Outline/Study Guide--Biotechnology
Course objective: Students will be able to explain major methods and
techniques used in molecular genetics to isolate, recombine, amplify, find
and study genes of interest.
Necessary for future material on: last five genetics labs. Helpful for
Directed Studies and Internships in research labs.
In genetic engineering and molecular characterization, what are the
physical obstacles to overcome? What techniques are used to
overcome these obstacles?
Restriction Mapping
• What is a restriction enzyme and why does it cut DNA at specific
sites? (What is the enzyme’s natural function?) What are sticky ends?
Why must two different pieces of DNA be cut with the same enzyme
in order for them to be cloned together?
• What is the function of electrophoresis and how does it separate
molecules?
• What is a restriction map? Could you determine a restriction map from
a DNA fingerprint on an electrophoresis gel?
Biotechnology, cont.
Blotting and Probing
• What is a Southern blot vs. a Northern blot? When would you
use one vs. the other?
• Why is hybridizing important? How does a probe “hybridize”?
• What specific sequence must a single stranded probe have in
order to identify the GOI?
How can one make many copies of the Gene of Interest?
What advantages and disadvantages exist in each?
• How does PCR work? What specific sequence must a PCR
primer have in order to amplify the right gene?
• What is a vector and what properties must it have in order to
be useful? Are all the vectors the same?
• What is the Lac Z system for? How does it work?
• What is a DNA library?
Uses of molecular genetic research
Basic research—understanding
living organisms at the molecular
level (e.g. fruit-fly lifecycle)
Applied research—for solving
specific biomedical problems
(e.g. gene therapy for
hemophilia or cystic fibrosis;
better livestock or agriculture)
Both involve use of recombinant
DNA technology (genetic
engineering)
Obstacles in Molecular Genetics
Goal: characterize the Gene of Interest (GOI)
•
“Macro-isolation” of Gene of Interest
– Restriction Digest of DNA
– Electrophoresis
– DNA Library
•
“Micro-isolation” of Gene of Interest (GOI)
– Southern Blot—Studying DNA (is a particular gene or sequence present in
this genome?)
– Hybridization (using complementarity to find the GOI on a Southern,
Northern, or in PCR)
– Sequencing
•
Amplification of GOI—need to make thousands of copies of one GOI in
order to characterize it.
– Using restriction sites to clone the GOI into a vector
– PCR—Polymerase Chain Reaction
•
Understanding the GOI
– Northern Blot—studying RNA (Which tissue expresses a particular RNA?)
– Western Blot—studying protein (does this tissue express a specific
protein?)
– RT PCR – how much expression of the GOI is found in this tissue?
:::::::
Restriction enzymes recognize
specific sequences (restriction
sites) and generate “sticky” or
“blunt” ends
:::::::
“sticky” ends (un-paired ss DNA)
How DNA from
different sources
is put together
(like Fig 19.1, Brooker)
Example of restriction sites within a double-stranded
DNA sequence
nt position
1
1
GATCACAGGTCTATCACCCTATTAACCACTCACGGGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGC
CTAGTGTCCAGATAGTGGGATAATTGGTGAGTGCCCTCGAGAGGTACGTAAACCATAAAAGCAGACCCCCCATACGTGCG
BanII
80
80
81
81
GATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTATTATTTA
CTATCGTAACGCTCTGCGACCTCGGCCTCGTGGGATACAGCGTCATAGACAGAAACTAAGGACGGAGTAGGATAATAAAT
Bsp1286I
160
160
161
161
TCGCACCTACGTTCAATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATAATAATA
AGCGTGGATGCAAGTTATAATGTCCGCTTGTATGAATGATTTCACACAATTAATTAATTACGAACATCCTGTATTATTAT
SspI
240
240
241
241
ACAATTGAATGTCTGCACAGCCACTTTCCACACAGACATCATAACAAAAAATTTCCACCAAACCCCCCCTCCCCCGCTTC
TGTTAACTTACAGACGTGTCGGTGAAAGGTGTGTCTGTAGTATTGTTTTTTAAAGGTGGTTTGGGGGGGAGGGGGCGAAG
320
320
321
321
TGGCCACAGCACTTAAACACATCTCTGCCAAACCCCAAAAACAAAGAACCCTAACACCAGCCTAACCAGATTTCAAATTT
ACCGGTGTCGTGAATTTGTGTAGAGACGGTTTGGGGTTTTTGTTTCTTGGGATTGTGGTCGGATTGGTCTAAAGTTTAAA
EaeI
400
400
401
401
TATCTTTTGGCGGTATGCACTTTTAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATACTACTAAT
ATAGAAAACCGCCATACGTGAAAATTGTCAGTGGGGGGTTGATTGTGTAATAAAAGGGGAGGGTGAGGGTATGATGATTA
480
480
Restriction Digested DNA
stained with Ethidium Bromide
on an Agarose Gel
Fragments of DNA
0 1
What DNA fragments
are generated when cut
by restriction digestion?
What would the
electrophoresis gel look
like if these fragments
were separated by size?
2
3
4
5
6
Restriction Digested DNA
stained with Ethidium Bromide
on an Agarose Gel
(Ethidium bromide
stains all DNA; DNA
probes only highlight
complementary
sequences)
But which fragment has
my gene of interest? How
can I find the right
fragment? (use a DNA
probe)
Obstacles in Molecular Genetics
Goal: characterize the Gene of Interest (GOI)
•
“Macro-isolation” of Gene of Interest
– Restriction Digest of DNA
– Electrophoresis
– DNA Library
•
“Micro-isolation” of Gene of Interest (GOI)
– Southern Blot—Studying DNA (is a particular gene or sequence present in
this genome?)
– Hybridization (using complementarity to find the GOI on a Southern,
Northern, or in PCR)
– Sequencing
•
Amplification of GOI—need to make thousands of copies of one GOI in
order to characterize it.
– Using restriction sites to clone the GOI into a vector
– PCR—Polymerase Chain Reaction
•
Understanding the GOI
– Northern Blot—studying RNA (Which tissue expresses a particular RNA?)
– Western Blot—studying protein (does this tissue express a specific
protein?)
– RT PCR – how much expression of the GOI is found in this tissue?
I need lots of copies of my gene of
interest. How can I do this?
Cloning into a vector
•Insert your GOI into
another organism’s
chromosome
•Hitch-hike replication
•May also get host
organism to express
protein of GOI.
PCR
•Cell free
•Test tube
•Fast
•Limited to already
known sequences
•Can’t directly make
a protein from PCR
Cloning of GOI
into vector, then
into host
organism
How do I know which
restriction enzyme to
use? (hint: the foreign
GOI must “fit” into the
vector)
Host organism
makes lots of copies
of the vector + GOI
What must a host
chromosome have in
order to be useful as
a vector?
What do you think would happen
if this vector did not have an ori?
Examples of different types of vectors and host
organisms
Genome of
Interest
Human
genome
Host
Organism
Vector
Size of
Insert
Yeast
YAC (Yeast
100 - 2000 kb
Artificial
Chromosome)
Worm
(nematode)
genome
Firefly
genome
Bacteria
Cosmid
< 45 kb
Virus
l phage
< 20 kb
Drosophila
genome
Bacteria
Plasmid
< 15 kb
How do you know when the vector actually has an insert? Use the
Lac Z-system
Chromosomal DNA
from human cells
ampR gene
Plasmid Vector
lacZ gene
Gene of interest
Unique
restriction
site
Origin of
replication
Cut the DNAs with same restriction enzyme.
Mix the DNAs together. Allow time for
sticky ends to base-pair. Add DNA ligase
to covalently link the DNA backbones.
Vector with
another fragment
of chromosomal DNA
or
Recircularized
vector
Vector with
GOI
or
Recombinant vectors
Brooker, fig 19.2
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Cloning a gene into a vector, cont.
Vector with the
gene of interest
Recircularized
vector
or
E. coli cell
(treated with
permeabilizing
agents)
Vector with
another fragment
of chromosomal DNA
or
Mix DNA with E.coli.
Permeable E. Coli take up DNA [Transformation]
Recircularized vector
without an insert
Plate cells on media containing
X-Gal, IPTG, and ampicillin.
Recombinant
vector
with an
insert
Blue colony
Brooker, fig 19.2
White colony
Each bacterial colony is derived from a single cell;
so all the cells in a colony are genetically identical.
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Lac Z System cont. White colonies contain vector with GOI.
Talk with your labmate or student next to you--predict
the types of colonies that would grow under these
conditions
Bacteria-no
vector
Bacteria”empty” vector
Bacteria-vector
with GOI
(no antibiotic)
(All with
Ampicillin)
Plain agar
Plain agar
X-Gal
Plain agar
X-Gal
Plain agar
Plain agar
X-Gal
Plain agar
X-Gal
Talk with your labmate or student next to you--predict
the types of colonies that would grow under these
conditions
Bacteria-no
vector
Bacteria”empty” vector
Bacteria-vector
with GOI
(no antibiotic)
Plain Agar
Bacteria-no
vector
(All with
Ampicillin)
Agar
ampicillin
Agar
X-Gal
Bacteria”empty” vector
Agar
Ampicillin
X-Gal
Agar
X-Gal
Bacteria-vector
with GOI
Agar
Ampicillin
X-Gal
Obstacles in Molecular Genetics
Goal: characterize the Gene of Interest (GOI)
•
“Macro-isolation” of Gene of Interest
– Restriction Digest of DNA
– Electrophoresis
– DNA Library
•
“Micro-isolation” of Gene of Interest (GOI)
– Southern Blot—Studying DNA (is a particular gene or sequence present in
this genome?)
– Hybridization (using complementarity to find the GOI on a Southern,
Northern, or in PCR)
– Sequencing
•
Amplification of GOI—need to make thousands of copies of one GOI in
order to characterize it.
– Using restriction sites to clone the GOI into a vector
– PCR—Polymerase Chain Reaction
•
Understanding the GOI
– Northern Blot—studying RNA (Which tissue expresses a particular RNA?)
– Western Blot—studying protein (does this tissue express a specific
protein?)
– RT PCR – how much expression of the GOI is found in this tissue?
Which fragment has my gene of
interest? How can I find the right
fragment?
Electrophoresis gel
Fig 18-5a Southern
blotting: blotting DNA
fragments onto a membrane
so that it can be probed
Which fragment has my gene of
interest? How can I find the right
fragment?
Electrophoresis gel
Southern blotting:
blotting DNA fragments
onto a membrane so that it
can be probed
DNA has now been transferred to
membrane.
Identifying the
gene of interest
(“probing” with a
labeled tag)
Which fragment will the genespecific probe bind to?
I want to identify
the b-globin gene.
And I want to
identify the
insulin gene.
Expose to film to “see”
the radioactive probe
How must the single stranded probes be
different in order for these two
investigators to identify their specific GOI?
Pretend this is a Southern Blot (membrane with DNA bound to it).
To which piece of ssDNA will the probe hybridize (bind)?
5’-CTAATGT-3’
5’-GATTACA-3’
Radioactively labeled
probe
3’-CGTTATA-5’
5’-CGATTAT-3’
5’-GATTACA-3’
3’-CTAATGT-5’
3’-GATTACA-5’
Membrane with bound
single stranded DNA
Pretend this is a Southern Blot (membrane with DNA bound to it).
To which piece of ssDNA will the probe hybridize (bind)?
5’-CTAATGT-3’
5’-GATTACA-3’
Radioactively labeled
probe
3’-CGTTATA-5’
5’-CGATTAT-3’
5’-GATTACA-3’
5’-GATTACA-3’
3’-CTAATGT-5’
3’-GATTACA-5’
Membrane with bound
single stranded DNA
Photography Film exposed to probed
Southern Blot
Bands of DNA that
bound weakly to the
radioactive probe
Bands of DNA that
bound to the
radioactive probe
Southern Blots Can Be Used For
Paternity Tests
Was Ronald Scott Kidnapped from the
Larsons?
“Northern blots show the
expression pattern of the GOI.”
Modern day “Southerns” and “Northerns”—
microarray analysis
Two distinct forms of large B-cell lymphoma are shown by the
expression pattern: GC B-like DLBCL (orange) and Activated B-like
DLBCL (blue)
ASH ALIZADEH et al. 2000
Screening a gene library for the GOI
• DNA (or Gene) Library—collection of host organisms containing
DNA vectors with GOI inserts from different parts of the Genome of
Interest
• Library allows smaller pieces of genome-of-interest to be replicated
inside organism and eventually selected based on size, sequence,
or sometimes functional protein.
XP
(colorless)
+
X
(black)
Pi
Alkaline phosphatase
Western Blots
Secondary antibody
Primary antibody
Protein of interest
(a) Interactions between the protein of interest and antibodies
Obstacles in Molecular Genetics
Goal: characterize the Gene of Interest (GOI)
•
“Macro-isolation” of Gene of Interest
– Restriction Digest of DNA
– Electrophoresis
– DNA Library
•
“Micro-isolation” of Gene of Interest (GOI)
– Southern Blot—Studying DNA (is a particular gene or sequence present in
this genome?)
– Hybridization (using complementarity to find the GOI on a Southern,
Northern, or in PCR)
– Sequencing
•
Amplification of GOI—need to make thousands of copies of one GOI in
order to characterize it.
– Using restriction sites to clone the GOI into a vector
– PCR—Polymerase Chain Reaction
•
Understanding the GOI
– Northern Blot—studying RNA (Which tissue expresses a particular RNA?)
– Western Blot—studying protein (does this tissue express a specific
protein?)
– RT PCR – how much expression of the GOI is found in this tissue?
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
C
A
C
C
G
T
A
A
G
G
A
C
T
G
(b) Output from automated sequencing
Figure 19.17b
19 - 77
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Sequence to
be analyzed
(target DNA)
Primer
Primerannealing
site
5′
Recombinant vector
Many copies of the recombinant vector, primer,
dNTPs, fluorescently labeled dideoxynucleotides,
and DNA polymerase are mixed together.
Incubate to allow the synthesis of DNA.
CACCGTAAGGACTddG
CACCGTAAGGACddT
CACCGTAAGGAddC
CACCGTAAGGddA
CACCGTAAGddG
CACCGTAAddG
CACCGTAddA
CACCGTddA
CACCGddT
CACCddG
CACddC
CAddC
CddA
ddC
Nucleotides added to primer
Separate newly made strands by
gel electrophoresis.
Sequence
deduced
from gel
Laser
beam
G
T
C
A
G
G
A
A
T
G
C
C
A
C
Fluorescence
detector
(a) Automated DNA sequencing
19 - 76
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
O
O–
P
O
O
–
O
P
O–
O
O
P
O–
Adenine
O
CH2
5′
4′
H
3′
H
O
H
1′
H
2′
H
H
2′, 3′-Dideoxyadenosine triphosphate (ddA)
Figure 19.16
19 - 73
Obstacles in Molecular Genetics
Goal: characterize the Gene of Interest (GOI)
•
“Macro-isolation” of Gene of Interest
– Restriction Digest of DNA
– Electrophoresis
– DNA Library
•
“Micro-isolation” of Gene of Interest (GOI)
– Southern Blot—Studying DNA (is a particular gene or sequence present in
this genome?)
– Hybridization (using complementarity to find the GOI on a Southern,
Northern, or in PCR)
– Sequencing
•
Amplification of GOI—need to make thousands of copies of one GOI in
order to characterize it.
– Using restriction sites to clone the GOI into a vector
– PCR—Polymerase Chain Reaction
•
Understanding the GOI
– Northern Blot—studying RNA (Which tissue expresses a particular RNA?)
– Western Blot—studying protein (does this tissue express a specific
protein?)
– RT PCR – how much expression of the GOI is found in this tissue?
(a) The outcome of a PCR experiment
Chromosomal DNA
Overview of
PCR
Primer binding near one
end of gene
A different primer binding
near other end of gene
Many PCR cycles
(b) The 3 steps of a PCR cycle
Many copies of GOI, flanked
by regions where primers
bind.
Site where forward
primer binds
Site where reverse
primer binds
Template DNA
Denature: Separate DNA
with high temperature.
Primer annealing: Lower temperature,
allows primers to bind template DNA.
Forward
primer
5′
3′
3′
5′
5′
3′
3′
5′
5′
3′
3′
5′
3′
3′
5′
Reverse
primer
5′
3′
5′
T GCA C CA GC A T C C GA T C
ACG T GGT C G T AGG C T AG
3′
Primer extension: Incubate at temperature
that allows DNA synthesis to occur.
5′
3′
3′
5′
3′
5′
3′
5′
Brooker Fig 19.4
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
5′
Reverse primer
Necessary items for PCR
Forward primer
Reverse primer
Free nucleotides
T A
G
G
T
C
C
C
A T
G
A
DNA Polymerase
Enzyme (“Taq”)
Template DNA
PCR: Amplifying DNA in a test tube
Reverse
primer
5’
3’
5’
3’
Template
DNA
3’
5’
3’
5’
3’
5’
5’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
5’
3’
3’
5’
3’
3’
5’
3’
5’
5’
3’
5’
5’
3’
3’
Forward
primer
3’
3’
5’
5’
3’
5’
3’
5’
3’
3’
5’
PCR: Amplifying DNA in a test tube
Reverse
primer
5’
3’
5’
3’
Template
DNA
3’
5’
3’
5’
3’
5’
5’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
5’
3’
3’
5’
3’
3’
5’
3’
5’
5’
3’
5’
5’
3’
3’
Forward
primer
3’
3’
5’
5’
3’
5’
3’
5’
3’
3’
5’
PCR: Amplifying DNA in a test tube
Reverse
primer
5’
3’
5’
3’
Template
DNA
3’
5’
3’
5’
3’
5’
5’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
5’
3’
3’
5’
3’
3’
5’
3’
5’
5’
3’
5’
5’
3’
3’
Forward
primer
3’
3’
5’
5’
3’
5’
3’
5’
3’
3’
5’
PCR: Amplifying DNA in a test tube
Reverse
primer
5’
3’
5’
3’
Template
DNA
3’
5’
3’
5’
3’
5’
5’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
5’
3’
3’
5’
3’
3’
5’
3’
5’
5’
3’
5’
5’
3’
3’
Forward
primer
3’
3’
5’
5’
3’
5’
3’
5’
3’
3’
5’
PCR: Amplifying DNA in a test tube
Reverse
primer
5’
3’
5’
3’
Template
DNA
3’
5’
3’
5’
3’
5’
5’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
5’
3’
3’
5’
3’
3’
5’
3’
5’
5’
3’
5’
5’
3’
3’
Forward
primer
3’
3’
5’
5’
3’
5’
3’
5’
3’
3’
5’
PCR: Amplifying DNA in a test tube
Reverse
primer
5’
3’
5’
3’
Template
DNA
3’
5’
3’
5’
3’
5’
5’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
5’
3’
3’
5’
3’
3’
5’
3’
5’
5’
3’
5’
5’
3’
3’
Forward
primer
3’
3’
5’
5’
3’
5’
3’
5’
3’
3’
5’
PCR: Amplifying DNA in a test tube
Reverse
primer
5’
3’
5’
3’
Template
DNA
3’
5’
3’
5’
3’
5’
5’
5’
5’
3’
5’
3’
5’
3’
5’
3’
5’
5’
3’
3’
5’
3’
3’
3’
3’
5’
5’
5’
3’
5’
5’
3’
3’
Forward
primer
3’
3’
5’
5’
3’
5’
3’
5’
3’
3’
5’
I want to amplify the
b-globin gene.
What specific thing must be different in these two
investigators’ PCR reactions?
And I want to
amplify the insulin
gene.
How do I design primers in order to detect
(or amplify) specific genes?
•
1.
2.
3.
4.
5.
6.
www.ncbi.nlm.nih.gov/omim/
search under “hemoglobin”
Click on HBB (hemoglobin-beta). 1419000?
Click to the map locus (chromosome 11p~~)
Click on [141900] on the chromosome itself
“RefSeq Gene” in right side bar
Use sequence information to design a
complementary primer for the 5’ end of the
gene, and 3’ end of gene.
Difference between “Genomic” and “cDNA”
Genomic—DNA exactly as found in the genome,
including introns and other non-coding portions of DNA
INTRONS-junk DNA
3’ untranslated
region
5’ untranslated region
EXONS-protein coding
cDNA—complementary DNA--made from mature mRNA and thus
containing only coding parts of gene
Obstacles in Molecular Genetics
Goal: characterize the Gene of Interest (GOI)
•
“Macro-isolation” of Gene of Interest
– Restriction Digest of DNA
– Electrophoresis
– DNA Library
•
“Micro-isolation” of Gene of Interest (GOI)
– Southern Blot—Studying DNA (is a particular gene or sequence present in
this genome?)
– Hybridization (using complementarity to find the GOI on a Southern,
Northern, or in PCR)
– Sequencing
•
Amplification of GOI—need to make thousands of copies of one GOI in
order to characterize it.
– Using restriction sites to clone the GOI into a vector
– PCR—Polymerase Chain Reaction
•
Understanding the GOI
– Northern Blot—studying RNA (Which tissue expresses a particular RNA?)
– Western Blot—studying protein (does this tissue express a specific
protein?)
– RT PCR – how much expression of the GOI is found in this tissue?
What does each technique “look like”?
1.)
Electrophoresis
2.)
Restriction Digest
3.)
PCR
4.)
Southern Blot
5.)
Screening a genomic library
6.)
Hybridization with radioactive probe
7.) Cloning of insert DNA into vector,
transformation into host organism
8.) Northern Blot
Which technique(s) do you use for each purpose (there may be
more than one way to solve a problem)?
1. Identifying specific sequences, regulatory regions or genes
(including introns).
2.)
Determining tissue-specific or stage-specific gene
expression.
3.)
Cutting of DNA at specific sequences
4.)
Separation of DNA fragments by size
5.)
Amplification of specific DNA sequences
6.)
Expressing an exogenous (foreign) protein in a host or
transformed organism.
Discuss with student next to you
From the techniques discussed so far, how
would you determine if Drosophila used
hemoglobin?
If they do, how would you determine when
[what stage] would they use it? Where in
their body would they use it?
Suppose that you just graduated from
college and have started working in a
biotechnology firm. Your first job
assignment is to clone the pig gene for the
hormone prolactin. Briefly explain a
strategy you might use to find and clone
the pig gene for prolactin.
Vectors require a selectable marker such as antibiotic
resistance so that:
a.
the host cell will replicate the vector and GOI [Gene-ofinterest] along with its own chromosome.
b.
host cells taking up vector can be identified against host
cells that have not.
c.
the host cell is able to have a GOI DNA fragment inserted
into it
d.
host cells with the vector are able to express the GOI.
e.
none of the above
Vectors require a selectable marker such as antibiotic resistance
so that:
a.
the host cell will replicate the vector and GOI [Gene-ofinterest] along with its own chromosome.
b.
host cells taking up vector can be identified against host
cells that have not.
c.
the host cell is able to have a GOI DNA fragment inserted
into it
d.
host cells with the vector are able to express the GOI.
e.
none of the above
Recombinant DNA Technology: practice questions
The following comprehension questions (at end of each chapter section) in
Brooker, Concepts of Genetics are recommended:
• Comprehension Questions (at end of each section): 19.1,19.2, 19.3.
Answers to Comprehension Questions are at the very end of every chapter.
• Solved Problems at end of chapter (answers included): S1, S2, S4
• Conceptual questions and Experimental/Application Questions at end of
chapter (answers found by logging into publisher’s website, or find them in
the book):
• Concepts—C1, C2, C4
• Application/Experimental Questions—E1, E2, E3, E4, E5, E6, E12,
E13, E14, E15, E16, E17, E24, E25,
• A little more challenging—E18, E19 (but first you have to understand
alternative splicing [in Ch 17], E21, E22