Transcript Using Genetic Markers

Key Concepts
Enzymes that cut DNA at specific locations and other enzymes
that piece DNA segments back together allow biologists to move
genes from one place to another.
Biologists can obtain many identical copies of a gene by (1)
inserting it into a bacterial cell that copies the gene each time the
cell divides or (2) by conducting a polymerase chain reaction.
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Key Concepts
The sequence of bases in a gene can be determined by dideoxy
sequencing.
If individuals with a certain phenotype also tend to share a genetic
marker (a known site in DNA that is unrelated to the phenotype),
the gene responsible for the phenotype is likely to be near that
marker.
Researchers are attempting to insert genes into humans to cure
genetic diseases. Efforts to insert genes into plants have been
much more successful.
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Introduction
• Manipulation of DNA sequences in organisms is known as genetic
engineering, and techniques used to engineer genes are called
recombinant DNA technology.
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The Effort to Cure Pituitary Dwarfism
• Pituitary dwarfism results from the lack of production of growth
hormone, encoded by the GH1 gene.
• Pituitary dwarfism type I is an autosomal recessive trait. Affected
individuals have two copies of the defective allele.
• Humans affected by pituitary dwarfism grow slowly, reaching a
maximum adult height of about 4 feet.
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© 2011 Pearson Education, Inc.
Why Did Early Efforts to Treat the Disease Fail?
• Early trials showed that people with pituitary dwarfism could be
treated successfully with growth hormone therapy, but only if the
protein came from humans.
• Growth hormone purified from the pituitary glands of human
cadavers is scarce and expensive.
• Human treatment with growth hormone from cadavers has been
banned due to possible contamination with prions—protein
particles that have been implicated as the cause of various
neurodegenerative disorders.
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Engineering a Safe Supply of Growth Hormone
• The recombinant DNA strategy for producing human growth
hormone involved cloning the human gene, introducing the gene
into bacteria, and having the recombinant cells produce the
hormone.
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Using Reverse Transcriptase to Produce cDNAs
• The enzyme reverse transcriptase can synthesize DNA from an
RNA template.
• Researchers used reverse transcriptase to make complementary
DNA (cDNA) from mRNA isolated from pituitary cells. (cDNA is
any DNA made from an RNA template.)
• They then used DNA cloning—the process of producing many
identical copies of a gene—to copy the cDNAs for analysis to
determine which encoded the growth hormone protein.
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© 2011 Pearson Education, Inc.
Using Plasmids in Cloning
• Plasmids are small, circular DNA molecules often found in
bacteria. They replicate independently of the chromosome.
• Plasmids can serve as a vector—a vehicle for transferring
recombinant genes to a new host.
• If a recombinant plasmid can be inserted into a bacterial or yeast
cell, the foreign DNA will be copied and transmitted to new cells as
the host cell grows and divides. In this way plasmids can be used to
produce millions of identical copies of specific genes.
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Transformation
If a recombinant plasmid can be inserted into a bacterial or yeast
cell, the foreign DNA will be copied and transmitted to new cells
as the host cell grows and divides. In this way, researchers can
obtain millions or billions of copies of specific genes.
• Plasmid vectors can be introduced into bacteria by
transformation, the process of taking up DNA from the
environment and incorporating it into the genome.
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Mass-Producing Growth Hormone
• Once the researchers found the human growth hormone cDNA,
they cloned it in a plasmid under the control of a bacterial
promoter.
• The transformed E. coli cells produced human growth hormone that
could be isolated and purified in large quantities.
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Ethical Concerns over Recombinant Growth Hormone
• The increased supply of growth hormone led to its use to treat
children who were short but not did not suffer from pituitary
dwarfism.
• The U.S. Food and Drug Administration has now approved use of
the hormone only for children projected to reach adult heights of
less than 5'3" for males and 4'11" for females.
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The Polymerase Chain Reaction
• The polymerase chain reaction (PCR) is an in vitro DNA
synthesis reaction in which a specific DNA sequence is replicated
over and over again.
• This technique generates many identical copies of a particular DNA
sequence.
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Requirements of PCR
• PCR is possible only when DNA sequence information surrounding
the gene of interest is available, because PCR requires primers that
match sequences on either side of the gene.
• One primer is complementary to a sequence on one strand upstream
of the target DNA and the other primer is complementary to a
sequence on the other strand downstream of the target.
• The primers will bind to single-stranded target DNA.
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© 2011 Pearson Education, Inc.
PCR in Action: Studying Fossil DNA
• Svante Pääbo and colleagues used PCR to compare DNA sequences
from a 30,000-year-old Homo neanderthalensis fossil with modern
Homo sapiens DNA to analyze how similar the two species are.
• Because the complete genomes of a wide array of organisms have
now been sequenced, researchers can find appropriate primer
sequences to use in cloning almost any target gene by PCR.
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How Was the Huntington's Disease Gene Found?
• Huntington’s disease is a rare but devastating neurodegenerative
disorder that is eventually fatal.
• An analysis of pedigrees from affected families suggested that the
trait results from a single, autosomal dominant allele.
– This means that sons or daughters of a Huntington's sufferer
have a 50 percent chance of receiving the disease allele and
developing the illness.
• Researchers set out to identify the gene or genes involved and to
document that one or more genes are altered in affected individuals.
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Locating Specific Genes
• To locate the gene or genes associated with a particular phenotype,
such as a disease, researchers traditionally started with a genetic
map (or linkage map or meiotic map).
• More recently, biologists have begun using a physical map of the
genome. A physical map records the absolute position of a gene—
in numbers of base pairs—along a chromosome.
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Using Genetic Markers
• Genetic maps are valuable because they contain genetic markers
(genes or other loci that have known locations).
• Each genetic marker provides a landmark at a position along a
chromosome that is known relative to other markers.
– Genetic markers must be polymorphic in order to be useful—
in other words, the phenotype associated with the marker must
be variable.
• DNA samples from affected families can be analyzed with genetic
markers to follow the inheritance of specific DNA regions.
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Using Genetic Markers
If you observe that a certain marker and a certain phenotype are
almost always inherited together, it is logical to conclude that the
genes involved are physically close to each other on the same
chromosome—meaning that they are closely linked.
• To locate specific disease genes, researchers must find a large
number of affected and unaffected people, and then locate a
genetic marker that occurs in the affected individuals but not in
the unaffected people.
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Using Genetic Markers
• Single nucleotide polymorphisms (SNPs) are sites in DNA where
some individuals in the population have different bases.
• SNPs can be used as genetic markers.
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Pinpointing the Huntington’s Disease Defect
• The researchers sequenced exons in the location of the Huntington's
disease gene from affected and unaffected individuals to pinpoint
specific bases that differ between the two groups.
• Individuals with Huntington's disease have an unusual number of
CAG codons at the 5' end of a particular gene.
• They called this gene IT15 and its protein product huntingtin.
Huntingtin is involved in the early development of nerve cells.
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Improved Understanding of the Phenotype
• Once a disease gene is found, the relationship between the gene and
the resulting disease phenotype is better understood.
• In Huntington's patients, huntingtin protein forms aggregates in the
brain, eventually causing neurons to die.
• These aggregates are thought to be a direct consequence of changes
in the number of CAG repeats in the IT15 gene.
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Therapy
• Therapies can be discovered by using transgenic animals that have
had the defective allele introduced into their genome.
• If the animals exhibit disease symptoms that parallel those of a
human disease they are said to provide an animal model of the
disease.
– Researchers can use animal models to test possible treatments.
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Three Types of Genetic Tests for Genetic Diseases
• Carrier testing can determine if an individual carries a defective
allele.
• Prenatal testing can determine if a fetus has a genetic disease by
analyzing some of its cells early in gestation.
• Adult testing can inform individuals if they are more likely to
develop certain diseases, such as breast cancer, due to a faulty gene.
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Ethical Concerns over Genetic Testing
• Genetic testing raises controversial ethical issues.
• These include whether a pregnancy should be terminated if a
debilitating disease is found in the fetus and whether health
insurance companies can deny coverage for individuals with a
genetic disease.
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The Potential of Gene Therapy
• Gene therapy is the introduction of a gene into affected cells to
replace or augment defective copies of the gene with normal alleles.
• In gene therapy, the healthy allele must be sequenced and well
understood, and then the DNA has to be introduced in a way that
ensures expression of the gene in the correct tissues, in the correct
amount, and at the correct time.
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Introducing Novel Alleles into Human Cells
• The current vector of choice in gene therapy are retroviruses.
– These are viruses with an RNA genome, including the enzyme
reverse transcriptase.
• If human genes are packaged into a retrovirus, the virus is capable
of inserting the human alleles into a chromosome in a target cell.
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Ethical Concerns over Gene Therapy
• Gene therapy is highly experimental, extremely expensive, and
intensely controversial.
• Although gene therapy holds great promise for the treatment of a
wide variety of devastating inherited diseases, fulfilling that
promise is almost certain to require many years of additional
research and testing, as well as the refinement of legal and ethical
guidelines.
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Biotechnology in Agriculture – Golden Rice
• Most strategies for genetic engineering in agriculture focus on one
of three objectives:
1. Reducing herbivore damage.
2. Making crops more resistant to herbicides.
3. Improving the quality of food products.
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Rice as a Target Crop
• Although half the world’s population depends on rice as its staple
food, this grain contains no vitamin A.
• Lack of vitamin A in the diet may cause blindness in children as
well as increased susceptibility to disease.
• However, rice does contain b-carotene, which is a precursor of
vitamin A.
• Scientists set out to develop rice enriched in b-carotene.
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Synthesizing b-Carotene in Rice
• The synthetic pathway for b-carotene has three enzymes.
• To produce transgenic rice strains capable of producing b-carotene,
the three genes that code for these enzymes had to be inserted into
rice plants.
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Using the Ti Plasmid to Produce Golden Rice
• To develop golden rice, researchers
modified Ti plasmids so that they
contained the genes for the three
enzymes needed to synthesize βcarotene. They then exposed plant
embryos to Agrobacterium cells
containing these genetically
modified Ti plasmids.
• A transgenic plant was produced
that is now called golden rice,
because its high concentration of bcarotene gives it a yellow color.
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Ethical Concerns
• Researchers and consumer advocates have expressed concerns
about the increasing numbers and types of genetically modified
foods available today.
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