Recombinant DNA Technology - Department of Biochemistry BMC, Sagar

Recombinant DNA
   Technology

 Department of Biochemistry
       BMC, Sagar

Recombinant DNA:
Cloning and Creation of Chimeric Genes

   Recombinant DNA (rDNA) is a form of artificial
    DNA that is created by combining two or more
    sequences that would not normally occur together
    through the process of gene splicing.

   Recombinant DNA technology is a technology
    which allows DNA to be produced via artificial
    means. The procedure has been used to change
    DNA in living organisms and may have even more
    practical uses in the future.

Recombinant DNA technology is
  one of the recent advances in
    biotechnology, which was
developed by two scientists named
    Boyer and Cohen in 1973.

Stanley N. Cohen , who
 received the Nobel Prize in
 Medicine in 1986 for his
 work on discoveries of
 growth factors.

Stanley N. Cohen (1935–) (top)
and Herbert Boyer (1936–)
(bottom), who constructed the
first recombinant DNA using
bacterial DNA and plasmids.

Recombinant DNA Technology

1. The basic procedures of recombinant
   DNA technology
2. Application of recombinant DNA
   technology

   Recombinant technology begins with the
    isolation of a gene of interest (target gene).
    The target gene is then inserted into the
    plasmid or phage (vector) to form replicon.
   The replicon is then introduced into host cells
    to be cloned and either express the protein or
    not.
   The cloned replicon is referred to as
    recombinant DNA. The procedure is called
    recombinant DNA technology. Cloning is
    necessary to produce numerous copies of the
    DNA since the initial supply is inadequate to
    insert into host cells.

   Cloning——In classical biology, a clone is a
    population of identical organisms derived
    from a single parental organism.
       For example, the members of a colony of
        bacterial cells that arise from a single cell on a
        petridish are clones. Molecular biology has
        borrowed the term to mean a collection of
        molecules or cells all identical to an original
        molecule or cell.

Six steps of Recombinant DNA
 1.   Isolating (vector and target gene)
 2.   Cutting (Cleavage)
 3.   Joining (Ligation)
 4.   Transforming
 5.   Cloning
 6.   Selecting (Screening)

Six basic steps are common to most
   recombinant DNA experiments
1. Isolation and purification of DNA.
   Both vector and target DNA molecules
   can be prepared by a variety of
   routine methods. In some cases, the
   target DNA is synthesized in vitro.

2. Cleavage of DNA at particular sequences. As
  we will see, cleaving DNA to generate
  fragments of defined length, or with specific
  endpoints, is crucial to recombinant DNA
  technology. The DNA fragment of interest is
  called insert DNA. In the laboratory, DNA is
  usually cleaved by treating it with
  commercially produced nucleases and
  restriction endonucleases.

3. Ligation of DNA fragments.
  A recombinant DNA molecule is usually
  formed by cleaving the DNA of interest to
  yield insert DNA and then ligating the insert
  DNA to vector DNA (recombinant DNA or
  chimeric DNA). DNA fragments are
  typically joined using DNA ligase (also
  commercially produced).
  – T4 DNA Ligase

4. Introduction of recombinant DNA into
  compatible host cells. In order to be
  propagated, the recombinant DNA
  molecule (insert DNA joined to vector
  DNA) must be introduced into a
  compatible host cell where it can replicate.
  The direct uptake of foreign DNA by a host
  cell is called genetic transformation (or
  transformation). Recombinant DNA can
  also be packaged into virus particles and
  transferred to host cells by transfection.

5. Replication and expression of
  recombinant DNA in host cells.
  Cloning vectors allow insert DNA to be
  replicated and, in some cases, expressed
  in a host cell. The ability to clone and
  express DNA efficiently depends on the
  choice of appropriate vectors and hosts.

6. Identification of host cells that contain
  recombinant DNA of interest. Vectors
  usually contain some genetic markers, or
  genes, that allow the selection of host
  cells that have taken up foreign DNA.
  The identification of a particular DNA
  fragment usually involves an additional
  step—screening a large number of
  recombinant DNA clones. This is almost
  always the most difficult step.

DNA
cloning in
a plasmid
vector
permits
amplificati
on of a
DNA
fragment.

First step:
           Isolating DNA

1. Vector
2. Target gene

How to get a target genes?
1.   Genomic DNA
2.   Artificial synthesis
3.   PCR amplification
4.   RT-PCR

Polymerase chain reaction (PCR)

   A technique called the polymerase chain
    reaction (PCR) has revolutionized
    recombinant DNA technology. It can
    amplify DNA from as little material as a
    single cell and from very old tissue such
    as that isolated from Egyptian mummies,
    a frozen mammoth, and insects trapped
    in ancient amber.

 method is used to
  amplify DNA
  sequences

 The polymerase
  chain reaction
  (PCR) can quickly
  clone a small        Initial
  sample of DNA in a   DNA
                       segment
  test tube

                                 Number of DNA
                                 molecules

PCR primers

RT-PCR
   Reverse transcription polymerase chain reaction
    (RT-PCR) is a variant of polymerase chain
    reaction (PCR.
    In RT-PCR, however, an RNA strand is first
    reverse transcribed into its DNA complement
    (complementary DNA, or cDNA) using the enzyme
    reverse transcriptase, and the resulting cDNA is
    amplified using traditional.
    – Template:RNA
    – Products: cDNA

Vectors- Cloning Vehicles
 Cloning   vectors can be plasmids,
 bacteriophage, viruses, or even small
 artificial chromosomes. Most vectors
 contain sequences that allow them to be
 replicated autonomously within a
 compatible host cell, whereas a minority
 carry sequences that facilitate integration
 into the host genome.

   All cloning vectors have in common at least
    one unique cloning site, a sequence that can
    be cut by a restriction endonuclease to allow
    site-specific insertion of foreign DNA. The
    most useful vectors have several restriction
    sites grouped together in a multiple cloning
    site (MCS) called a polylinker.

Types of vector
1. Plasmid Vectors
2. Bacteriophage Vectors
3. Virus vectors
4. Shuttle Vectors--can replicate in either
   prokaryotic or eukaryotic cells.
5. Yeast Artificial Chromosomes as
   Vectors

Plasmid Vectors
 Plasmids are circular, double-stranded
  DNA (dsDNA) molecules that are separate
  from a cell’s chromosomal DNA.
 These extra chromosomal DNAs, which
  occur naturally in bacteria and in lower
  eukaryotic cells (e.g., yeast), exist in a
  parasitic or symbiotic relationship with
  their host cell.

Plasmid

   Plasmids can replicate autonomously within
    a host, and they frequently carry genes
    conferring resistance to antibiotics such as
    tetracycline, ampicillin, or kanamycin. The
    expression of these marker genes can be
    used to distinguish between host cells that
    carry the vectors and those that do not

pBR322
   pBR322 was one of the first versatile plasmid
    vectors developed; it is the ancestor of many of the
    common plasmid vectors used in biochemistry
    laboratories.
   pBR322 contains an origin of replication (ori) and
    a gene (rop) that helps regulate the number of
    copies of plasmid DNA in the cell. There are two
    marker genes: confers resistance to ampicillin,
    and confers resistance to tetracycline. pBR322
    contains a number of unique restriction sites that
    are useful for constructing recombinant DNA.

pBR322
1. Origin of
   replication
2. Selectable
   marker
3. unique
   restriction
   sites

Enzymes
   1.   Restriction endonuclease, RE
   2.   DNA ligase
   3.   Reverse transcriptase
   4.   DNA polymerase, DNA pol
   5.   Nuclease
   6.   Terminal transferase

Restriction Enzymes and DNA Ligases Allow
  Insertion of DNA Fragments into Cloning Vectors

Restriction enzymes cleave DNA
               The same sequence of bases is
                found on both DNA strands, but
                in opposite orders. GAATTC
                                  CTTAAG
               This arrangement is called a
                palindrome. Palindromes are
                words or sentences that read the
                same forward and backward.
                form sticky ends: single
                stranded ends that have a
                tendency to join with each
                other ( the key to
                recombinant DNA)

Restriction Enzymes Cut DNA Chains at
           Specific Locations
 Restriction enzymes are endonucleases
  produced by bacteria that typically
  recognize specific 4 to 8bp sequences,
  called restriction sites, and then cleave both
  DNA strands at this site.
 Restriction sites commonly are short
  palindromic sequences; that is, the
  restriction-site sequence is the same on
  each DNA strand when read in the 5′ → 3′
  direction.

Cut out the gene

  Restriction enzymes

Restriction enzymes
   Restriction enzymes are named after the
    bacterium from which they are isolated
    –     For example, Eco RI is from Escherichia coli,
        and Bam HI is from Bacillus amyloliquefaciens .
        The first three letters in the restriction enzyme
        name consist of the first letter of the genus (E)
        and the first two letters of the species (co). These
        may be followed by a strain designation (R) and
        a roman numeral (I) to indicate the order of
        discovery (eg, EcoRI, EcoRII).

Blunt ends or sticky ends
 Each enzyme recognizes and cleaves a
  specific double-stranded DNA sequence that
  is 4–7 bp long. These DNA cuts result in
  blunt ends (eg, Hpa I) or overlapping (sticky)
  ends (eg, BamH I) , depending on the
  mechanism used by the enzyme.
 Sticky ends are particularly useful in
  constructing hybrid or chimeric DNA
  molecules .

Results of restriction endonuclease digestion.
Digestion with a restriction endonuclease can result
in the formation of DNA fragments with sticky, or
cohesive ends (A) or blunt ends (B). This is an
important consideration in devising cloning
strategies.

Inserting DNA Fragments into Vectors
   DNA fragments with either sticky ends or blunt
    ends can be inserted into vector DNA with the
    aid of DNA ligases.
    For purposes of DNA cloning, purified DNA
    ligase is used to covalently join the ends of a
    restriction fragment and vector DNA that have
    complementary ends . The vector DNA and
    restriction fragment are covalently ligated
    together through the standard 3 → 5
    phosphodiester bonds of DNA.
   DNA ligase “pastes” the DNA fragments
    together

Ligation of restriction fragments
with complementary sticky ends.

insertional inactivation

A method of screening recombinants for inserted DNA fragments.
Using the plasmid pBR322, a piece of DNA is inserted into the unique
PstI site. This insertion disrupts the gene coding for a protein that
provides ampicillin resistance to the host bacterium. Hence, the
chimeric plasmid will no longer survive when plated on a substrate
medium that contains this antibiotic. The differential sensitivity to
tetracycline and ampicillin can therefore be used to distinguish clones
of plasmid that contain an insert.

Screening (Strategies)
1. Gel Electrophoresis Allows Separation of
   Vector DNA from Cloned Fragments
2. Cloned DNA Molecules Are Sequenced
   Rapidly by the Dideoxy Chain-Termination
   Method
3. The Polymerase Chain Reaction Amplifies a
   Specific DNA Sequence from a Complex
   Mixture
4. Blotting Techniques Permit Detection of
   Specific DNA Fragments and mRNAs with
   DNA Probes

A      B            C          M

                                               bp
                                            —1534
                                            — 994

                                            — 695

                                            — 515
                                            — 377
                                            — 237

         Gel Electrophoresis
    negative charged DNA run to the anode

Southern blot technique can detect a specific DNA
fragment in a complex mixture of restriction fragments.

                                     Hybridization

                                              Radioactive isotope

Types of blotting techniques
   Southern blotting
       Southern blotting techniques is the first nucleic acid
        blotting procedure developed in 1975 by Southern.
       Southern blotting is the techniques for the specific
        identification of DNA molecules.
   Northern blotting
       Northern blotting is the techniques for the specific
        identification of RNA molecules.
   Western blotting
       Western blotting involves the identification of proteins.
       Antigen + antibody

Applications of Recombinant
     DNA Technology

1. Analysis of Gene Structure and
   Expression
2. Pharmaceutical Products
  – Drugs
  – Vaccines
3. Genetically modified organisms (GMO)
  – Transgenic plants
  – Transgenic animal
4. Application in medicine
5. 

Analysis of Gene Structure and
                   Expression
   Using specialized recombinant DNA techniques,
    researchers have determined vast amounts of DNA
    sequence including the entire genomic sequence of
    humans and many key experimental organisms.
    This enormous volume of data, which is growing at
    a rapid pace, has been stored and organized in two
    primary data banks:
       the GenBank at the National Institutes of Health,
        Bethesda, Maryland,
       and the EMBL Sequence Data Base at the European
        Molecular Biology Laboratory in Heidelberg, Germany.

Pharmaceutical Products
   Some pharmaceutical applications of DNA
    technology:
     Large-scale production of human hormones
      and other proteins with therapeutic uses
     Production of safer vaccines
   A number of therapeutic gene products —
    insulin, the interleukins, interferons, growth
    hormones, erythropoietin, and coagulation
    factor VIII—are now produced
    commercially from cloned genes

 Pharmaceutical
  companies already are
  producing molecules
  made by recombinant
  DNA to treat human
  diseases.
 Recombinant bacteria
  are used in the
  production of human
  growth hormone and
  human insulin

Use recombinant cells to mass produce proteins
   – Bacteria
   – Yeast
   – Mammalian

• Growth hormone
• Insulin                       deficiency
  – Hormone required to         – Faulty pituitary and
    properly process sugars       regulation
    and fats
                                – Had to rely on cadaver
  – Treat diabetes                source
  – Now easily produced by      – Now easily produced by
    bacteria                      bacteria

Subunit Herpes Vaccine

Not always used for good...
            • High doses of HGH can
              cause permanent side
              effects
              – As adults normal growth
                has stopped so excessive GH
                can thicken bones and
                enlarge organs

Genetically modified organisms (GMO)
          Use of recombinant plasmids in
           agriculture
            – plants with genetically desirable
              traits
               • herbicide or pesticide resistant corn
                 & soybean
                  – Decreases chemical insecticide use
                  – Increases production
               • “Golden rice” with beta-carotene
                  – Required to make vitamin A, which in
                    deficiency causes blindness

   Crops have been
    developed that are
    better tasting, stay
    fresh longer, and are
    protected from disease
    and insect infestations.

                               “Golden rice” has been
                               genetically modified to
                               contain beta-carotene

Genetic Engineering of Plants
   Plants have been bred for millennia to
    enhance certain desirable characteristics in
    important food crops.
   Transgenic plants.

The luciferase gene from a
firefly is transformed into
tobacco plant using the Ti
plasmid. Watering the plant
with a solution of luciferin
(the substrate for firefly
luciferase) results in the
generation of light by all
plant tissues.

Insect-resistant tomato plants
The plant on the left contains a gene that encodes a
bacterial protein that is toxic to certain insects that
feed on tomato plants. The plant on the right is a
wild-type plant. Only the plant on the left is able to
grow when exposed to the insects.

Transgenic animals

A transgenic
mouse

Mouse on right is
normal; mouse on
left is transgenic
animal expressing
rat growth hormone

   Trangenic plants and animals
    have genes from other
    organisms.

   These transgenic sheep
    carry a gene for a
    human blood protein
     – This protein may help in
       the treatment of cystic
       fibrosis

Other benefits of GMOs
   Disease resistance
       There are many viruses, fungi, bacteria that cause plant
        diseases
       “Super-shrimp”
   Cold tolerance
       Antifreeze gene from cold water fish introduced to
        tobacco and potato plants
   Drought tolerance & Salinity tolerance
       As populations expand, potential to grow crops in
        otherwise inhospitable environments

Downsides???
   Introduce allergens?
   Pass trans-genes to
    wild populations?
    –   Pollinator transfer
   R&D is costly
    – Patents to insure
      profits
         • Patent infringements
         • Lawsuits
         • potential for capitalism
           to overshadow
           humanitarian efforts

Application in medicine
 Human   Gene Therapy
 Diagnosis of genetic disorders
 Forensic Evidence

Human Gene Therapy
   Human gene therapy seeks to repair the damage
    caused by a genetic deficiency through
    introduction of a functional version of the
    defective gene. To achieve this end, a cloned
    variant of the gene must be incorporated into the
    organism in such a manner that it is expressed
    only at the proper time and only in appropriate
    cell types. At this time, these conditions impose
    serious technical and clinical difficulties.

   Gene therapy is the alteration of an afflicted
    individual’s genes
   Gene therapy holds great potential for treating
    disorders traceable to a single defective gene
   Vectors are used for delivery of genes into cells
   Gene therapy raises ethical questions, such as
    whether human germ-line cells should be treated to
    correct the defect in future generations

   Many gene therapies have received approval from
    the National Institutes of Health for trials in
    human patients, including the introduction of gene
    constructs into patients. Among these are
    constructs designed to cure ADA- SCID (severe
    combined immunodeficiency due to adenosine
    deaminase [ADA] deficiency), neuroblastoma, or
    cystic fibrosis, or to treat cancer through
    expression of the E1A and p53 tumor suppressor
    genes.

Cloned gene
                            Insert RNA version of normal allele
                            into retrovirus.

                          Viral RNA

                            Let retrovirus infect bone marrow cells
 Retrovirus                 that have been removed from the
 capsid                     patient and cultured.

                                                                      Somatic cells
                                                                      Only!
                                  Viral DNA carrying the normal
                                  allele inserts into chromosome.     Not for
                                                                      reproductive
 Bone
 marrow                                                               cells !!
 cell from
 patient

                                                    Bone
    Inject engineered                               marrow
    cells into patient.