• The Molecular Revolution:
• Biotechnology and Beyond
• Ch 20 (p. 410-413) = End with Adding
genes to an organism
• Bioskills 5 (p.26-28)
• Bioskills 8 (p.32-35)
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Learning Objectives:
1. Define key terms: biotechnology, ligation, transformation, digestion,
restriction enzyme, recombinant DNA [Knowledge]
2. Describe some of the basic techniques used in recombinant DNA
technology and their significance: restriction enzymes, agarose gel
electrophoresis, polymerase chain reaction [Knowledge, Comprehension].
3. Describe the general steps involved in creating recombinant DNA. [Knowledge,
Comprehension]
4. Describe the key components of a plasmid and the role of each in the cloning
process.
5. Describe the general steps involved in the PCR reaction and the
components required [Knowledge, Comprehension].
6. Compare PCR to DNA replication. [Comprehension]
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Chapter 20 Opening Roadmap
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Introduction to The Molecular Revolution
• Biologists have learned how to sequence and manipulate DNA
– Called genetic engineering
– Mixing and matching sequences of DNA in a lab is called recombinant
DNA technology
• The human genome—all the DNA of a person— was sequenced by 2001
• Genomics is the effort to sequence, interpret, and compare whole genomes.
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Using Plasmids in Cloning
• Producing many copies of a gene or
other DNA sequence is called DNA
cloning
• Cloning is often done by inserting a
piece of DNA into a plasmid
– Small, circular DNA molecule
– Often found in bacteria
– Replicate independently of the
chromosome
– Contain a gene for antibiotic
resistance or other traits that allow a
cell to grow in a hostile environment
Plasmids 101: What is a plasmid?
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Cloning vectors
• Plasmids can serve as cloning
vectors
– A piece of foreign DNA is
spliced into a plasmid
– The plasmid is inserted into a Plasmids 101: What is a plasmid?
bacteria cell
– The plasmid is replicated and
transmitted to daughter cells as
the host cell grows and divides
– Produces billions of identical
copies of the DNA
• The DNA is harvested by breaking
the bacteria open and isolating the
plasmids Bacterial Transformation Workflow | Thermo Fisher Scientific
- CA
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Using Restriction Endonucleases and DNA
Ligase to Cut and Paste DNA
• A restriction endonuclease is a bacterial enzyme that cuts DNA at specific
base sequences
– Sequences are called recognition sites
– Over 800 restriction endonucleases are known
• The cuts are often staggered, resulting in sticky ends
– Complementary single-stranded ends
– Joining of DNA fragments is promoted by sticky ends
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Genes Can Be Inserted into Plasmids for Cloning
Using Restriction Endonucleases and
DNA Ligase to Cut and Paste DNA
• One way of inserting DNA into
plasmids:
– Step 1: A plasmid and a foreign
DNA have the same recognition
site
– Step 2: Both are cut by a
restriction endonuclease
– Step 3: Complementary sticky
ends are produced
– Step 4: DNA ligase is used to
attach the pieces of DNA
– Step 5: Recombinant plasmids
are introduced into
E. coli and allowed to grow
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Transformation: Introducing Recombinant Plasmids
into Bacterial Cells
• Plasmid vectors can be introduced
into bacteria by transformation
– Chemicals or an electrical
shock increase the
permeability of the cell’s
plasma membrane
– Cells are spread onto plates
under conditions that allow
only cells with plasmid to
grow
Typically an antibiotic is
added to the plates
Plasmids contain an
antibiotic-resistance gene
Cells without the plasmid
are killed
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Using Reverse Transcriptase to Produce
cDNAs
• The enzyme reverse transcriptase
synthesizes DNA from an RNA template
– Called complementary DNA, or cDNA
• cDNAs can be used to identify which genes
are expressed in a particular cell type
– Isolate mRNA from a type of cell
– Make double-stranded cDNA from them
– Clone and analyze the cDNA to learn
which genes are expressed in those
cells
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Adding Genes to an Organism
• Biotechnology is the engineering of genes, cells, and organisms for research
• Used in agriculture to improve crops
– Transgenic plants are created by inserting foreign DNA into a plasmid
– Transform the plasmid into A. tumefaciens
– Allow it to infect plants
• Creates a genetically modified (GM) crop
• Roundup Ready crop plants have a bacterial gene that makes them
– Tolerant to the herbicide Roundup
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Golden Rice
• Transgenic crops containing extra nutrients
• Food biofortification
• Golden rice has added genes that encode enzymes needed to
synthesize β-carotene
• Precursor for vitamin A
• Deficiency in vitamin A results in children dying or going blind.
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BioSkill 8: Using Molecular Biology Tools and Techniques
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How does PCR work?
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BioSkill 5: Separating and Visualizing Molecules
Using Electrophoresis to Separate Molecules
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Why Do Separated Molecules Form Bands?
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Capillary Tube Electrophoresis
Using Thin Layer Chromatography to Separate Molecules
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