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Genetic Engineering and Biotechnology

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Title: Genetic Engineering and Biotechnology


1
Genetic Engineering and Biotechnology
  • Topic 4.4

2
Recombinant DNA Technology
  • 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
    sourceseven 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.

3
Recombinant DNA Technology
  • 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.

4
Recombinant DNA Technology
  • 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.

5
Recombinant DNA Technology
  • 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.

6
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7
Restriction Enzymes
  • 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 cells own DNA is protected from
    restriction enzymes through chemical modification
    by other enzymes.

8
Restriction Enzymes
  • 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.

9
Restriction enzymes
  • Creating recombinant DNA using a restriction
    enzyme and DNA ligase (Figure 12.2)
  • 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 hydrogen-bonded base pairs with
    complementary single-stranded stretches of DNA.

10
Restriction Enzymes
  • 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.

11
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12
Cloning Genes in Recombinant Plasmids
  • 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.

13
Cloning Genes in Recombinant Plasmids
  • 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.

14
Cloning Genes in Recombinant Plasmids
  • 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.

15
Cloning Genes in Recombinant Plasmids
  • 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.

16
Cloning Genes in Recombinant Plasmids
  • 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.
  • In our example, the biologist will grow a cell
    clone large enough to produce protein V in
    marketable quantities.

17
Cloning Genes in Recombinant Plasmids
  • 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.

18
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19
Genomic Library
  • 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.
  • 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.

20
Genomic Library
21
Genomic Library
  • 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.

22
Reverse transcriptase
  • 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
    single-stranded 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.

23
Reverse transcriptase
  • 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.

24
Mass-Produced Gene Products
  • 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.

25
Mass-Produced Gene Products
  • 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)

26
Mass-Produced Gene Products
  • Although there are many advantages to using
    bacteria, it is sometimes desirable or necessary
    to use eukaryotic cells to produce a protein
    product.
  • 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.

27
Mass-Produced Gene Products
  • The cells of choice for making some gene products
    come from mammals.
  • 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.

28
Mass-Produced Gene Products
  • Recently, pharmaceutical researchers have been
    exploring the mass production of gene products by
    whole animals or plants rather than cultured
    cells.
  • 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 genes product is secreted in the
    animals milk.
  • Sheep are being used to carry a gene for a human
    blood protein that is a potential treatment for
    cystic fibrosis.

29
DNA technology and the pharmaceutical industry
and medicine
  • DNA technology and gene cloning are widely used
    to produce medicines and to diagnose disease
  • Therapeutic hormones
  • Human insulin and human growth hormone
  • Diagnonsis and Treatment of disease
  • Pinpoint genetic disease alleles
  • Diagnosis HIV
  • Vaccines
  • Hepatitis B

30
Nucleic Acid Probes
  • Often the most difficult task in gene cloning is
    finding the right shelf in a genomic
    librarythat is, identifying a bacterial or phage
    clone containing a desired gene from among all
    those created.
  • 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.

31
Nucleic Acid Probe
  • 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.

32
Nucleic Acid Probe
  • Refer to p. 238 Figure 12.8 for the procedure of
    how a probe works.

33
DNA Microarray
  • 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
  • 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.

34
DNA microarray
  • Refer to p. 238 Figure 12.9 for the procedure of
    DNA microarray

35
Gel Electrophoresis
  • 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.

36
Gel Electrophoresis
  • http//learn.genetics.utah.edu/content/labs/gel/

37
RFLPs
  • Unless you have an identical twin, your DNA is
    different from everyone elses 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.

38
RFLPs
  • 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)

39
RFLPs
  • How Restriction Fragments Reflect DNA Sequence
  • 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.

40
RFLPs
41
Forensic Science
42
Forensic Science
  • 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 victims 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.

43
Forensic Science
  • 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 suspects
    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.

44
Forensic Science
  • DNA fingerprinting can also be used to establish
    family relationships.
  • 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.

45
Forensic Science
  • 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.

46
Forensic Science
  • How reliable is DNA fingerprinting?
  • 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.

47
Forensic Science
DNA Fingerprints From a Murder Case
48
Forensic Science
  • http//www.pbs.org/wgbh/nova/sheppard/analyze.html

49
Gene Therapy
  • Techniques for manipulating DNA have the
    potential for treating a variety of diseases by
    gene therapy- alteration of an afflicted
    individuals genes.
  • Theoretically, people with disorders traceable to
    a single defective gene should be able to replace
    or supplement the gene with a normal allele.
  • 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 persons 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.

50
Gene Therapy
  • 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 patients life and
    produce the missing protein. The patient will be
    cured!

51
Gene Therapy
  • 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.

52
Gene Therapy
  • 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?

53
Gene Therapy
  • 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 genes
    insertion does not harm some other necessary cell
    function?

54
PCR
  • 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.
  • 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.

55
PCR
  • 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.
  • 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.

56
PCR
57
PCR
  • Devised in 1985, PCR has had a major impact on
    biological research and biotechnology.
  • 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.

58
Human Genome Project
  • 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

59
Human Genome Project
  • 1. Genetic (linkage) mapping
  • 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.

60
Human Genome Project
  • 2. Physical mapping
  • 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.

61
Human Genome Project
  • 3. DNA Sequencing
  • 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

62
Human Genome Project
  • 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.
  • Venters 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
    consortiums 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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Human Genome Project
  • The potential benefits of having a complete map
    of the human genome are great
  • 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, Alzheimers
    disease, and cancer.
  • Hundreds of disease-associated genes have already
    been identified as a result of the project.

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Human Genome Project
  • 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.
  • This challenge and the applications of the new
    knowledge should keep scientists busy well into
    the twenty-first century.

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Human Genome- not just genes!
  • 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?
  • 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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Human Genome- not just genes!
  • 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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Human Genome- not just genes!
  • 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 endscalled
    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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Human Genomenot just genes!
  • 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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Genomics
  • 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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Genomics
  • Why map so many genomes?
  • 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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Proteomics
  • 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
  • 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 proteinsthat 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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Genetically Modified Organisms
  • 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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Genetically Modified Organisms
  • 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.
  • 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 deficiencyand
    resulting blindnessamong the half of the worlds
    people who depend on rice as their staple food.

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Genetically Modified Organisms
  • Agricultural researchers are also making
    transgenic animals.
  • 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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Genetically Modified Organisms
  • Transgenic animals
  • 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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Genetically Modified Organisms
  • Social concerns
  • 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.
  • 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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Genetically Modified Organisms
  • 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
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Cloning
  • Cloning provides strong evidence that
    differentiated cells retain their full genetic
    potential.
  • Animal cloning is achieved through a procedure
    called nuclear transplantation.
  • 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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Cloning
  • Reproductive cloning
  • 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 nucleusa clone
    of the donor.
  • This type of cloning results in the birth of a
    new individual

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Cloning
  • Therapeutic cloning
  • 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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Cloning
  • 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 conditionssuch as the presence of
    certain growth factorscan 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 patients
    own cells.

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Cloning
  • 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 cells.

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