Download Genetic Engineering and Biotechnology

Topic 4.4
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Most DNA technology methods depend on bacteria,
more specifically E. coli.
In fact, research into the genetics of E.coli during the
1970s led to the development of recombinant DNA
technology, a set of laboratory techniques for combining
genes from different sources—even different species–
into a single DNA molecule.
It is now widely used to alter the genes of many types of
cells for practical purposes.
For example, scientists have genetically engineered
bacteria to mass-produce many useful chemicals, from
cancer drugs to pesticides. Furthermore, genes have been
transferred from bacteria into plants and from humans
to farm animals.
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To manipulate genes in the laboratory,
biologists often use bacterial plasmids, which
are small, circular DNA molecules that replicate
separately from the much larger bacterial
chromosome.
Because plasmids can carry virtually any gene
and replicate in bacteria, they are key tools for
gene cloning, the production of multiple
identical copies of a gene-carrying piece of
DNA.
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Overview of gene cloning:
 1. the procedure begins when a plasmid is isolated from a
bacterium and
 2.DNA carrying a gene of interest is obtained from another cell.
 The gene of interest could be, for instance, a human gene
encoding a protein of medical value or a plant gene conferring
resistance to pests.
 3. A piece of DNA containing the gene is inserted into the
plasmid. The resulting plasmid now consists of recombinant
DNA, DNA in which genes from two different sources are
combined in vitro into the same DNA molecule.
 4. Next, a bacterial cell takes up the plasmid through
transformation.
 5. This recombination bacterium then reproduces to form a clone
of cells (a group of identical cells descended from a single
ancestral cell), each carrying a copy of the gene.
 Cloned genes can be used directly or to manufacture protein products.
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Gene-cloning methods are central to genetic
engineering, the direct manipulation of genes
for practical purposes.
Genetic engineering has launched a revolution
in biotechnology, the use of organisms or their
components to make useful products.
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For the gene cloning procedure to occur, a piece of DNA
containing the gene of interest must be cut out of a
chromosome and “pasted” into a bacterial plasmid.
The cutting tools are bacterial enzymes called restriction
enzymes.
In nature, these enzymes protect bacterial cells against
intruding DNA from other organisms or viruses.
They work by chopping up the foreign DNA, a process
that restricts foreign DNA from surviving in the cell.
 The bacterial cell’s own DNA is protected from
restriction enzymes through chemical modification by
other enzymes.
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Hundreds of different restriction enzymes have
been identified and isolated. Each restriction
enzyme is very specific, recognizing a particular
short DNA sequence (usually four to eight
nucleotides long).
Once the DNA sequence is recognized, the
restriction enzyme cuts both DNA strands at
specific points within the sequence.
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Creating recombinant DNA using a restriction
enzyme and DNA ligase (Figure 12.2):
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1. we start with a piece of DNA containing one
recognition sequence for a particular restriction
enzyme from E.coli. In this case, the restriction
enzyme will cut the DNA strands between the bases
A and G within the sequence, producing pieces of
DNA called restriction fragments.
2. The staggered cuts yield two double-stranded DNA
fragments with single-stranded ends, called “sticky
ends.” Sticky ends are the key to joining DNA
restriction fragments originating from different
sources. These short extensions can form hydrogenbonded base pairs with complementary singlestranded stretches of DNA.
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3. a “foreign” piece of DNA from another source is now added. This
“foreign” piece of DNA has single-stranded ends identical in base
sequence to the sticky ends on the original DNA.
 The “foreign” DNA has ends with this particular base sequence
because it was cut from a larger molecule by the same restriction
enzyme used to cut the original DNA.
4. The complementary ends on the original and “foreign” fragments
allow them to stick together by base-pairing.
 The union between foreign and original DNA fragments is made
permanent by the “pasting” enzyme DNA ligase.
 This enzyme, which the cell normally uses in DNA replication,
catalyzes the formation of covalent bonds between adjacent
nucleotides, sealing the breaks in the DNA strands.
5. The final outcome is a stable molecule of recombinant DNA.
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Consider a typical genetic engineering challenge:
a molecular biologist at a pharmaceutical
company has identified a human gene that codes
for a valuable product: a hypothetical substance
called protein V that kills certain human viruses.
The biologist wants to set up a system for
making large amounts of the gene so that the
protein can by manufactured on a large scale.
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Steps to a way to make many copies of the gene using
the techniques of recombinant DNA technology:
1. The biologist isolates two kinds of DNA: the bacterial
plasmid that will serve as the vector (gene carrier), and the
human DNA containing gene V.
 In this example, the DNA containing the gene of interest
comes from human tissue cells that have been growing
in laboratory culture. The plasmid comes from the
bacterium E.coli.
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2. The researcher treats both the plasmid and the human
DNA with the same restriction enzyme.
An enzyme is chosen that cleaves the plasmid in only
one place.
The human DNA, with thousands of restriction sites, is
cut into many fragments, one of which carries gene V. In
making the cuts, the restriction enzyme creates sticky
ends on both the human DNA fragments and the
plasmid.
The figure on p. 234 shows the processing of just one
human DNA fragment and one plasmid, but actually
millions of plasmids and human DNA fragments (most
of which do not contain gene V) are treated
simultaneously.
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3. The human DNA is mixed with the cut
plasmid. The sticky ends of the plasmid base-pair
with the complementary sticky ends of the
human DNA fragment.
4. the enzyme DNA ligase joins the two DNA
molecules by covalent bonds, and the result is a
recombinant DNA plasmid containing gene V.
5. The recombinant plasmid is added to a
bacterium. Under the right conditions, the
bacterium takes up the plasmid DNA by
transformation.
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6. This step is the actual gene cloning. The
bacterium is allowed to reproduce, forming a
clone of cells that all carry the recombinant
plasmid.
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In our example, the biologist will grow a cell clone
large enough to produce protein V in marketable
quantities.
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This cloning procedure, which uses a mixture
of fragments from the entire genome of an
organism, is referred to as the “shotgun”
approach.
Thousands of different recombinant plasmids
are produced in step 3, and a clone of each is
made during steps 5 and 6.
The complete set of plasmid clones, each
carrying copies of a particular segment from
the initial genome, is a type of library.
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Each bacterial clone from the procedure we
previously discussed consists of identical cells
with recombinant plasmids carrying one
particular fragment of human DNA.
The entire collection of all the cloned DNA
fragments from a genome is called a genomic
library.
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Various DNA segments represent thousands of
“books” that are “shelved” in plasmids inside
bacterial cells.
A typical cloned DNA fragment is big enough to
carry one or a few genes, and together the fragments
include the entire genome of the organism from
which the DNA was derived.
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Bacterial plasmids are one type of vector that can be
used in the cloning of genes, but not the only type.
Phages can also serve as vectors.
When a phage is used, the DNA fragments are inserted
into phage DNA molecules. The recombinant phage
DNA can then be introduced into a bacterial cell through
the normal infection process.
Inside the cell, the phage DNA replicates and produces
new phage particles, each carrying the foreign DNA.
A collection of phage clones can constitute a second type
of genomic library.
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Rather than starting with an entire eukaryotic
genome, a researcher can focus on the genes
expressed in a particular kind of cell by using its
mRNA as the starting material.:
1. the chosen cells transcribe their genes and
 2. process transcripts to produce mRNA.
 3. the researcher isolates the mRNA and makes singlestranded DNA transcripts from it using the enzyme
reverse transcriptase, which is obtained from
retroviruses.
 4. enzymes are added to break down the mRNA and
 5. DNA polymerase is used to synthesize a second DNA
strand.
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Complementary DNA (cDNA) is the DNA that
results from this procedure.
It represents only the subset of genes that were
transcribed into mRNA in the starting cells.
 Among other purposes, a cDNA library is useful for
studying the genes responsible for the specialized
functions of a particular cell type, such as brain or
liver cells.
 And because cDNAs lack introns, they are shorter
than the full versions of the genes, and therefore
easier to work with.
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Recombinant cells and organisms constructed by
DNA technology are used to manufacture many
useful products, chiefly proteins.
Most of these products are made by cells grown
in culture.
By transferring the gene for a desired protein in
a bacterium, yeast, or other kind of cell that is
easy to grow, one can produce large quantities of
proteins that are present naturally in only
minute amounts.
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Bacteria are often the best organisms for
manufacturing a protein product.
Major advantages of bacteria include the plasmids and
phages available for use as gene-cloning vectors and
the fact that bacteria can be grown rapidly and
cheaply in large tanks.
 Furthermore, bacteria can be readily engineered to
produce large amounts of particular proteins and in
some cases to secrete the protein products into their
growth medium, which simplifies the task of
collecting and purifying the products.
 A number of proteins of importance in human
medicine and agriculture are made by E. coli (refer to
table 12.6 on p. 236)
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Although there are many advantages to using
bacteria, it is sometimes desirable or necessary to
use eukaryotic cells to produce a protein
product.
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Often times, the yeast Saccharomyces cerevisae, which
is used in making bread and beer, is the first-choice
eukaryotic organism for protein production.
Yeast are easy to grow, and can take up foreign DNA
and integrate it into their genomes like E.coli.
Also have plasmids that can be used as gene vectors,
and are often better than bacteria at synthesizing and
secreting eukaryotic proteins.
S.cerevisiae is currently used to produce a number of
proteins.
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The cells of choice for making some gene
products come from mammals.
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Genes fro these products are often cloned in bacteria
as a preliminary step.
For example, the genes for two proteins that affect
blood clotting, Factor VIII and TPA, are cloned in a
bacterial plasmid before transfer to mammalian cells
for large-scale production.
 Many proteins that mammalian cells secrete are
glycoproteins, proteins with chains of sugars attached.
 Because only mammalian cells can attach the sugars
correctly, mammalian cells must be used to make these
products.
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Recently, pharmaceutical researchers have
been exploring the mass production of gene
products by whole animals or plants rather
than cultured cells.
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For example, using recombinant DNA technology,
genetic engineers can add a gene for a desired
human protein to the genome of a mammal in such a
way that the gene’s product is secreted in the
animal’s milk.
Sheep are being used to carry a gene for a human
blood protein that is a potential treatment for cystic
fibrosis.
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DNA technology and gene cloning are widely
used to produce medicines and to diagnose
disease:
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Therapeutic hormones
 Human insulin and human growth hormone
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Diagnonsis and Treatment of disease
 Pinpoint genetic disease alleles
 Diagnosis HIV
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Vaccines
 Hepatitis B
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Often the most difficult task in gene cloning is
finding the right “shelf” in a genomic library—
that is, identifying a bacterial or phage clone
containing a desired gene from among all those
created.
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If bacterial clones containing a specific gene actually
translate the gene into protein, they can be identified
by testing for the protein product.
However, this is not always the case. Fortunately,
researchers can also test directly for the gene itself.
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Methods for detecting genes directly depend on base pairing
between the gene and a complementary sequence on another
nucleic acid molecule, either DNA or RNA.
When at least part of the nucleotide sequence of a gene is already
known or can be guessed, this information can be used to
advantage.
 For example, if we know that a hypothetical gene contains the
sequence TAGGCT, a biochemist can synthesize a short single
strand of DNA with the complementary sequence (ATCCGA)
and label it with a radioactive isotope or fluorescent dye.
 This labeled, complementary molecule is called a nucleic acid
probe because it is used to find a specific gene or other
nucleotide sequence within a mass of DNA.
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Refer to p. 238 Figure 12.8 for the procedure of
how a probe works.
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Besides hunting for one specific gene, nucleic
acid probes can be used to perform large-scale
analyses that determine which of many genes
are active (transcribed) in particular cells at
particular times.
This technique relies on DNA microarrays:
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DNA microarray is a glass slide carrying thousands of
different kinds of single-stranded DNA fragments
arranged in an array (grid).
Each DNA fragment is obtained from a particular
gene; a single microarray thus carries DNA from
thousands of genes.
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Refer to p. 238 Figure 12.9 for the procedure of
DNA microarray
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Gel electrophoresis is a technique that uses gel ( a thin slab of
jellylike material) as a molecular sieve to separate nucleic acids or
proteins on the basis of size or electrical charge.
How gel electrophoresis would be used to separate the various DNA
molecules in three different mixtures:
 A sample of each mixture is placed in a well at one end of a flat,
rectangular gel.
 A negatively charged electrode from a power supply is attached
near the DNA-containing end of the gel, and a positive electrode
is attached near the other end.
 Because DNA molecules have negative charge owing to their
phosphate groups, they all travel through the gel toward the
positive pole.
 As they move, a thicket of polymer fibers within the gel impedes
longer molecules more than it does shorter ones, separating them
by length.
 Thus, gel electrophoresis separates a mixture of linear DNA
molecules into bands, each consisting of DNA molecules of the
same length, with shorter molecules toward the bottom.
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http://learn.genetics.utah.edu/content/labs/g
el/
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Unless you have an identical twin, your DNA is
different from everyone else’s; its total nucleotide
sequence is unique.
Some of your DNA consists of genes, and even more of
it is composed of noncoding stretches of DNA.
Whether a segment of DNA codes for amino acids or
not, it is inherited just like any other part of a
chromosome. For this reason, geneticists can use any
DNA segment that varies from person to person as a
genetic marker, a chromosomal landmark whose
inheritance can be studied. And just like a gene, a
noncoding segment of DNA is more likely to be an exact
match to the comparable segment in a relative than to
the segment in an unrelated individual.
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Restriction fragment analysis is a method for
detecting differences in nucleotide sequence
between homologous samples of DNA, usually
from two different individuals.
In restriction fragment analysis, two of the
methods we have discussed are used in succession:
DNA fragments produced by restricted enzymes
are sorted by gel electrophoresis. ***The number of
restriction fragments and their sizes reflect the specific
sequence of nucleotides in the starting DNA.
The differences in restriction fragments produced
in this way are called restriction fragment length
polypmorphisms (RFLPs, produced “rif-lips”)
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How Restriction Fragments Reflect DNA
Sequence
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For example, if a forensic scientists were trying to
identify a match between two DNA samples: one
obtained from a crime scene and one obtained from a
suspect.
To detect the differences between the collections of
restriction fragments, we need to separate the restriction
fragments in the two mixtures and compare their
lengths.
We can accomplish these things through gel
electrophoresis.
Then you can compare the bands, and check the
similarities and differences between the base sequences
in DNA from two individuals.
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Forensic science is the scientific analysis of evidence for
crime scene and other legal investigations, and DNA
technology now plays an important role.
In violent crimes, body fluids or small pieces of tissue
may be left at the crime scene or on the clothes of the
victim or assailant.
If rape has occurred, semen may be recovered from the
victim’s body.
With enough tissue or semen, forensic scientists can
determine the blood type or tissue type using older
methods that test for proteins.
However, such tests require fresh samples in relative
large amounts.
Also, because many people have the same blood or
tissue type , this approach can only exclude a suspect; it
cannot provide strong evidence of guilt.
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DNA testing can identify the guilty individual with a high degree of
certainty because the DNA sequence of every person is unique (except
for identical twins).
RFLP analysis is one major type of DNA testing .
It is a powerful method for comparing DNA samples and requires
only about 1,000 cells.
In a murder case, for example, such analysis can be used to compare
DNA samples from the suspect, the victim, and bloodstains on the
suspect’s clothes.
Radioactive probes mark the electrophoresis bands that contain
certain markers.
Usually about a dozen markers are tested; in other words, only a few
selected portions of DNA are compared.
However, even such a small set of markers from an individual can
provide a DNA fingerprint, or specific pattern of bands, that is of
forensic use, because the pattern of bands, that is of forensic use,
because the probability that two people would have exactly the same
set of markers is very small.
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DNA fingerprinting can also be used to
establish family relationships.
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A comparison of the DNA or a mother, her child,
and the purported father can conclusively settle a
question of paternity.
Sometimes paternity is of historical interest: DNA
fingerprinting provide strong evidence that Thomas
Jefferson or one of his close male relatives fathered at
least one child with his slave Sally Hemings.
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Today, the markers most often used in DNA
fingerprinting are inherited variations in the
lengths of repetitive DNA.
These repetitive sequences are highly variable from
person to person, providing even more markers than
RFLPs.
 For example, one person may have nucleotides ACA
repeated 65 times at one genome locus and 118 times
at a second locus, whereas another person is likely to
have different numbers of repeats at these loci.
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How reliable is DNA fingerprinting?
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In most legal cases, the probability of two people
having identical DNA fingerprints is between one
chance in 10,000 and one in a billion. The exact figure
depends on how many markers are in the population.
For this reason, DNA fingerprints are now accepted as
compelling evident by legal experts and scientists
alike.
In fact, DNA analysis on stored forensic samples has
provided the evidence needed to solve many “cold
cases” in recent years. DNA fingerprinting has also
exonerated many wrongly convicted people, some of
whom were on death row.
DNA Fingerprints From a Murder Case
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http://www.pbs.org/wgbh/nova/sheppard/
analyze.html
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Techniques for manipulating DNA have the
potential for treating a variety of diseases by gene
therapy- alteration of an afflicted individual’s
genes.
Theoretically, people with disorders traceable to a
single defective gene should be able to replace or
supplement the gene with a normal allele.
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The new allele could be inserted into somatic cells of the
tissue affected by the disorder
To be permanent, the normal allele would have to be
transferred to cells that multiply throughout a person’s
life.
 Bone marrow cells, which include the stem cells that give
rise to all the cells of the blood and immune system, are
primate candidates.
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One possible procedure for gene therapy in an individual
whose bone marrow cells do not produce a vital protein
product because of a defective gene:
 1. The normal gene is cloned and then inserted into the
nucleic acid of a retrovirus vector that has been
rendered harmless.
 2. Bone marrow cells are taken from the patient and
infected with the virus.
 3. the virus inserts its nucleic acid, including the
human gene, in the cells’ DNA.
 4. The engineered cells are then injected back into the
patient.
 *If the procedure succeeds, the cells will multiply
throughout the patient’s life and produce the missing
protein. The patient will be cured!
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Although the concept of gene therapy remains
promising, very little scientifically strong
evidence of effective gene therapy has yet
appeared.
Active research into human gene therapy, with
new, tougher safety guidelines, continues.
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Human gene therapy raises both techinical and ethical issues.
Ethical issues:
 Who will have access to it? The procecures now being tested are
expensive and require expertise and equipment found only in
major medical centers.
 Should gene therapy be reserved for treating serious diseases?
 And, what about its potential use for enhancing athletic ability,
physical appearance, and even intelligence?
 Should we try to eliminate genetic defects in children and their
descendants?
 From a biological perspective, the elimination of unwanted
alleles from the gene pool could backfire.
 Genetic variation is a necessary ingredient for the survival of a
species as environmental conditions change with time.
 Genes that are damaging under some conditions may be
advantageous under others (one example is the sickle-cell allele)
 Are we willing to risk making genetic changes that could be
detrimental to our species in the future?
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Technical issues:
 How can researchers build in gene control mechanisms to
ensure that cells with the transferred gene make appropriate
amounts of the gene product at the right time and in the right
parts of the body?
 And how can they be sure that the gene’s insertion does not
harm some other necessary cell function?
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DNA cloning in cells is often the best method for
preparing large quantities of a particular gene.
However, when the source of DNA is scanty or
impure, the polymerase chain reaction (PCR) is a
much better method.
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In this technique, any specific target segment within a
DNA molecule can be quickly amplified (copied many
times) in a test tube.
Starting with a single DNA molecule, automated PCR
can generate 100 billion similar molecules in a few
hours.
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PCR, in principle, is simple.
 A DNA sample is mixed with the DNA replication enzyme DNA
polymerase, nucleotide monomers, and a few other ingredients.
 The solution is then exposed to cycles of heating (to separate the
DNA strands) and cooling.
 During each cycle, the DNA is replicated, doubling the amount of
DNA.
 For PCR to work, only minute amounts of DNA need be present
in the starting material, and this DNA can be in a partially
degraded state.
 From such a scant starting sample, PCR can produce enough DNA for
restriction fragment analysis or other DNA technologies.
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However, occasional errors during PCR replication impose limits
on the number of good copies that can be made by this method.
 So, PCR cannot replace gene cloning in cells when large amounts of
DNA are needed.
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Devised in 1985, PCR has had a major impact
on biological research and biotechnology.
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It has been used to amplify DNA from a wide variety
of sources:
 fragments of ancient DNA from a 40,000 year old frozen
woolly mammoth
 DNA from fingerprints or from tiny amounts of blood,
tissue, or semen found at crime scenes
 DNA from single embryonic cells for rapid prenatal
diagnosis of genetic disorders
 DNA of viral genes from cells infected with such
difficult-to-detect viruses such as HIV.
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The Human Genome Project (HGP) is an effort to map the human
genome in total detail by determining the entire nucleotide
sequence of human DNA.
Begun in 1990, this ambitious project was expected to take 15 years
but was largely finished several years ahead of schedule.
The project was organized by an international, publicly funded
consortium of researchers and proceeded through three stages that
provided progressively more detailed views of the human genome:
 1. Genetic (linkage) mapping
 2. Physical mapping
 3.DNA sequencing
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1. Genetic (linkage) mapping
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Geneticists combined pedigree analysis of large
families with DNA technology to map over 5,000
genetic markers.
The resulting low-resolution linkage map provided a
framework for mapping other markers and for
arranging later, more detailed maps of particular
regions.
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2. Physical mapping
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To create a physical map, researchers determined the
number of base pairs between markers.
This is done by cutting the DNA of each chromosome
into a number of restriction fragments, cloning them,
and then figuring out the original order of the
fragments.
The key is to make fragments that overlap and then
use probes or automated nucleotide sequencing of
the ends to find overlaps. In this way, more and more
fragments can be assigned to a sequential order that
corresponds to their order in a chromosome.
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3. DNA Sequencing
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The most arduous part of the project is determining
the nucleotide sequences of a set of DNA fragments
covering the entire genome, the fragments already
mapped in stage 2.
Advances in automatic DNA sequencing have been
crucial to this endeavor. Sequencing machines can
handle DNA molecules up to about 800 nucleotides
in length
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This three-stage approach is logical and thorough.
However, in the mid 1990s, J. Craig Venter, a former government
scientist, proposed an alternative strategy and set up the company
Celera Genomics to implement it.
Venter’s “whole genome shotgun” approach was essentially to
proceed directly to the sequencing of small, random DNA
fragments, relying on software to determine the order of the pieces.
Celera actually made significant use of the consortium’s data from
stages 1 and 2, but the competition between the two groups
hastened the progress.
In February 2001, Celera announced the sequencing of over 90% of
the human genome.
At the same time, HGP researchers made a similar announcement.
Sequencing of the human genome is now virtually complete,
although some gaps remain to be mapped because certain parts of
the chromosomes resist mapping by the usual methods.
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The potential benefits of having a complete map
of the human genome are great:
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For basic science, the info is already providing insight
into such fundamental mysteries as embryonic
development and evolution.
For human health, the identification of genes will aid
in the diagnosis, treatment, and possibly prevention of
many of our more common ailments, including heart
disease, allergies, diabetes, schizophrenia, alcoholism,
Alzheimer’s disease, and cancer.
Hundreds of disease-associated genes have already
been identified as a result of the project.
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The DNA sequences from the HGP are
deposited in a database available to researchers
all over the world via the Internet.
Scientists use software to analyze the sequences
Then comes the most exciting challenge:
figuring out the functions of the genes and how
they work together to direct the structure and
function of a living organism.
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This challenge and the applications of the new
knowledge should keep scientists busy well into the
twenty-first century.
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The biggest surprise from the HGP is the small
number of human genes. The current estimate
is about 20,000 – 25,000 genes, only one and a
half to two times the number found in the fruit
fly and nematode worm.
How, then, to account for human complexity?
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Part of the answer may lie in alternative RNA
splicing scientists think that a typical human gene
probably specifies several polypeptides.
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In addition to genes, humans, like most complex
eukaryotes, have a huge amount of noncoding
DNA, about 97% of the total.
Some noncoding DNA is made up of gene control
sequences such as promoters and enhancers.
 The remaining DNA includes introns (whose total
length may be ten times greater than the exons of a
gene) and noncoding DNA located between genes.
 Much of the DNA between genes consists of
repetitive DNA, nucleotide sequences present in
many copies in the genome.
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In one type of repetitive DNA, a unit of just a few
nucleotide pairs is repeated many times in a row.
Stretches of DNA with thousands of such repetitions are
prominent at the centromeres and ends of chromosomes,
suggesting that this DNA plays a role in chromosome
structure.
Recent research supports the idea that the repetitive DNA
at chromosome ends—called telomeres– also have a
protective function; a significant loss of telomeric DNA
quickly leads to cell death.
Furthermore, abnormal lengthening of this DNA may help
“immortal” cancer cells evade normal cell aging.
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In the second main type of repetitive DNA, each
repeated unit is hundreds of nucleotides long,
and the copies are scattered around the genome.
Most of these sequences seem to be associated
with transposons (“jumping genes”), DNA
segments that can move or be copied from one
location to another in a chromosome and even
between chromosomes.
Transposons can land in the middle of other
genes and disrupt them. Reasearchers believe
that transposons, through their copy-and-paste
mechanism, are responsible for the proliferation
of dispersed repetitive DNA in the human
genome.
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Now that sequences of many entire genomes
are available, scientists can study whole sets of
genes and their interactions, an approach called
genomics.
Genomics is yielding new insights into
fundamental questions about genome
organization, regulation of gene expression,
growth and development, and evolution.
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Why map so many genomes?
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Comparative analysis with the genes of other species
also helps scientists interpret the human genome.
Also allows us to evaluate the evolutionary
relationships between those species.
 The more similar in sequence, the more closely related
those species are by their evolutionary history.
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The success in sequencing genomes and studying whole
genomes is encouraging scientists to attempt similar
systematic study of the full protein sets (proteomes)
encoded by genomes, an approach called proteomics.
The number of proteins in humans far exceeds the
number of genes.
And since proteins, not genes, actually carry out the
activities of the cell, scientists must study when and
where proteins are produced in an organism and how
they interact in order to understand the functioning of
cells and organisms.
Assembling and analyzing proteomes pose many
experimental challenges, but ongoing advances are
providing the tools to continue the investigation.
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Genomics and proteomics are enabling biologists
to approach the study of life from an
increasingly global perspective.
Biologists are now in a position to compile
catalogs of genes and proteins—that is, a listing
of all the “parts” that contribute to the operation
of cells, tissues, and organisms.
With such catalogs in hand, researchers are
shifting their attention from the individual parts
to how they function together in biological
systems.
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Scientists concerned with feeding the growing human
population are using DNA technology to make
genetically modified organisms for use in agriculture.
A GM organism (GMO) is one that has acquire one or
more genes by artificial means rather than by
traditional breeding methods. (The new gene may or
may not be from another species).
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To make genetically modified plants, researchers can
manipulate the DNA of a single somatic cell and then
grow a plant with a new trait from the engineered cell.
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Already in commercial use are a number of crop plants carrying
new genes for desirable traits, such as delayed ripening and
resistance to spoilage and disease.
The majority of the American soybean and cotton crops are
genetically modified.
Many plants have received bacterial genes that make them
resistant to herbicides.
Health benefits include “Golden rice” which produces grains
containing beta-carotene, which our body used to make vitamin
A.
 This could help prevent Vitamin A deficiency—and resulting
blindness—among the half of the world’s people who depend on rice
as their staple food.
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Agricultural researchers are also making
transgenic animals.
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To do this, scientists first remove egg cells from a
female and fertilize them in vitro.
They then inject a previously cloned gene directly into
the nuclei of the fertilized eggs.
Some of the cells integrate the foreign DNA into their
genomes.
The engineered embryos are then surgically
implanted in a surrogate mother.
If an embryo develops successfully, the result is a
transgenic animal, containing a gene from a third
“parent” that may even be of another species.
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Transgenic animals
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The goal is, for example, to make sheep with better
quality wool or a cow that will mature in a shorter
time.
Scientists might identify and clone a gene that causes
the development of larger muscles (which make up
most of the meat we eat) in one variety of cattle and
transfer it to other cattle or even sheep.
Also may be used as pharmaceutical “factories” to
produce otherwise rare biological substances for
medical use
 For example, manipulating chicken eggs.
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Social concerns:
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Early concerns focused on the possibility that
recombinant DNA technology might create new
pathogens.
 One safety measure is a set of strict laboratory
procedures designed to protect researchers from infection
by engineered microbes and to prevent the microbes
from accidentally leaving the laboratory.
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Today, most public concern about possible hazards
centers not on recombinant microbes but on
genetically modified (GM) crops.
 Advocates of a cautious approach fear that some crops
carrying genes from other species might be hazardous to
human health or the environment.
 One specific concern is that genetic engineering could
transfer allergens to plants people eat.
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Today, governments and regulatory agencies
throughout the world are grappling with how
to facilitate the use of biotechnology in
agriculture, industry, and medicine while
ensuring that new products and procedures are
safe.
In the US, all projects are evaluated for
potentials risks by regulatory agencies such as
the FDA, EPA, and NIH, and Department of
Agriculture.
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Cloning provides strong evidence that
differentiated cells retain their full genetic
potential.
Animal cloning is achieved through a procedure
called nuclear transplantation.
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Involves replacing the nucleus of an egg cell or zygote
with the nucleus of adult somatic cell.
The egg cell may then begin to divide.
About 5 days later, repeated cell divisions form a
blastocyst, a ball of cells.
At this point, the blastocyst may be used for different
purposes.
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Reproductive cloning
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If the animal to be cloned is a mammal, further
development requires implanting the blastocyst into
the uterus of a surrogate mother.
The resulting animal will be genetically identical to
the donor of the nucleus—a “clone” of the donor.
This type of cloning results in the birth of a new
individual
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Therapeutic cloning
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Embryonic stem cells (ES cells) are harvested from
the blastocyst.
In nature, embryonic stem cells give rise to all the
different kinds of specialized cells of the body.
In the laboratory, embryonic stem cells are easily
grown in culture, where, given the right conditions,
they can perpetuate themselves indefinitely.
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Therapeutic cloning applications:
 Therapeutic cloning produces ES cells that in the early animal
embryo differentiate to give rise to all the cell types in the body.
 When grown in laboratory culture, ES cells can divide
indefinitely (like cancer cells)
 But the right conditions—such as the presence of certain growth
factors—can induce changes in gene expression that cause
differentiation into a particular cell type.
 If scientists can discover the right conditions, they will be able to
grow cells for the repair of injured or diseased organs.
 Such cells could be made by inserting a cell nucleus from a
patient into an ES cell from which the nucleus has been removed.
 When implanted in the patient, these cells would not be rejected
by the immune system because they would be genetically
identical to the patient’s own cells.
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ES cells raise both ethical and technical problems.
 Human ES cells must be obtained by destroying human embryos
(such as ones donated by patients undergoing infertility treatment).
 This might be avoided by using adult stem cells, cells present in
adult tissues that generate replacements for nondividing
differentiated cells.
 Unlike ES cells, adult stem cells are part way along the road to
differentiation.
 They can often give rise to multiple types of specialized cells, but
it is not clear whether they can give rise to all types of cells.
 Like ES cells, adult stem cells can be grown in culture and
induced to differentiate into a range of cell types.
 For example, adult stem cells in bone marrow generate all types
of blood cells.
 Perhaps adult stem cells, ethically less problematic to obtain than
ES cells, may provide the answer to human tissue and organ
replacement.
 However, ES cells are currently more promising than adult stem