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Recombinant DNA and Biotechnology

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Title: Recombinant DNA and Biotechnology


1
Recombinant DNA and Biotechnology
2
16 Recombinant DNA and Biotechnology
  • 16.1 How Are Large DNA Molecules Analyzed?
  • 16.2 What Is Recombinant DNA?
  • 16.3 How Are New Genes Inserted into Cells?
  • 16.4 What Are the Sources of DNA Used in Cloning?
  • 16.5 What Other Tools Are Used to Manipulate DNA?
  • 16.6 What Is Biotechnology?

3
16.1 How Are Large DNA Molecules Analyzed?
  • Naturally occurring enzymes that cleave and
    repair DNA are used in the laboratory to
    manipulate and recombine DNA.

4
16.1 How Are Large DNA Molecules Analyzed?
  • Restriction enzymes (restriction endonucleases)
    cut double-stranded DNA into smaller pieces.
  • Bacteria use these as defense against DNA from
    bacteriophage.
  • DNA is cut between the 3' hydroxyl group of one
    nucleotide and the 5' phosphate group of the
    nextrestriction digestion.

5
Figure 16.1 Bacteria Fight Invading Viruses with
Restriction Enzymes
6
16.1 How Are Large DNA Molecules Analyzed?
  • There are many restriction enzymes that cut DNA
    at specific base sequencesthe recognition
    sequence, or restriction site.

7
16.1 How Are Large DNA Molecules Analyzed?
  • Restriction enzymes do not cut bacterias own DNA
    because the recognition sequences are modified.
  • Methylases add methyl groups after replication
    makes sequence unrecognizable by restriction
    enzyme.

8
16.1 How Are Large DNA Molecules Analyzed?
  • Bacterial restriction enzymes can be isolated
    from cells.
  • DNA from any organism will be cut wherever the
    recognition site occurs.
  • EcoRI (from E. coli) cuts DNA at this sequence

9
16.1 How Are Large DNA Molecules Analyzed?
  • The sequence is palindromicit reads the same in
    both directions from the 5' end.
  • EcoRI occurs about once every four genes in
    prokaryotes. DNA can be chopped into small pieces
    containing a few genes.

10
16.1 How Are Large DNA Molecules Analyzed?
  • The EcoRI sequence does not occur anywhere in the
    genome of the phage T7. Thus it can survive in
    its host, E. coli.

11
16.1 How Are Large DNA Molecules Analyzed?
  • After DNA is cut, fragments of different sizes
    can be separated by gel electrophoresis.
  • Mixture of fragments is place on a well in a
    porous gel. An electric field is applied across
    the gel. Negatively charged DNA fragments move
    towards positive end.
  • Smaller fragments move faster than larger ones.

12
Figure 16.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 1)
13
Figure 16.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 2)
14
Figure 16.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 3)
15
16.1 How Are Large DNA Molecules Analyzed?
  • Electrophoresis provides information on
  • Size of fragments. Fragments of known size
    provide comparison.
  • Presence of specific sequences. These can be
    determined using probes.
  • DNA is denatured while in the gel, then
    transferred to a nylon filter to make a blot.

16
Figure 16.3 Analyzing DNA Fragments by Southern
Blotting
17
16.1 How Are Large DNA Molecules Analyzed?
  • DNA fingerprinting uses restriction analysis and
    electrophoresis to identify individuals.
  • Works best with genes that are polymorphichave
    multiple alleles.

18
16.1 How Are Large DNA Molecules Analyzed?
  • Two types of polymorphisms
  • Single nucleotide polymorphisms (SNPs) inherited
    variation involving a single base
  • Short tandem repeats (STRs) moderately
    repetitive sequences side by side

19
16.1 How Are Large DNA Molecules Analyzed?
  • STRs are recognizable if they lie between two
    restriction sites.
  • Several different STRs can be used to determine
    the unique pattern for an individual.

20
Figure 16.4 DNA Fingerprinting with Short Tandem
Repeats
21
16.1 How Are Large DNA Molecules Analyzed?
  • DNA fingerprinting requires at least 1 µg of DNA
    (amount in about 100,000 human cells).
  • This is not always available, so amplification by
    PCR is used.

22
16.1 How Are Large DNA Molecules Analyzed?
  • DNA fingerprinting is used in forensics.
  • It is more often used to prove innocence than
    guilt.
  • Only a small portion of the genome is examined
    there is the possibility that two people could
    have the same sequence.

23
16.1 How Are Large DNA Molecules Analyzed?
  • DNA fingerprinting has been used to analyze
    historical events.
  • The skeletal remains of Russian Tsar Nicholas II
    and his family were identified from DNA in bone
    fragments.
  • DNA also showed relationships with living
    descendents of the Tsar.

24
Figure 16.5 DNA Fingerprinting the Russian Royal
Family
25
16.1 How Are Large DNA Molecules Analyzed?
  • DNA technology can be used to identify species.
  • A proposal to identify all known species and look
    for unknowns has been put forth by the Consortium
    for the Barcode of Life (CBOL)
  • Use a short sequence from a gene (cytochrome
    oxidase) as a barcode for each species.

26
Figure 16.6 A DNA Barcode
27
16.1 How Are Large DNA Molecules Analyzed?
  • The barcode project could contribute to
  • Evolution research
  • Species diversity issues
  • Identification of new species
  • Identification of undesirable microbes or
    bioterrorism agents

28
16.2 What Is Recombinant DNA?
  • DNA fragments can be rejoined by DNA ligase.
  • Any two DNA sequences can be spliced.
  • First done in 1973 with two E. coli plasmids
    Recombinant DNA was born

29
Figure 16.7 Making Recombinant DNA (Part 1)
30
Figure 16.7 Making Recombinant DNA (Part 2)
31
16.2 What Is Recombinant DNA?
  • Some restriction enzymes cut both DNA strands
    exactly opposite each other.
  • Others (such as EcoRI) make a staggered cut.
    Results in single-stranded tails at the ends of
    fragments.
  • Tails are called sticky endscan bind by base
    pairing to other sticky ends.

32
Figure 16.8 Cutting and Splicing DNA
33
16.2 What Is Recombinant DNA?
  • Sticky ends of fragments that were cut by the
    same restriction enzyme are all the samethus
    fragments from different species can be joined.
  • When temperature is lowered, the fragments
    annealjoin by hydrogen bonding. Must be
    permanently spliced by DNA ligase.

34
16.3 How Are New Genes Inserted into Cells?
  • Recombinant DNA technology can be used to clone,
    or make exact copies of genes.
  • The gene can be used to make a proteinbut it
    must first be inserted, or transfected, into host
    cells.
  • The altered host cell is called transgenic.

35
16.3 How Are New Genes Inserted into Cells?
  • To determine which of the host cells contain the
    new sequence, the recombinant DNA is often tagged
    with reporter genes.
  • Reporter genes have easily observed phenotypes or
    genetic markers.

36
16.3 How Are New Genes Inserted into Cells?
  • The first host cells used were bacteria,
    especially E. coli.
  • Yeasts (Saccharomyces) are commonly used as
    eukaryotic hosts.
  • Plant cells are also usedthey have totipotency,
    the ability of any differentiated cell to develop
    into a new plant.

37
16.3 How Are New Genes Inserted into Cells?
  • The new DNA must also replicate as the host cell
    divides. It must become a segment with an origin
    of replicationa replicon or replication unit.

38
16.3 How Are New Genes Inserted into Cells?
  • New DNA can become part of a replicon in two
    ways
  • Inserted near an origin of replication in host
    chromosome.
  • It can be part of a carrier sequence or vector
    that already has an origin of replication.

39
16.3 How Are New Genes Inserted into Cells?
  • A vector should have four characteristics
  • Ability to replicate independently of the host
    cell
  • A recognition sequence for a restriction enzyme
  • A reporter gene
  • Small size in comparison with hosts chromosomes

40
16.3 How Are New Genes Inserted into Cells?
  • Plasmids have all these characteristics.
  • Plasmids are small, many have only one
    restriction site.
  • Genes for antibiotic resistance can be used as
    reporter genes.
  • And they have an origin of replication and can
    replicate independently.

41
Figure 16.9 Vectors for Carrying DNA into Cells
(A)
42
16.3 How Are New Genes Inserted into Cells?
  • Plasmids can be used for genes of 10,000 bp or
    less. Most eukaryote genes are larger than this.
  • Viruses can be used as vectorse.g.,
    bacteriophage. The genes that cause host cell to
    lyse can be cut out and replaced with other DNA.

43
16.3 How Are New Genes Inserted into Cells?
  • Bacterial plasmids dont work for yeasts because
    the origins of replication use different
    sequences.
  • A yeast artificial chromosome (YAC) has been
    created contains yeast origin of replication,
    plus yeast centromere and telomere sequences.
  • Also contains artificial restriction sites and
    reporter genes

44
Figure 16.9 Vectors for Carrying DNA into Cells
(B)
45
16.3 How Are New Genes Inserted into Cells?
  • A plasmid from the soil bacterium Agrobacterium
    tumefaciens is used as a vector for plant cells.
  • Plasmid Ti (tumor inducing) causes crown gall.
  • Plasmid has a region called T DNA, which inserts
    copies of itself into chromosomes of infected
    plants.

46
16.3 How Are New Genes Inserted into Cells?
  • T DNA has several restriction sites, where new
    DNA can be inserted.
  • With altered T DNA, plasmid no longer causes
    tumors, but can still insert itself into host
    chromosomes.

47
Figure 16.9 Vectors for Carrying DNA into Cells
(C)
48
16.3 How Are New Genes Inserted into Cells?
  • Usually only a small proportion of host cells
    take up the vector, and they may not have the
    appropriate sequence.
  • Host cells with the desired sequence must be
    identifiable.

49
16.3 How Are New Genes Inserted into Cells?
  • One method
  • E. coli is host pBR322 plasmid is the vector.
  • Plasmid has genes for resistance to ampicillin
    and tetracycline.
  • Plasmid has only one restriction site for enzyme
    BamHI, within the gene for tetracycline
    resistance.

50
16.3 How Are New Genes Inserted into Cells?
  • If new DNA is inserted at that restriction site,
    it inactivates the gene for tetracycline
    resistance.
  • Plasmid then has gene for ampicillin resistance,
    but not for tetracycline. This can be used to
    select for host cells with new DNA.

51
Figure 16.10 Marking Recombinant DNA by
Inactivating a Gene
52
16.3 How Are New Genes Inserted into Cells?
  • Other reporter genes
  • Artificial vectors with restriction sites within
    the lac operon. If new DNA is inserted there,
    vector no longer carries its original function
    into the host cell.
  • Green fluorescent protein, which normally occurs
    in the jellyfish Aequopora victoriana.

53
16.4 What Are the Sources of DNA Used in Cloning?
  • DNA fragments used for cloning come from three
    sources
  • Gene libraries
  • Reverse transcription from mRNA
  • Artificial synthesis or mutation of DNA

54
16.4 What Are the Sources of DNA Used in Cloning?
  • Human chromosomes contain an average of 80
    million bp each.
  • The DNA is cut into fragments by restriction
    enzymes, the fragments are stored as a gene
    library.
  • Each fragment is inserted into a vector, which
    goes into a host cell.

55
Figure 16.11 Constructing a Gene Library
56
16.4 What Are the Sources of DNA Used in Cloning?
  • If phage ? is used as a vector, about 50,000
    volumes are required to store the library.
  • One petri plate can hold 80,000 phage colonies,
    or plaques.
  • DNA in the plaques is screened using specific
    probes.

57
16.4 What Are the Sources of DNA Used in Cloning?
  • Smaller DNA libraries can be made from
    complementary DNA (cDNA).
  • mRNA is extracted from a tissue and the poly A
    tails allowed to hybridize with oligo dTa string
    of thymine bases.
  • Oligo dT serves as a primer for reverse
    transcriptase to synthesize a complementary DNA
    strand.

58
Figure 16.12 Synthesizing Complementary DNA
59
16.4 What Are the Sources of DNA Used in Cloning?
  • cDNA libraries are made from particular tissues
    at particular times and represent a snapshot of
    the mRNA present at that time.
  • Used to compare gene expression in different
    tissues at different stages of development.
  • cDNA is also used to clone eukaryotic genes.

60
16.4 What Are the Sources of DNA Used in Cloning?
  • DNA can be synthesized if the amino acid sequence
    of a protein is known.
  • This process is now automated, and labs can make
    custom DNA sequences overnight.
  • Flanking sequences for transcription initiation,
    termination, and regulation and start and stop
    codons are also added.

61
16.4 What Are the Sources of DNA Used in Cloning?
  • Synthetic DNA can be used to create specific
    mutations in order to study the consequences of
    the mutation.
  • Called mutagenesis techniques.
  • These techniques have revealed many
    cause-and-effect relationships, e.g., determining
    signal sequences.

62
16.5 What Other Tools Are Used to Manipulate DNA?
  • Three additional ways of manipulating DNA
  • Knockout experiments
  • Gene silencing
  • DNA chips

63
16.5 What Other Tools Are Used to Manipulate DNA?
  • A knockout experiment involves homologous
    replication to replace a gene with an inactive
    gene, and determine results in a living organism.
  • The normal allele of a gene is inserted into a
    plasmid restriction enzymes are used to insert a
    reporter gene in the middle of the normal gene.

64
16.5 What Other Tools Are Used to Manipulate DNA?
  • The gene is thus inactivated.
  • The plasmid is then transfected into a stem cell
    of a mouse embryo.
  • Stem cell undifferentiated cell that divides and
    differentiates to form different tissues.

65
16.5 What Other Tools Are Used to Manipulate DNA?
  • Much of the normal gene is still present, so
    homologous recognition takes place between the
    normal allele and the inactive allele on the
    plasmid.
  • Recombination can occur, and inactive allele is
    swapped for the normal allele.
  • The transfected stem cell is then inserted into
    an early mouse embryo.

66
Figure 16.13 Making a Knockout Mouse (Part 1)
67
Figure 16.13 Making a Knockout Mouse (Part 2)
68
16.5 What Other Tools Are Used to Manipulate DNA?
  • Translation of mRNA can be blocked by
    complementary micro RNAsantisense RNA.
  • Antisense RNA can be synthesized, and added to
    cells to prevent translationthe effects of the
    missing protein can then be determined.

69
16.5 What Other Tools Are Used to Manipulate DNA?
  • Interference RNA (RNAi) is a rare natural
    mechanism that blocks translation.
  • Short, double stranded RNA is unwound and binds
    to complementary mRNA by a protein complex, which
    also catalyzes the breakdown of the mRNA.
  • Small interfering RNA (siRNA) can be synthesized
    in the laboratory.

70
Figure 16.14 Using Antisense RNA and RNAi to
Block Translation of mRNA
71
16.5 What Other Tools Are Used to Manipulate DNA?
  • Antisense RNA and RNAi are also used to study
    cause-and-effect relationships.
  • Example Antisense RNA is used to block
    translation of proteins essential for growth of
    cancer cellsthe cells revert to normal phenotype.

72
16.5 What Other Tools Are Used to Manipulate DNA?
  • DNA chip technology provides a large array of
    sequences for hybridization experiments.
  • A series of DNA sequences are attached to a glass
    slide in a precise order.
  • The slide has microscopic wells which each
    contain thousands of copies of sequences up to 20
    nucleotides long.

73
Figure 16.15 DNA on a Chip
74
16.5 What Other Tools Are Used to Manipulate DNA?
  • To analyze mRNA, it is incubated with reverse
    transcriptase to make cDNA.
  • The cDNA is amplified using PCR.
  • Technique is called RT-PCR.
  • Amplified cDNA is tagged with a fluorescent dye
    and used as a probe of the DNA on the chip.

75
16.5 What Other Tools Are Used to Manipulate DNA?
  • DNA chip technology has been developed to
    identify gene expression patterns in women with a
    propensity for breast cancer tumors to recura
    gene expression signature.

76
16.6 What Is Biotechnology?
  • Biotechnology is the use of living cells to
    produce materials useful to people.
  • Examples use of yeasts to brew beer and wine,
    use of bacteria to produce cheese, yogurt, etc.
  • Use of microbes to produce antibiotics such as
    penicillin, alcohol, and other products.

77
16.6 What Is Biotechnology?
  • Gene cloning is now used to produce proteins in
    large amounts.
  • Almost any gene can be inserted into bacteria or
    yeasts, and the resulting cells induced to make
    large quantities of the product.
  • Requires specialized vectors.

78
16.6 What Is Biotechnology?
  • Expression vectors are synthesized that include
    sequences needed for expression of the transgene
    in the host cell.

79
Figure 16.16 An Expression Vector Allows a
Transgene to Be Expressed in a Host Cell
80
16.6 What Is Biotechnology?
  • Expression vectors can be modified by
  • Inducible promoters enhancers can also be added
    so that protein synthesis takes place at high
    rates.
  • Tissue-specific promoters
  • Signal sequencese.g., a signal to secrete the
    product to the extracellular medium.

81
Table 16.1
82
16.6 What Is Biotechnology?
  • Example of a medical application
  • After wounds heal, blood clots are dissolved by
    plasmin. Plasmin is stored as an inactive form
    called plasminogen.
  • Conversion of plasminogen is activated by tissue
    plasminogen activator (TPA).
  • TPA can be used to treat strokes and heart
    attacks, but large quantities are neededcan be
    made using recombinant DNA technology.

83
Figure 16.17 Tissue Plasminogen Activator From
Protein to Gene to Drug (Part 1)
84
Figure 16.17 Tissue Plasminogen Activator From
Protein to Gene to Drug (Part 2)
85
16.6 What Is Biotechnology?
  • Pharming production of medically useful proteins
    in milk.
  • Transgenes for a protein are inserted into the
    egg of a domestic animal, next to the promoter
    for lactoglobulina protein in milk. The
    transgenic animal then produces large quantities
    of the protein in its milk.

86
Figure 16.18 Pharming
87
16.6 What Is Biotechnology?
  • Through cultivation and selective breeding,
    humans have been altering the traits of plants
    and animals for thousands of years.
  • Recombinant DNA technology has several
    advantages
  • Specific genes can be targeted.
  • Any gene can be introduced into any other
    organism.
  • New organisms are generated quickly.

88
Table 16.2
89
16.6 What Is Biotechnology?
  • Crop plants have been modified to produce their
    own insecticides
  • The bacterium Bacillus thuringiensis produces a
    protein that kills insect larvae.
  • Dried preparation of B. thuringiensis are sold as
    a safe alternative to synthetic insecticides. The
    toxin is easily biodegradable.

90
16.6 What Is Biotechnology?
  • Genes for the toxin have been isolated, cloned,
    and modified, and inserted into plant cells using
    the Ti plasmid vector.
  • Transgenic corn, cotton, soybeans, tomatoes, and
    other crops are being grown. Pesticide use is
    reduced.

91
16.6 What Is Biotechnology?
  • Some transgenic crops are resistant to
    herbicides.
  • Glyphosate (Roundup) is widely used to kill
    weeds.
  • Expression vectors have been used to make plants
    that synthesize so much of the target enzyme of
    glyphosate that they are unaffected by the
    herbicide.

92
16.6 What Is Biotechnology?
  • The gene has been inserted into corn, soybeans,
    and cotton.
  • About half of U.S. crops of these plants contain
    this gene.

93
16.6 What Is Biotechnology?
  • Crops with improved nutritional characteristics
  • Rice does not have ß-carotene, but does have a
    precursor molecule.
  • Genes for enzymes that synthesize ß-carotene from
    the precursor are taken from daffodils and
    inserted into rice by the Ti plasmid.

94
16.6 What Is Biotechnology?
  • The transgenic rice is yellow, and can supply
    ß-carotene to improve the diets of many people.
  • ß-carotene is converted to vitamin A in the body.

95
Figure 16.19 Transgenic Rice Is Rich in ß-Carotene
96
16.6 What Is Biotechnology?
  • Recombinant DNA is also used to adapt a crop
    plant to an environment.
  • Example plants that are salt-tolerant
  • Genes from a protein that moves sodium ions into
    the central vacuole were isolated from
    Arabidopsis and inserted into tomato plants.

97
Figure 16.20 Salt-Tolerant Tomato Plants
98
16.6 What Is Biotechnology?
  • Concerns over biotechnology
  • Genetic manipulation is an unnatural interference
    in nature.
  • Genetically altered foods are unsafe to eat.
  • Genetically altered crop plants are dangerous to
    the environment.

99
16.6 What Is Biotechnology?
  • Advocates of biotechnology point out that all
    crop plants have been manipulated by humans.
  • Advocates say that since only single genes for
    plant function are inserted into crop plants,
    they are still safe for human consumption.
  • Genes that affect human nutrition may raise more
    concerns.

100
16.6 What Is Biotechnology?
  • Concern over environmental effects centers on
    escape of transgenes into wild populations
  • For example, if the gene for herbicide resistance
    made its way into the weed plants.
  • Beneficial insects can also be killed from eating
    plants with B. thuringiensis genes.
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