Chapter 9:

Biotechnology and
Recombinant DNA
Fundamental Tools Used in Biotechnology

 Restriction enzymes
• Recognize and cut 4 to 6 base-
pair nucleotide sequence
• Typically palindrome
• Generates restriction fragments
• Complementary “sticky” ends
can anneal
• DNA ligase joins molecules
• Allows creation of recombinant
DNA molecules
Fundamental Tools Used in Biotechnology

 Restriction enzymes (continued…)

• Name represents bacterium from which isolated
Fundamental Tools Used in Biotechnology

 Gel electrophoresis separates DNA based on size

• Run current; (-) DNA migrates toward (+) electrode
• Dye to visualize; can also separate RNA, proteins
Fundamental Tools Used in Biotechnology

 CRISPR systems enable gene editing using Cas9

cutting enzyme to locate, alter specific sites in DNA
• Uses single-stranded RNA guide to recognize DNA
sequence
Applications of Genetic Engineering

 Numerous uses for genetically

engineered bacteria
• Protein production
• DNA production
• Research tools
• Relies on DNA cloning
Applications of Genetic Engineering

 DNA cloning
• Isolate DNA
• Cut with restriction enzymes
• Join DNA (insert) with plasmid
(vector) to generate
independently replicating
recombinant molecule
• Can use high-copy-number
vector
• Introduce into host (e.g., E. coli)
Applications of Genetic Engineering

 Protein production safer, more economical

• Gene for human insulin cloned into bacteria
• Previously insulin extracted from pancreatic glands of
cattle and pigs, sometimes caused allergic reactions
• Vaccine production: clone gene for specific proteins
• E.g. vaccines for hepatitis B and cervical cancer
• Foot-and-mouth disease of domestic animals
• Cheese production: chymosin (rennin)
• Enzyme from stomachs of calves
Applications of Genetic Engineering

 DNA production aids in research

• Clone DNA segment into bacterium (e.g., E. coli)
• Allows easy production of DNA for study
• Random samples of DNA from environment can be
cloned and then sequenced
• Termed “shotgun cloning”
• First step of metagenomics
• Aids in study of ~99% of bacteria that have not been
grown in culture
Applications of Genetic Engineering

 Researching Gene Function and Regulation

• Gene fusion of gene being
studied and a reporter gene
• Encodes observable
product
– E.g., green fluorescent
protein (GFP)
Applications of Genetic Engineering
 Genetically Engineered Eukaryotes
• Yeasts can be engineered like bacteria; provide model
• Engineered plants and animals termed transgenic
• Ti plasmid from Agrobacterium tumefaciens used to
generate corn, cotton, potatoes that produce Bt-toxin
from Bacillus thuringiensis
– Toxic only to insects and their larvae
• Soybean, cotton, corn engineered to resist
biodegradable herbicide glyphosate (Roundup)
– Allows reduced tilling and thus less erosion
• Plants with improved nutritional value
– Rice containing β-carotene; iron
Techniques Used in Genetic Engineering

 DNA library
• A collection of clones that
together contain entire genome
• Process: isolate genome;
restriction digest
• Insert fragments into vectors
• Clone fragments into
population of E. coli cells
• Other techniques then used to
find gene of interest within the
library
Techniques Used in Genetic Engineering

 Must remove introns from eukaryotic cells

• Isolate mRNA
• Reverse
transcribe
to cDNA
Techniques Used in Genetic Engineering

 Generating a Recombinant DNA Molecule

• Vector is usually modified plasmid or bacteriophage
• Has origin of replication
• Must have restriction site(s)
– Multiple-cloning site useful
• Selectable marker
– Ampicillin common
• Second marker helpful
– Disrupted by insertional
inactivation
– Distinguishes
recombinant plasmids
from intact vector
Techniques Used in Genetic Engineering

 Genetic markers
• pUC18 as example
• Selectable marker: ampicillin R
• Cells with either vector grow
• Second marker: lacZ’ gene
• Product cleaves X-gal,
Forms blue compound
• Multiple-cloning site in gene
• Insert interrupts lacZ’
• Intact vector 
blue colonies
• Recombinant molecule 
white colonies
Concerns Regarding Genetic Engineering

 New technologies need to be scrutinized for safety

• Recombinant DNA Advisory Committee (RAC) formed by
NIH nearly three decades ago
• Numerous advances have been made
• Technologies could be used for malicious purposes
• Concerns over sequencing human DNA
• Genetically modified (GM) organisms hold promises
• Concerns over possible allergens
• Unintended effects on environment
– Pollen from Bt-plants may or may not harm butterflies
– Herbicide-resistance genes may transfer to weeds
• CRISPR/Cas9 technologies may be difficult to detect
DNA Sequencing

 Human Genome Project produced great advances

• Rapid growth in field of genomics
• Sequencing of numerous organisms
• Spawned new field of bioinformatics to analyze data
• Allows determination of amino acid sequences
• Comparisons of different proteins, various organisms
• Evolutionary relatedness of organisms
 National Microbiome Initiative aims to study
microbiomes across different ecosystems
DNA Sequencing

 Dideoxy chain termination most common method

• Automated and fast
• In vitro DNA synthesis
• Template DNA
• DNA polymerase
• Primer
• Deoxynucleotides
• dNTPs
• Dideoxynucleotides
• ddNTPs
DNA Sequencing

 Dideoxy chain termination most common method

• Dideoxynucleotides (ddNTPs) act as chain terminators
• Lack 3’ OH, so synthesis stops when incorporated
• Yields mixture of DNA of different lengths
• Denature DNA
• Separate ssDNA via
electrophoresis
• Read fluorescent label
on ddNTPs to obtain
sequence
DNA Sequencing

 Separate molecules via gel electrophoresis

• Laser reads color
• Determines sequence
DNA Sequencing

 High-Throughput sequencing methods

• Next generation (“next-gen” methods)
• Highly automated; fast, lower costs
• Use millions of overlapping small sequences
• Computers align and merge data to assemble sequence
• Easier using reference genome
• Errors common so genomes analyzed multiple times
Polymerase Chain Reaction (PCR)

 Creates billions of copies of DNA in just hours

• Products can be visualized via gel electrophoresis
• Allows detection of
specific sequences
• Can detect organisms
without culturing
– E.g., pathogens
• Requires template DNA,
polymerase, primers,
deoxynucleotide triphosphates
(dATP, dGTP, dCTP, dTTP)
Polymerase Chain Reaction (PCR)

 Three-step cycle
• DNA denatured by heating
(~95°C)
• Temperature lowered (~50°C)
to allow primers to anneal
• Temperature raised (~70°C)
to allow DNA synthesis
• DNA doubled in each cycle,
so exponential increase
• Heat-stable Taq polymerase
from Thermus aquaticus
critical
Polymerase Chain Reaction (PCR)

 Primers define length of PCR product

Polymerase Chain Reaction (PCR)

 Exponential
amplification of DNA
• Increasing numbers
of product of correctly
defined length
• ~30-35 cycles typical
Polymerase Chain Reaction (PCR)

 Variations of conventional PCR

• RT-PCR (reverse-transcription PCR)
• Reverse transcriptase used to synthesize cDNA
from mRNA template in a sample
• cDNA is used as template for amplification
• qPCR (quantitative PCR) or real-time PCR
• Fluorescent marker detects PCR product
• Tracks amplification in real time
• Determines relative amount of target DNA in sample
Science Takes the Witness Stand

 Amplification of short tandem repeats (STRs)

• CODIS database contains patterns of 20 different STR
loci, allows unique identification
Probe Technologies

 DNA probes locate specific nucleotide sequence

• Probe is single-stranded piece of DNA
• Will hybridize to complementary sequence
• Labeled with marker
• Numerous different
probe technologies
Probe Technologies

 Colony Blotting
• Detects specific DNA sequences in colonies
• Commonly used to identify which clones in a
collection contain sequence of interest
Probe Technologies

 Fluorescence in situ hybridization (FISH)

• Probe hybridizes to nucleotide sequences in intact cells
fixed to microscope slide
• Cells visualized via fluorescence microscope
• Probe often binds to rRNA
sequences since numerous
• Rapid identification in
specimen without culturing
• Detects related organisms
or specific species
• Two different markers can
detect two different groups
in a sample
Probe Technologies

 DNA Microarrays allow study of gene expression

• Array has many oligonucleotides that act as probes
• Specific to each gene of interest
• mRNA is isolated, used to synthesize labeled cDNA
• Two fluorescent dyes
• cDNA represents genes
that were being expressed
• cDNA anneals to
oligonucleotides of array
• Detector reads colors
• Allows comparisons of
conditions