shorter DNA fragments move through the gel pores more quickly and travel farther through the gel

DNA fragments appear as bands, visualized through staining or detecting radioactivity or fluorescence

Each band is a collection of DNA molecules of the same length


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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 WorldTrade Center

Species identification: Evidence for sale of products from endangered species


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Genomics

Genomics is the scientific study of whole genomes

Genomics is the study of an organisms complete set of genes and their interactions

Initial studies focused on prokaryotic genomes

Many eukaryotic genomes have since been investigated

Evolutionary relationships can be elucidated

Genomic studies showed a ti6% similarity in DNA sequences between chimpanzees and humans

Functions of human disease-causing genes have been determined by comparisons to similar genesin yeast


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The Human Genome Project revealed that most of the human genome does not consist ofgenes

Goals of the Human Genome Project (HGP):

To determine the nucleotide sequence all DNA in the human genome

To identify the location and sequence of every human gene

Results of the Human Genome Project

Humans have 21,000 genes in 3.2 billion nucleotide pairs

Only 1.5% of the DNA codes for proteins, tRNAs, orrRNAs

The remaining 88.5% of the DNA contains

Control regions such as promoters and enhancers

Unique noncoding DNA

Repetitive DNA

Found in centromeres and telomeres

Found dispersed throughout the genome,related to transposable elements that canmove or be copied from one location toanother

Proteomics is the scientific study of the full set of proteins encoded by a genome

Proteomics: studies the proteome, the complete set of proteins specified by a genome


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Investigates protein functions and interactions

The human proteome may contain 100,000 proteins

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, generegulating brain size, FOXP2 gene involved with speech and vocalization

Cells are organized into tissues, organs, and systems

Tissues are made of groups of specialized cells.

Groups of tissues combine to form organs.

Organs work together to form the bodys systems.

Stem cellsare immature cells that can divide and differentiate into specialized cell types.


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Stem cells keep tissues healthy

Most body tissues contain stem cells that help regenerate those tissues and organs.

Using stem cells to build new organs

In order to build a new organ, a biopsy of the organ tissue is taken from the patient.

The stem cells from each tissue are grown in culture, allowing the cells to divide and create largenumbers of cells.

The stem cells are layered onto a biodegradable scaffold in the shape of the organ, and the scaffold isplaced in a chamber with nutrients and chemicals to support and encourage cell growth.

Surgeons then implant the newly grown organ into the patients body.

Tissues grown from a persons own cells pose no risk of rejection because the tissue is genetically

related to the donor.


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Adult stem cells

Specific stem cells are responsible for specific tissue types.

Adult stem cells, or somatic stem cells, are stem cells located in tissues that help maintain andregenerate those tissues.


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Specialized cells express different genes

Every cell in a persons body contains the same genes, or genome, but each type of cell has a uniquepattern of gene expression. This means that each type of cell makes a unique set of proteins and has aunique function in the body.

Cellular differentiation

In addition to dividing, cells formed from stem cells must go through cellular differentiationthe

process by which a cell specializes to carry out a specific role.

Cells become specialized through differential gene expressionthe process by which genes areturned on in different cell types.


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Regenerative medicine

A drawback to engineering new organs is that surgeons have to operate on the patients to obtain stemcells and again to repair the damaged organ.

Regenerative medicine would allow damaged organs to be repaired from within by stimulating stemcells to divide and differentiate when they otherwise would not.

Cells in the body continuously divide to replace damaged or aging cells.

Regenerative medicine can use therapeutic drugs to stimulate specific stem cells in the body to growand differentiate.

Regenerative medicine can also involve removing stem cells from the body, chemically inducing them

to reproduce and differentiate, and then re-implanting a small sample of differentiated cells into apatient with a damaged tissue or organ.

Regenerative medicine seeks to manipulate stem cells into cell types that they wouldnt differentiateinto on their own.


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Not all stem cells are created equal

Adult stem cells typically can differentiate only into one or a few cell typesthey are multipotentbecause they can give rise to a limited number of cell types.

Some stem cells arepluripotentthey can differentiate into nearly any cell type in the body

Some stem cells are totipotentthey can differentiate into any of the bodys cell types.

Chapters 7, 8, And 13 - Biotechnology

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