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Recombinant DNA and Biotechnology


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Title: Recombinant DNA and Biotechnology

Recombinant DNA and Biotechnology
Recombinant DNA and Biotechnology
  • Cleaving and Rejoining DNA
  • Getting New Genes into Cells
  • Sources of Genes for Cloning
  • Some Additional Tools for DNA Manipulation
  • Biotechnology Applications of DNA Manipulation

Cleaving and Rejoining DNA
  • Recombinant DNA technology is the manipulation
    and combination of DNA molecules from different
  • Recombinant DNA technology uses the techniques of
    sequencing, rejoining, amplifying, and locating
    DNA fragments, making use of complementary base

Cleaving and Rejoining DNA
  • Bacteria defend themselves against invasion by
    viruses by producing restriction enzymes which
    catalyze the cleavage of DNA into small
  • The enzymes cut the bonds between the 3 hydroxyl
    of one nucleotide, and the 5 phosphate of the
  • There are many such enzymes, each of which
    recognizes and cuts a specific sequence of bases,
    called a recognition sequence or restriction site
    (4 to 6 base pairs long).

Figure 16.1 Bacteria Fight Invading Viruses with
Restriction Enzymes
Cleaving and Rejoining DNA
  • Host DNA is not damaged due to methylation of
    certain bases at the restriction sites this is
    performed by enzymes called methylases.
  • The enzyme EcoRI cuts DNA with the following
    paired sequence
  • 5 ... GAATTC ... 3
  • 3 ... CTTAAG ... 5
  • Notice that the sequence is palindromic It reads
    the same in the 5-to-3 direction on both

Cleaving and Rejoining DNA
  • Using EcoRI on a long stretch of DNA would create
    fragments with an average length of 4,098 bases.
  • Using EcoRI to cut up small viral genomes may
    result in only a few fragments.
  • For a eukaryotic genome with tens of millions of
    base pairs, the number of fragments will be very
  • Hundreds of restriction enzymes have been
    purified from various organisms, and these
    enzymes serve as knives for genetic surgery.

Cleaving and Rejoining DNA
  • The fragments of DNA can be separated using gel
    electrophoresis. Because of its phosphate groups,
    DNA is negatively charged at neutral pH.
  • When DNA is placed in a semisolid gel and an
    electric field is applied, the DNA molecules
    migrate toward the positive pole.
  • Smaller molecules can migrate more quickly
    through the porous gel than larger ones.
  • After a fixed time, the separated molecules can
    then be stained with a fluorescent dye and
    examined under ultraviolet light.

Figure 16.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 1)
Figure 16.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 2)
Figure 16.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 3)
Cleaving and Rejoining DNA
  • Electrophoresis gives two types of information
  • Size of the DNA fragments can be determined by
    comparison to DNA fragments of known size added
    to the gel as a reference.
  • A specific DNA sequence can be determined by
    using a complementary labeled single-stranded DNA
  • The specific fragment can be cut out as a lump of
    gel and removed by diffusion into a small volume
    of water.

Figure 16.3 Analyzing DNA Fragments
Cleaving and Rejoining DNA
  • Some restriction enzymes cut DNA strands and
    leave staggered ends of single-stranded DNA, or
    sticky ends, that attract complementary
  • If two different DNAs are cut so each has sticky
    ends, fragments with complementary sticky ends
    can be recombined and sealed with the enzyme DNA
  • These simple techniques, which give scientists
    the power to manipulate genetic material, have
    revolutionized biological science in the past 30

Figure 16.4 Cutting and Splicing DNA
Getting New Genes into Cells
  • The goal of recombinant DNA work is to produce
    many copies (clones) of a particular gene.
  • To make protein, the genes must be introduced, or
    transfected, into a host cell.
  • The host cells or organisms, referred to as
    transgenic, are transfected with DNA under
    special conditions.
  • The cells that get the DNA are distinguished from
    those that do not by means of genetic markers,
    called reporter genes.

Getting New Genes into Cells
  • Bacteria have been useful as hosts for
    recombinant DNA.
  • Bacteria are easy to manipulate, and they grow
    and divide quickly.
  • They have genetic markers that make it easy to
    select or screen for insertion.
  • They have been intensely studied and much of
    their molecular biology is known.

Getting New Genes into Cells
  • Bacteria have some disadvantages as well.
  • Bacteria lack splicing machinery to excise
  • Protein modifications, such as glycosylation and
    phosphorylation, fail to occur as they would in a
    eukaryotic cell.
  • In some applications, the expression of the new
    gene in a eukaryote (the creation of a transgenic
    organism) is the desired outcome.

Getting New Genes into Cells
  • Saccharomyces, bakers and brewers yeast, are
    commonly used eukaryotic hosts for recombinant
    DNA studies.
  • In comparison to many other eukaryotic cells,
    yeasts divide quickly, they are easy to grow, and
    have relatively small genomes (about 20 million
    base pairs).

Getting New Genes into Cells
  • Plants are also used as hosts if the goal is to
    make a transgenic plant.
  • It is relatively easy to regenerate an entire
    plant from differentiated plant cells because of
    plant cell totipotency.
  • The transgenic plant can then reproduce naturally
    in the field and will carry and express the gene
    on the recombinant DNA.

Getting New Genes into Cells
  • New DNA can be introduced into the cells genome
    by integration into a chromosome of the host
  • If the new DNA is to be replicated, it must
    become part of a segment of DNA that contains an
    origin of replication called a replicon, or
    replication unit.

Getting New Genes into Cells
  • New DNA can be incorporated into the host cell by
    a vector, which should have four characteristics
  • The ability to replicate independently in the
    host cell
  • A recognition sequence for a restriction enzyme,
    permitting it to form recombinant DNA
  • A reporter gene that will announce its presence
    in the host cell
  • A small size in comparison to host chromosomes

Getting New Genes into Cells
  • Plasmids are ideal vectors for the introduction
    of recombinant DNA into bacteria.
  • A plasmid is small and can divide separately from
    the hosts chromosome.
  • They often have just one restriction site, if
    any, for a given restriction enzyme.
  • Cutting the plasmid at one site makes it a linear
    molecule with sticky ends.
  • If another DNA is cut with the same enzyme, it is
    possible to insert the DNA into the plasmid.
  • Plasmids often contain antibiotic resistance
    genes, which serve as genetic markers.

Figure 16.5 (a) Vectors for Carrying DNA into
Getting New Genes into Cells
  • Only about 10,000 base pairs can be inserted into
    plasmid DNA, so for most eukaryotic genes a
    vector that accommodates larger DNA inserts is
  • For inserting larger DNA sequences, viruses are
    often used as vectors.
  • If the genes that cause death and lysis in E.
    coli are eliminated, the bacteriophage l can
    still infect the host and inject its DNA.
  • The deleted 20,000 base pairs can be replaced by
    DNA from another organism, creating recombinant
    viral DNA.

Getting New Genes into Cells
  • Bacterial plasmids are not good vectors for yeast
    hosts because prokaryotic and eukaryotic DNA
    sequences use different origins of replication.
  • A yeast artificial chromosome, or YAC, has been
    made that has a yeast origin of replication, a
    centromere sequence, and telomeres, making it a
    true eukaryotic chromosome.
  • YACs have been engineered to include specialized
    single restriction sites and selectable markers.
  • YACs can accommodate up to 1.5 million base pairs
    of inserted DNA.

Figure 16.5 (b) Vectors for Carrying DNA into
Getting New Genes into Cells
  • Plasmid vectors for plants include a plasmid
    found in the Agrobacterium tumefaciens bacterium,
    which causes the tumor-producing disease, crown
    gall, in plants.
  • Part of the tumor-inducing (Ti) plasmid of A.
    tumefaciens is T DNA, a transposon, which inserts
    copies of itself into the host chromosomes.
  • If T DNA is replaced with the new DNA, the
    plasmid no longer produces tumors, but the
    transposon still can be inserted into the host
    cells chromosomes.
  • The plant cells containing the new DNA can be
    used to generate transgenic plants.

Figure 16.5 (c) Vectors for Carrying DNA into
Getting New Genes into Cells
  • When a population of host cells is treated to
    introduce DNA, just a fraction actually
    incorporate and express it.
  • In addition, only a few vectors that move into
    cells actually contain the new DNA sequence.
  • Therefore, a method for selecting for transfected
    cells and screening for inserts is needed.
  • A commonly used approach to this problem is
    illustrated using E. coli as hosts, and a plasmid
    vector with genes for resistance to two

Figure 16.6 Marking Recombinant DNA by
Inactivating a Gene
Getting New Genes into Cells
  • Other methods have since been developed for
  • The gene for luciferase, the enzyme that makes
    fireflies glow in the dark, has been used as a
    reporter gene.
  • Green fluorescent protein, which is the product
    of a jellyfish gene, glows without any required
  • Cells with this gene in the plasmid grow on
    ampicillin and glow when exposed to ultraviolet

Sources of Genes for Cloning
  • Gene libraries contain fragments of DNA from an
    organisms genome.
  • Restriction enzymes are used to break chromosomes
    into fragments, which are inserted into vectors
    and taken up by host cells.

Figure 16.7 Construction of a Gene Library
Sources of Genes for Cloning
  • Using plasmids for insertion of DNA, about one
    million separate fragments are required for the
    human genome library.
  • Phage l, which carries four times as much DNA as
    a plasmid, is used to hold these random
  • It takes about 250,000 different phage to ensure
    a copy of every sequence.
  • This number seems large, but just one growth
    plate can hold as many as 80,000 phage colonies.

Sources of Genes for Cloning
  • A smaller DNA library can be made from
    complementary DNA (cDNA).
  • Oligo dT primer is added to mRNA tissue where it
    hybridizes with the poly A tail of the mRNA
  • Reverse transcriptase, an enzyme that uses an RNA
    template to synthesize a DNARNA hybrid, is then
  • The resulting DNA is complementary to the RNA and
    is called cDNA. DNA polymerase can be used to
    synthesize a DNA strand that is complementary to
    the cDNA.

Figure 16.8 Synthesizing Complementary DNA
Sources of Genes for Cloning
  • If the amino acid sequence of a protein is known,
    it is possible to synthesize a DNA that can code
    for the protein.
  • Using the knowledge of the genetic code and known
    amino acid sequences, the most likely base
    sequence for the gene may be found.
  • Often sequences are added to this sequence to
    promote expression of the protein.
  • Human insulin has been manufactured using this

Sources of Genes for Cloning
  • With synthetic DNA, mutations can be created and
  • Additions, deletions, and base-pair substitutions
    can be manipulated and tracked.
  • The functional importance of certain amino acid
    sequences can be studied.
  • The signals that mark proteins for passage
    through the ER membrane were discovered by
    site-directed mutagenesis.

Some Additional Tools for DNA Manipulation
  • Homologous recombination is used to study the
    role of a gene at the level of the organism.
  • In a knockout experiment, a gene inside a cell is
    replaced with an inactivated gene to determine
    the inactivated genes effect.
  • This technique is important in determining the
    roles of genes during development.

Figure 16.9 Making a Knockout Mouse (Part 1)
Figure 16.9 Making a Knockout Mouse (Part 2)
Some Additional Tools for DNA Manipulation
  • The emerging science of genomics has to contend
    with two difficulties
  • The large number of genes in eukaryotic genomes
  • The distinctive pattern of gene expression in
    different tissues at different times
  • To find these patterns, DNA sequences have to be
    arranged in an array on some solid support.
  • DNA chip technology provides these large arrays
    of sequences for hybridization.

Figure 16.10 DNA on a Chip
Some Additional Tools for DNA Manipulation
  • Analysis of cellular mRNA using DNA chips
  • In a process called RT-PCR, cellular mRNA is
    isolated and incubated with reverse transcriptase
    (RT) to make complementary DNA (cDNA). The cDNA
    is amplified by PCR prior to hybridization.
  • The amplified cDNA is coupled to a fluorescent
    dye and then hybridized to the chip.
  • A scanner detects glowing spots on the array. The
    combinations of these spots differ with different
    types of cells or different physiological states.

Some Additional Tools for DNA Manipulation
  • DNA chip technology can be used to detect genetic
    variants and to diagnose human genetic diseases.
  • Instead of sequencing the entire gene, it is
    possible to make a chip with 20-nucleotide
    fragments including every possible mutant
  • Hybridizing that sequence with a persons DNA may
    reveal whether any of the DNA hybridized to a
    mutant sequence on the chip.

Some Additional Tools for DNA Manipulation
  • Base-pairing rules can also be used to stop mRNA
  • Antisense RNA is complementary to a sequence of
  • The antisense RNA forms a double-stranded hybrid
    with an mRNA, which inhibits translation.
  • These hybrids are broken down rapidly in the
    cytoplasm, so translation does not occur.
  • In the laboratory, antisense RNA can be made and
    added to cells to block translation.

Figure 16.11 Using Antisense RNA and RNAi to
Block Translation of mRNA
Some Additional Tools for DNA Manipulation
  • A related technique uses interference RNA (RNAi)
    which inhibits mRNA translation in the inactive X
    chromosome of mammals.
  • Scientists can synthesize a small interfering RNA
    (siRNA) to inhibit translation of any known gene.

Some Additional Tools for DNA Manipulation
  • The two-hybrid system allows scientists to test
    for protein interactions within a living cell.
  • A two-hybrid system uses a transcription factor
    that activates the transcription of an easily
    detectable reporter gene.
  • This transcription factor has two domains one
    that binds to DNA at the promoter, and another
    that binds to the transcription complex to
    activate transcription.
  • An example is the yeast two-hybrid system.

Figure 16.12 The Two-Hybrid System
Biotechnology Applications of DNA Manipulation
  • Biotechnology is the use of microbial, plant, and
    animal cells to produce materialssuch as foods,
    medicines, and chemicalsthat are useful to
  • The use of yeast to create beer and wine and
    bacterial cultures to make yogurt and cheese are
    examples of centuries-old biotechnology.
  • Gene cloning techniques of modern molecular
    biology have vastly increased the number of these
    products beyond those that are naturally made by

Biotechnology Applications of DNA Manipulation
  • Expression vectors are typical vectors, but they
    also have extra sequences needed for the foreign
    gene to be expressed in the host cell.
  • An expression vector might have an inducible
    promoter, which can be stimulated into expression
    by responding to a specific signal such as a
  • A tissue-specific promoter is expressed only in a
    certain tissue at a certain time.
  • Targeting sequences are sometimes added to direct
    the protein product to an appropriate destination.

Figure 16.13 An Expression Vector Allows a
Foreign Gene to Be Expressed in a Host Cell
Biotechnology Applications of DNA Manipulation
  • Many medical products have been made using
    recombinant DNA technology.
  • For example, tissue plasminogen activator (TPA),
    is currently being produced in E. coli by
    recombinant DNA techniques.
  • TPA is an enzyme that converts blood plasminogen
    into plasmin, a protein that dissolves clots.
  • Recombinant DNA technology has made it possible
    to produce the naturally occurring protein in
    quantities large enough to be medically useful.

Figure 16.14 Tissue Plasminogen Activator From
Protein to Gene to Drug
Table 16.1 Some Medically Useful Products of
Biotechnology Applications of DNA Manipulation
  • Selective breeding has been used for centuries to
    improve plant and animal species to meet human
  • Molecular biology is accelerating progress in
    these applications.
  • There are three major advantages over traditional
  • Specific genes can be affected.
  • Genes can be introduced from other organisms.
  • Plants can be regenerated much more quickly by
    cloning than by traditional breeding.

Biotechnology Applications of DNA Manipulation
  • Insecticides tend to be nonspecific, killing both
    pest and beneficial insects. They can also be
    blown or washed away to contaminate and pollute
    non-target sites.
  • Bacillus thuringiensis bacteria produce a protein
    toxin that kills insect larvae pests and is
    80,000 times more toxic than the typical chemical
  • Transgenic tomato, corn, potato, and cotton
    plants have been made that produce a toxin from
    B. thuringiensis.

Biotechnology Applications of DNA Manipulation
  • The process of producing pharmaceuticals using
    agriculture is nicknamed pharming.
  • Transgenic sheep are being used to produce human
    a-1-antitrypsin (a-1-AT) in their milk this
    protein inhibits the enzyme elastase, which
    breaks down connective tissue in the lungs.
    Treatment with a-1-AT alleviates symptoms in
    people suffering from emphysema.
  • Other products of pharming include blood
    clotting factors and antibodies for treating
    colon cancer.

Biotechnology Applications of DNA Manipulation
  • Crops that are resistant to herbicides
  • Glyphosate (Roundup) is a broad-spectrum
    herbicide that inhibits an enzyme system in
    chloroplasts that is involved in the synthesis of
    amino acids.
  • A bacterial gene, which confers resistance to
    glyphosate, is inserted into useful food crops
    (corn, cotton, soybeans) to protect them from the
    herbicide, which otherwise would kill them along
    with the weeds.

Biotechnology Applications of DNA Manipulation
  • Grains with improved nutritional characteristics
  • Genes from bacteria and daffodil plants are
    transferred to rice using the vector
    Agrobacterium tumefaciens.
  • Now a genetically modified strain of rice
    produces b-carotene, a molecule that is converted
    to vitamin A in animals.

Biotechnology Applications of DNA Manipulation
  • Crops that adapt to the environment
  • A gene was recently discovered in the thale cress
    (Arabidopsis thaliana) that allows it to thrive
    in salty soils.
  • When this gene is added to tomato plants, they
    can grow in soils four times as salty as the
    normal lethal level.
  • This finding raises the prospect of growing
    useful crops on previously unproductive soils
    with high salt concentration.
  • Biotechnology may allow us to adapt plants to
    different environments.

Biotechnology Applications of DNA Manipulation
  • There is public concern about biotechnology
  • Genetically modified E. coli might share their
    genes with the E. coli bacteria that live
    normally in the human intestines.
  • Researchers now take precautions against this.
    For example, the strains of E. coli used in the
    lab have a number of mutations that make their
    survival in the human intestine impossible.

Biotechnology Applications of DNA Manipulation
  • There are concerns that genetic manipulation
    interferes with nature, that genetically altered
    foods are unsafe, and that genetically altered
    plants might allow transgenes to escape to other
    species and thus threaten the environment.
  • Regarding safety for human consumption, advocates
    of genetic engineering note that typically only
    single genes specific for plant function are
  • As plant biotechnology moves from adding genes to
    improve plant growth to adding genes that affect
    human nutrition, such concerns will become more

Biotechnology Applications of DNA Manipulation
  • The risks to the environment are more difficult
    to assess.
  • Transgenic plants undergo extensive field testing
    before they are approved for use, but the
    complexity of the biological world makes it
    impossible to predict all potential environmental
    effects of transgenic organisms.
  • Because of the potential benefits of agricultural
    biotechnology, most scientists believe we should
    proceed, but with caution.

Biotechnology Applications of DNA Manipulation
  • With the exception of identical twins, each human
    being is genetically distinct from all other
    human beings.
  • Characterization of an individual by DNA base
    sequences is called DNA fingerprinting.

Biotechnology Applications of DNA Manipulation
  • Scientists look for DNA sequences that are highly
  • Sequences called VNTRs (variable number of tandem
    repeats) are easily detectable if they are
    between two restriction enzyme recognition sites.
  • Different individuals have different numbers of
    repeats. Each gets two sequences of repeats, one
    from the mother and one from the father.
  • Using PCR and gel electrophoresis, patterns for
    each individual can be determined.

Figure 16.17 DNA Fingerprinting
Biotechnology Applications of DNA Manipulation
  • The many applications of DNA fingerprinting
    include forensics and cases of contested
  • DNA from a single cell is sufficient to determine
    the DNA fingerprint because PCR can amplify a
    tiny amount of DNA in a few hours.
  • PCR is used in diagnosing infections in which the
    infectious agent is present in small amounts.
  • Genetic diseases such as sickle-cell anemia are
    now diagnosable before they manifest themselves.