Now what?

Questions/Concerns/Review

Essential Question:
When does biotechnology become unethical? Where do we draw the line?
 AP Biology
Jan 11, 2022
 RW View: Virology has played major roles in the biological revolutions of the last
century. Viruses have been at the centers of science, agriculture, and medicine for
millennia, and some of our greatest challenges/triumphs have involved virology.
Current research includes disease treatment using viral gene transfer and vaccines.

 Scripture: “At least there is hope for a tree: If it is cut down, it will sprout again, and its new
shoots will not fail.”—Job 14:7

 Application: Scripture (God’s word) must remain the foundation of one’s decision
and ultimately actions. History shows despite great scientific experiments and
intelligent human thought processes, the “unknown” (e.g., “black box”) still exists.

 Review: Chapters 17-18 Examination

 Class Focus: Chapter 20, Concepts 1-2

 Interactive Development: Read/review lecture concepts; complete concept checks

 Chapter 20

DNA Tools and

Biotechnology

Lecture Presentations by
Nicole Tunbridge and
© 2017 Pearson Education, Inc.
Kathleen Fitzpatrick
Chapter 20 Key Concepts
 20.1, DNA sequencing and DNA cloning  genetic engineering

 20.2, DNA technology used to study gene expression

• 20.3, Cloned organisms and stem cells useful for research and application

• 20.4, Practical application of DNA-based biotechnology and affect on society

DNA Toolbox
 Recently, genome sequences of 2 extinct species (Neanderthals and

wooly mammoths) completed  evolution connection

‒ Sequencing technique advances making genome sequencing faster

and less expensive
 DNA technology: DNA sequencing and manipulating
 Biotechnology: manipulation of organisms or components to
make useful products via technology

© 2017 Pearson Education, Inc.

Concept 20.1: DNA sequencing and cloning valuable
tools for genetic engineering and biological inquiry

 DNA hybridization: based on DNA strand complementarity

‒ Base pairing of 1 strand complementary sequence on another strand

 Genetic engineering
‒ Direct gene manipulation for practical purposes

 DNA Sequencing
‒ Gene’s complete nucleotide sequence determined via DNA sequencing

‒ First automated procedure based on technique called dideoxy or chain

termination sequencing developed by Frederick Sanger

‒ In last 15 years, “next-generation sequencing” techniques have been

developed which is much faster

© 2017 Pearson Education, Inc.

Synthesis Sequencing (fig 20.3)

 Many DNA fragments copied—“high-throughput” technology

‒ Specific strand of each fragment immobilized

‒ Complementary strand synthesized 1 nucleotide at a time

‒ 1000s -100,000 of fragments ~300 nucleotides long can be in parallel

 “Third-generation sequencing” even faster less $

 In some methods, single long DNA molecule sequenced as it
moves through a very small pore (i.e., nanopore)
‒ Each base identified by way it interrupt electrical current as it passes
through pore

© 2017 Pearson Education, Inc.

Fig 20.3 Technique

1 Genomic DNA is fragmented.

Results

No. of nucleotides in sequence

2 Each fragment is isolated with a 4
A
bead. T
G
3 TTCTGCGAA
C
3 Using PCR, 106 copies of each
fragment are made, each attached 2
to the bead by the 5′ end.
1

4 The bead is placed into a well with

DNA polymerases and primers.
Template strand
of DNA 3′
5′ 3′ 5′ A T GC
Primer

5 Solutions of each of the four nucleotides are added

to all wells sequentially and then washed off.
The entire process is then repeated.

A TGC A TGC A TGC A TGC

Template
C strand C C C
C of DNA C C C
A A dTTP A dGTP A dCTP
A dATP A A A
T T T T
G G G GC PPi
TA PPi TA TA TA
DNA GC GC GC GC
polymerase GC GC GC GC
AG Primer AG AG AG
TA TA TA TA

6 If a nucleotide is joined to a 7 If a nucleotide is not 8 The process is repeated until every

growing strand, PPi is complementary to the fragment has a complete complementary
released, causing a flash next template base, strand. The pattern of flashes reveals the
of light that is recorded. no PPi is released, and sequence.
no flash of light is recorded.
© 2017 Pearson Education, Inc.
Making Multiple Copies of Gene or DNA Segment

 DNA cloning
‒ Use to work directly with specific genes
‒ Scientists prepare defined DNA segments in multiple identical copies

 Plasmids
‒ Small, circular DNA molecules that replicate separately from bacterial
chromosome

 Gene cloning: production of multiple copies of single gene

‒ Researchers insert DNA into plasmid to produce recombinant DNA
molecule containing DNA from 2 different sources
‒ Reproduction of a recombinant plasmid in a bacterial cell results in
cloning of the plasmid including the foreign DNA
‒ Figure 20.4
© 2017 Pearson Education, Inc.
Figure 20.4 Bacterium 1 Gene inserted Cell containing gene
into plasmid of interest
(a cloning vector)

Bacterial Plasmid
chromosome
DNA of
Recombinant Gene of chromosome
DNA (plasmid) interest (“foreign” DNA)
2 Plasmid put into
bacterial cell

Recombinant
bacterium

3 Host cell grown in culture to form a clone of

cells containing the “cloned” gene of interest

Gene of
interest Protein expressed
from gene of interest
Copies of gene Protein harvested

Gene for pest resistance 4 Basic research Human growth hormone

inserted into plants and various treats stunted growth
applications

Gene used to alter bacteria Protein dissolves blood clots

for cleaning up toxic waste in heart attack therapy

© 2017 Pearson Education, Inc.

Gene cloning (cont’d)

 Cloning vector: plasmid used to clone foreign gene

 Bacterial plasmids
‒ Widely used as cloning vectors

‒ Readily obtained, easily manipulated, and easily introduced

‒ Once in bacteria they multiply rapidly

 Gene cloning useful for amplifying genes to produce

protein product for research and/or medical purposes

© 2017 Pearson Education, Inc.

Making Recombinant DNA Plasmids (fig 20.5)

 Bacterial restriction enzymes cut DNA at restriction sites

‒ “Molecular scissors”

‒ Makes many cuts, yielding restriction fragments

 Useful restriction enzymes cut DNA in staggered way

‒ Producing fragments with at least 1 single-stranded end (sticky end)

‒ Sticky ends can bond with complementary sticky ends

‒ DNA ligase: enzyme that seals bonds between restriction fragments

‒ Enables researchers to join 2 DNA fragments from different sources

© 2017 Pearson Education, Inc.

Figure 20.5
Bacterial
plasmid

Restriction site
5′ 3′
DNA GA AT T C
CT T AAG
3′ 5′
1 Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
3′ 5′
5′ A AT T C 3′
G
C T TA A G
5′ 3′
3′ Sticky 5′
end
5′
2 Base pairing of sticky A AT T C 3′
G
ends produces various G
CT TA A
combinations. 3′
5′
Fragment from different
DNA molecule cut by the
same restriction enzyme
5′ 3′5′ 3′5′ 3′
G AAT T C G AAT T C
C TTAA G C TTAA G
3′ 5′3′ 5′3′ 5′
One possible combination
3 DNA ligase
seals the strands.

5′ 3′

3′ Recombinant DNA molecule 5′

Recombinant
plasmid

© 2017 Pearson Education, Inc.

Animation: Restriction Enzymes

© 2017 Pearson Education, Inc.

Video: Biotechnology Lab

© 2017 Pearson Education, Inc.

Gel Electrophoresis
 Mechanism to
separate and
visualize DNA
fragments
‒ Technique uses
gel to separate
mixture of
nucleic acids or
proteins
‒ Electrical
current passed
through gel
‒ Separated by
size, charge or
other physical
properties
© 2017 Pearson Education, Inc.
Polymerase Chain Reaction (PCR)
 Used to amplify DNA sequence
‒ Produces many copies specific target DNA
sequence

 Three-step cycle (fig 20.7)

‒ Heating (denaturing)
‒ Cooling (annealing)
‒ Replication (extension)
 Chain reaction produces exponentially
growing identical DNA molecules
 Materials
‒ Thermocycler
‒ Taq polymerase: heat-stable DNA
polymerase
‒ Specific primers ID amplification sequence
© 2017 Pearson Education, Inc.
Animation: Cloning a Gene

© 2017 Pearson Education, Inc.

Expressing Cloned Eukaryotic Genes

 After gene cloned, protein produced in larger amounts

for research or practical applications
‒ Cloned genes can be expressed in either bacterial or eukaryotic cells

 Bacterial Expression Systems

‒ Several technical difficulties hinder expression of cloned eukaryotic
genes in bacterial host cells—promoters + control sequence differences

‒ To overcome difficulties, scientists employ expression vector

• Expression vector: cloning vector with highly active bacterial promoter

‒ Additionally, presence of eukaryotic gene intron must be overcome

• Researchers can avoid problem by using cDNA

• cDNA: DNA complementary to the mRNA (contains only exons)

© 2017 Pearson Education, Inc.

Eukaryotic DNA Cloning and Expression Systems
 Molecular biologists can avoid eukaryote-bacterial
incompatibility issues by using eukaryotic cells

‒ Example: use yeast (unicellular eukaryote) as host for gene cloning/expressing

 However, yeasts may not be able to modify expressed

mammalian proteins properly

 Alternative method: cultured mammalian or insect

cells used to express and study proteins

© 2017 Pearson Education, Inc.

Methods to Incorporate Recombinant DNA into Host Cell

 Electroporation

‒ Method to introduce recombinant DNA into eukaryotic cells

‒ Apply brief electrical pulse  temporary holes in plasma membranes

 Microscopic needle injection

‒ DNA inserted into cells using microscopically thin needles

 Once inside cell, DNA incorporated into host cell’s DNA via natural
genetic recombination

© 2017 Pearson Education, Inc.

Cross-Species Gene Expression and Evolutionary
Ancestry
 Remarkable ability of bacteria to express eukaryotic proteins
underscores shared evolutionary ancestry of living species
‒ Example: Pax-6 (gene directing vertebrate eye formation) same gene for insect eye
formation
‒ Same gene sequence, but very different phenotypic eye
 Insect & vertebrate Pax-6 genes can substitute for each other

© 2017 Pearson Education, Inc.

Concept 20.2: Biologists use DNA technology to
study gene expression and function
 Analysis of when/where gene expressed provides
important clues about gene function
 Analyzing Gene Expression
‒ Most straight forward way to discover which genes expressed in
certain cells to identify mRNAs being made

 Studying the Expression of Single Genes

‒ mRNA detected by nucleic acid hybridization with complementary
molecules
‒ Complementary molecules (either DNA or RNA) are nucleic acid probes

 In situ hybridization: use of fluorescent dyes attached to

probes to ID location of specific mRNAs in intact organism
‒ Technique depicted in Figure 20.9
© 2017 Pearson Education, Inc.
Figure 20.9 3′ 5′ 3′ 5′
TA A C G G T T C C A G TCAAGTTGCTCT
···A U U G C CAA G G U C··· · · · A G U U C A A C G A G A· · ·
5′ 3′ 5′ 3′
wg mRNA en mRNA
Cells Cells
expressing expressing
the wg gene the en gene

Head Thorax Abdomen

50 µm T1 T2 T3 A1 A2 A3 A4 A5
Segment
boundary

Head Thorax Abdomen

© 2017 Pearson Education, Inc.
Reverse Transcriptase-PCR (RT-PCR)
 Useful for comparing amounts of specific mRNAs in
several samples simultaneously

 Procedure (fig 20.10)

‒ Reverse transcriptase added to mRNA  complementary DNA (cDNA)

‒ cDNA serves as template for PCR amplification of gene of interest

‒ Products run on gel; complement of mRNA of interest identified (fig 20.11)

‒ RT-PCR enhancement called quantitative RT-PCR (qRT-PCR)

‒ Uses fluorescent dye that glows when bound to ds PCR product

‒ Newer methods of this type do not require gel electrophoresis

‒ Used to discover which tissue producing specific mRNA

© 2017 Pearson Education, Inc.
Figure 20.10 DNA in
nucleus
mRNAs in
cytoplasm

Reverse
transcriptase Poly-A tail
mRNA
5′ A A A A A A 3′
3′ T T T T T
5′
DNA Primer
strand (poly-dT)
5′ AAA AAA 3′
3′ 5′
T T T T T

5′ 3′
3′ 5′
DNA
polymerase
5′ 3′
3′ 5′
cDNA
© 2017 Pearson Education, Inc.
Figure 20.11
Technique
1 cDNA synthesis mRNAs

cDNAs
Primers
2 PCR amplification

Specific
gene

Results Embryonic stages

1 2 3 4 5 6
3 Gel electrophoresis

© 2017 Pearson Education, Inc.

Studying Expression of Interacting Gene Groups

 Gene system approach

‒ Study of expression of thousands of genes at one time
‒ Aids identify networks of gene expression across entire genome

 DNA microarrays compare gene expression patterns (fig 20.12) :

‒ In different tissues
‒ At different times
‒ Under different conditions

 Microarray (fig 20.12)

‒ Tiny amounts of
numerous single-stranded
genes fixed to a glass slide

© 2017 Pearson Education, Inc.

Figure 20.12
cDNA
Each dot is a well
containing identical Genes expressed
copies of DNA in first tissue.
fragments that carry
a specific gene.

Genes expressed
in second tissue.

Genes expressed
in both tissues.

Genes expressed
in neither tissue.

© 2017 Pearson Education, Inc.

RNA Sequencing (RNA-seq)
 More rapid and inexpensive sequencing method
‒ Scientists sequence cDNA samples from different tissues and/or
embryonic stages to determine gene expression differences

 Procedure (fig 20.13)

1) RNAs isolated

2) Cut into short, similar-sized fragments

3) Converted into cDNAs

4) Sequenced

 Scientists can now measure expression of thousands of

genes at one time
 Information provide grander view of how ensembles of genes interact
to form an organism and maintain its vital systems
© 2017 Pearson Education, Inc.
Figure 20.13 1 mRNAs are
isolated from the
tissue being studied.
2 mRNAs are cut into
similar-sized, small
fragments.

3 mRNAs are reverse-

transcribed into cDNAs
of the same size.

4 cDNAs are GGAGAAGTCT CCGTTACTGC

sequenced. AGTCTGCCGT
CCCTGTGGGG GAGAAGTCTG

5 The short sequences are GGAGAAGTCT

CCCTGTGGGG
mapped by computer onto GAGAAGTCTG

the genome sequence. CCGTTACTGC

AGTCTGCCGT

···GGAGAAGTCTGCCGTTACTGCCCTGTGGGGC···

Genome sequence
© 2017 Pearson Education, Inc.
Determining Gene Function
 Disable gene  observe consequences

 Editing Genes and Genomes

‒ in vitro mutagenesis: specific mutations introduced into cloned gene

‒ Observe altering or destroying of gene function

‒ When mutated gene returned to cell, normal gene’s function might be

determined by examining mutant’s phenotype

 CRISPR-Cas9 System

‒ Powerful new technique for gene editing in living cells and organisms

© 2017 Pearson Education, Inc.

CRISPR-Cas9 System (fig 20.14)
 Acts with “guide RNA” made from CRISPR bacterial
system region
 Cuts both strands of any DNA sequence complementary
to guide RNA
 Guide RNA engineered to complement target gene 
target DNA cut
 When cut DNA is repaired, nucleotides may be
introduced or removed, causing gene to be inactivated
 Researchers have also modified the technique so gene
with mutation in it can be repaired
 Done by introducing segment of wild-type gene, which
may be used as template to repair target DNA

© 2017 Pearson Education, Inc.

Figure 20.14
Cas9 active sites NUCLEUS
Guide RNA
complementary
sequence

Guide RNA engineered to 2 5′ 3′

Cas9 protein 5′
“guide” the Cas9 protein 3′ 5′
to a target gene

Part of the
5′ target gene
3′
Complementary Resulting cut
sequence that can in target gene
Active sites that bind to a target gene
can cut DNA
Cas9–guide RNA complex

1
3
Normal
(functional)
gene for use as
a template by
repair enzymes
(a) Scientists can disable (b) If the target gene has
(“knock out”) the target gene a mutation, it can be
CYTOPLASM to study its normal function. repaired.

NUCLEUS
Random nucleotides Normal nucleotides

© 2017 Pearson Education, Inc.

Other Methods for Studying Gene Function
 RNA interference (RNAi)
‒ Additional method for silencing gene expression

‒ Procedure: Synthetic dsRNA molecules matching sequence of

particular gene used to breakdown/block gene’s mRNA

‒ In humans, researchers analyze genomes of many people with certain

genetic condition to try to find nucleotide changes specific to condition

 Genome-wide association studies test for genetic

markers (i.e., sequences that vary among individuals)
‒ SNPs (single nucleotide polymorphisms) and single nucleotide variants among most
useful genetic markers (fig 20.15)

‒ SNPs found frequently associated with particular inherited disorder

alert researchers to most likely location for disease-causing gene

‒ SNPs rarely involved in disease; most often in noncoding regions

© 2017 Pearson Education, Inc.
Figure 20.15

SNP linked to normal allele SNP linked to disease-associated allele

A C
DNA
T G
Normal allele Disease-associated
allele

© 2017 Pearson Education, Inc.

 Helpful Biotechnology Video(s)

Bozeman Science, “Molecular Biology”

https://www.youtube.com/watch?v=yYIZgS-L5Sc
Keep it going…

 Homework

 Review Chapter 20, Concepts 1-2 in textbook

 Laboratory: Gel electrophoresis

 Examination: Chapter 20-21 examination TBD