000_Front Matter_GenSci_001-036 DNA Sci Chap 01 10/3/12 3:36 PM Page vii
Preface
The DNA Learning Center’s effort to develop lab experiments for high school and colleges dates to 1985, when Dave Micklos teamed with Greg Freyer to develop a
sequence of experiments to make and analyze a recombinant DNA molecule. The initial testing was done in Rich Roberts’ lab, well before he won the Nobel Prize, and
incorporated key insights on inserting DNA into bacteria from the dean of transformation, Doug Hanahan. The lab sequence was initially a slim lab manual called
“Recombinant DNA for Beginners,” whose title was derived from the Graham Nash
album “Songs for Beginners.” It expressed perfectly our ideal of making complicated
gene technology accessible to novices.
Within a year, we were training teachers across the country on summer tours of
one, then two, Vector vans. These were customized Ford Econoline vans that packed
enough equipment, reagents, and supplies to convert any general science lab into a
molecular genetics lab. Along the way, we developed a complementary minitext that
presented the concepts behind the labs, as well as extensions of recombinant DNA
technology in basic and applied research. The much expanded work was formally
published in 1991 as the Cold Spring Harbor Laboratory Press book DNA Science.
That name, which Jim Watson threw out over lunch one day, seemed to capture the
excitement of a new science based on the ability to manipulate and understand the
DNA molecule.
DNA Science, now in its second edition, has sold more than 90,000 copies and is
credited with helping to catalyze the movement to bring hands-on experiments with
DNA into high school and beginning college classrooms. Two experiments found their
way into the Advanced Placement biology curriculum, giving these experiments a
nationwide audience. Stand-alone kits, developed with Carolina Biological Supply
Company, reach well over 100,000 students per year.
The experiments in DNA Science are based exclusively on bacterial genetic systems. Now, Genome Science aims to take students to a higher level of biological and
technological integration—to study the function of eukaryotic genes and genomes.
Nineteen laboratories focus on four revolutionary technologies—polymerase chain
reaction, DNA sequencing, RNA interference (RNAi), and bioinformatics—across
three eukaryotic systems: humans, plants, and Caenorhabditis elegans. All labs stress
the modern synthesis of molecular biology and computation, integrating in vitro
experimentation with in silico bioinformatics. In addition to well-tested biochemical
methods, Genome Science introduces DNA Subway, an intuitive bioinformatics platform that makes easy work of gene and genome analysis.
vii
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viii / Preface
The four major techonologies are organized into stand-alone chapters with
extensive text introductions that place related labs into a common historical and conceptual framework. This modular approach provides options to develop new courses
or to integrate labs into existing courses or student research. We especially hope that
these protocols will help educators to extend research to classroom settings and distribute experiments in which multiple classes analyze and contribute to common data
sets. DNA barcoding is especially amenable to “campaigns” in which many students
contribute to understanding diversity within a common biogeographical unit.
Genome Science borrows many user-friendly features from its predecessor, including flow charts, marginal notes, reagent recipes, and extensive instructor information.
To ease implementation, most labs are available as ready-to-use kits from Carolina
Biological Supply Company. In addition, like its predecessor, Genome Science aims to
help beginners use modern tools to explore the unseen world of genes and genomes.
In contemplating the cosmos in 1927, the great mathematical geneticist J.B.S.
Haldane famously said, “My own suspicion is that the universe is not only queerer
than we suppose, but queerer than we can suppose.” Had he been alive today, Haldane
would almost certainly have the same suspicion about the genomes of higher organisms. In this sense, genome scientists are the new cosmologists of biology, uncovering
the strange and beautiful structure of the genetic material that runs through all life.
Happy explorations.
DAVID MICKLOS
BRUCE NASH
UWE HILGERT
Cold Spring Harbor, New York
March 2012
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LABORATORY
Culturing and Observing C. elegans
4.1
▼ OBJECTIVES
This laboratory demonstrates several important concepts of modern biology. During
the course of this laboratory, you will
•
Learn about the use of model organisms in research.
•
Observe development and identify specific developmental stages in Caenorhabditis
elegans.
•
Explore the relationship between genotype and phenotype.
In addition, this laboratory utilizes several experimental methods in modern biological
research. You will
•
Use sterile technique to isolate and grow pure cultures of bacteria and C. elegans.
•
Use antibiotic selection to maintain a recombinant bacterial culture.
•
Use dissecting microscopes to observe and analyze cultures of bacteria and C. elegans.
INTRODUCTION
Sydney Brenner won a
Nobel prize for establishing
C. elegans as a model
organism.
(Photo courtesy of Matthew
Meselson.)
A human is a complicated organism, and most molecular genetic experiments would
be either technically difficult or unethical to perform on human subjects. For these
reasons, biologists often use simpler “model” organisms that are easy to culture and
manipulate in the laboratory. Despite obvious physical differences, model organisms
and humans share many key biochemical and physiological functions that have been
conserved (preserved) during evolution. The nematode worm C. elegans is one of several organisms commonly studied by biological researchers today.
C. elegans is a microscopic roundworm. Although some roundworms are parasitic, C. elegans is a free-living worm that feeds on soil bacteria. These worms grow
quickly, developing from embryo to adult in 3 d. C. elegans is a simple animal with
only ~1000 cells, and scientists know exactly how each of those cells develops from the
fertilized egg. C. elegans was the first multicellular organism to have its entire genome
sequenced, with the surprising finding that 40% of its genes have human matches.
Mating animals, isolating genes, and introducing foreign DNA are much easier in C.
elegans than in more complicated animals. All of these features make C. elegans a great
model for understanding how cells divide, develop, and take on specialized tasks in
higher (eukaryotic) organisms. Recently, the discovery that any of the organism’s
genes can be “silenced” using a technique called RNA interference (RNAi) has made
C. elegans an ideal organism to quickly determine the functions of genes identified by
sequencing the genome.
551
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Embryonic development in C. elegans. This series of images shows
different stages during embryonic development. Fertilization to
hatching takes just 14 h.
(Reprinted, with permission, from O’Rourke M, Bowerman B. 2005. Nature
434: 444–445; ©Macmillan.)
This laboratory introduces C. elegans and describes methods required for its culture; these techniques and familiarity with C. elegans are prerequisites for RNAi experiments. Included are techniques for growing Escherichia coli cells and preparing plates
to feed C. elegans. The most common strain of bacteria used to feed worms is the E.
coli strain OP50, which is grown on standard LB plates or in LB broth. For RNAi
experiments, specialized strains of bacteria, each containing a plasmid that expresses
a gene-specific double-stranded RNA (dsRNA), are fed to worms to trigger gene
silencing.
A technique for spreading bacteria onto standard plates to isolate single cells from
one another is described. Each cell then reproduces to form a visible colony composed
of genetically identical clones. Small-scale suspension cultures of E. coli are then
grown by overnight incubation using cells derived from a single colony, which minimizes the chance of using a cell mass contaminated with a foreign microorganism.
These overnight cultures are used to inoculate, or seed, specialized “NGM (nematode
growth medium)-lite” agar plates on which the worms are grown. E. coli OP50 is seeded to NGM-lite plates. In addition, RNAi strains expressing dsRNA corresponding to
three genes, dpy-11, bli-1, and unc-22, are seeded to NGM-lite plates with ampicillin
and isopropyl-β-D-thiogalactopyranoside (IPTG). Ampicillin selects for bacteria carrying the RNAi plasmid, and IPTG triggers dsRNA expression from the plasmid in the
bacteria. In Laboratory 4.2, the function of these three genes is assayed when RNAi is
induced by culturing worms on these plates.
Two methods to propagate worms are described. Worms growing on an NGM-lite
plate are transferred by cutting out a section of the medium and placing it on the surface of a new plate. This “chunking” method is a rapid way to move multiple worms
from a plate where the E. coli food source has been consumed to a plate with a fresh
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Culturing and Observing C. elegans / 553
lawn of bacteria. Individual worms can be transferred to fresh plates using the flattened tip of a platinum wire; this technique for “picking” individual worms is the
starting point for genetic crosses and RNAi experiments.
Finally, wild-type C. elegans hermaphrodites are observed using microscopy, and
their morphology, behavior, and life cycle are analyzed. Abnormal morphology and
behavior of mutant worms are also observed.
FURTHER READING
Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics 77: 71–94.
C. elegans Sequencing Consortium. 1998. Genome sequence of the nematode C. elegans: A platform
for investigating biology. Science 282: 2012–2018.
De Melo JV, De Souza W, Peixoto CA. 2002. Ultrastructural analyses of the Caenorhabditis elegans
DR 847 bli-1(n361) mutant which produces abnormal cuticle blisters. J Submicrosc Cytol Pathol
34: 291–297.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. 1998. Potent and specific genetic
interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806–811.
Ko FCF, Chow KL. 2002. A novel thioredoxin-like protein encoded by the C. elegans dpy-11 gene is
required for body and sensory organ morphogenesis. Development 129: 1185–1194.
Kramer JM, French RP, Park EC, Johnson JJ. 1990. The Caenorhabditis elegans rol-6 gene, which
interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen.
Mol Cell Biol 10: 2081–2089.
Moerman DG, Benian GM, Waterston RH. 1986. Molecular cloning of the muscle gene unc-22 in
Caenorhabditis elegans by Tc1 transposon tagging. Proc Natl Acad Sci 83: 2579–2583.
Moerman DG, Benian GM, Barstead RJ, Schriefer LA, Waterston RH. 1988. Identification and intracellular localization of the unc-22 gene product of Caenorhabditis elegans. Genes Dev 2: 93–105.
551-564_Lab04.1_GenSci_Genome Science 10/10/12 4:20 PM Page 554
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PLANNING AND PREPARATION
The following table will help you to plan and integrate the different experimental
methods.
Experiment
Day
Time
Activity
2 or more d
before Part I
90 min
Prelab:
Prepare LB, LB/amp, NGM-lite, and NGM-lite/amp
+ IPTG plates.
1–2 d
before Part I
30 min
Prelab:
Streak starter plates for OP50 and RNAi feeding strains
(dpy-11, bli-1, unc-22).
1
20 min
Prelab:
Set up student stations.
Stage A: Culturing E. coli
I. Streak E. coli to obtain
single colonies
II. Grow E. coli overnight
30 min
Lab:
Streak plates.
15–20 h
Postlab:
Incubate plates.
15 min
Prelab:
Aliquot LB and LB/amp.
15 min
Lab:
Prepare overnight cultures.
12–48 h
Postlab:
Incubate cultures.
30 min
Lab:
Seed NGM-lite and NGM-lite/amp + IPTG plates.
24–36 h
Postlab:
Incubate plates.a
6
30 min
Prelab:
Chunk wild-type worms to OP50-seeded NGM-lite
plates.b
8
20 min
Prelab:
Set up student stations.
15 min
Lab:
Chunk worms to OP50-seeded NGM-lite plates.
Incubate for 48 h.
20 min
Prelab:
Make worm picks.
30 min
Lab:
Pick L4 worms to fresh OP50-seeded NGM-lite plates.
20 min
Prelab:
Set up student stations.
45 min
Lab:
Study the morphology, behavior, and life cycle of
wild-type worms under a dissecting microscope.
30 min
Prelab:
Chunk wild-type and mutant C. elegans strains (rol-6,
bli-1, unc-22, dpy-11) to OP50-seeded NGM-lite
plates.
2
cultures
III. Seed NGM-lite and NGM-
Set up student stations.
4
lite/amp + IPTG plates
with E. coli
Stage B: Culturing C. elegans
I. Chunk wild-type C.
elegans
II. Pick individual C.
10
elegans
Set up student stations.
Stage C: Observing Wild-Type and Mutant C. elegansc
I. Observe the C.
12–13
elegans life cycle
II. Observe C. elegans
mutants
9
Set up student stations.
12–13
aAfter
45 min
Lab:
Examine wild-type and mutant worms; identify differences in development rate, morphology, or movement.
incubation, seeded plates from Part III of Stage A can be stored in sealed containers for several weeks at 4°C.
enough plates for each student are available, this extra round of chunking may be skipped. (It is a good idea to prepare extra plates,
because some may become contaminated during preparation or student work.)
cBoth procedures in Stage C require wild-type worms from Parts I or II of Stage B. If you are not completing Parts I or II of Stage C immediately after Stage B, transfer small chunks of wild-type worms to OP50-seeded NGM-lite plates 2 d before completing Stage C.
bIf
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OVERVIEW OF EXPERIMENTAL METHODS IN STAGE A: CULTURING E. COLI
I. STREAK E. COLI TO OBTAIN SINGLE COLONIES
FLAME
loop
COOL
loop
FLAME
loop
COOL
loop
SCRAPE UP
cell mass
STREAK 2
COOL
loop
STREAK 1
FLAME
loop
COOL
loop
STREAK 4
INCUBATE
15–20 h
FLAME
loop
STREAK 3
37°C
II. GROW E. COLI OVERNIGHT CULTURES
FLAME
pipette
REMOVE
cap and
flame
tube
mouth
REMOVE
cap and
flame tube
mouth
EXPEL
sample
REMOVE
cap and
flame tube
mouth
DROP
tip-first
into tube
WITHDRAW
sample
REFLAME
and
replace
cap
REFLAME
and
replace
cap
REFLAME
and
replace
cap
SCRAPE UP
cell mass with
pipette tip
INCUBATE
12–24 h
with
shaking
37°C
III. SEED NGM-LITE AND NGM-LITE/AMP + IPTG PLATES WITH E. COLI
FLAME
pipette
DROP
sample on
center of
agar
REMOVE
cap and
flame
tube
mouth
WITHDRAW
sample
INCUBATE
24–36 h at room temperature
REFLAME
and
replace
cap
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STAGE A: CULTURING E. COLI
◗ I. Streak E. coli to Obtain Single Colonies
REAGENTS, SUPPLIES, & EQUIPMENT
For each group
Bunsen burner
Inoculating loop
LB agar plate
3 LB + ampicillin <!> (LB/amp) plates
Permanent marker (red)
To share
“Bio bag” or heavy-duty trash bag
CAUTION! When using an
open flame, take appropriate precautions. Make sure
that any loose clothing is
secured and tie long hair
back. Do not lean over the
flame.
Make it a habit to always
flame the loop one last
time.
Bleach (10%) <!> or disinfectant (e.g., Lysol)
E. coli OP50 culture
E. coli RNAi feeding strain cultures (dpy-11,
bli-1, and unc-22)
Incubator set at 37°C
See Cautions Appendix for appropriate handling of materials marked with <!>.
1. Use a red permanent marker to label the bottom of the LB agar plate with your
group number, the date, and “OP50.”
2. Hold the inoculating loop like a pencil and sterilize the loop in the Bunsen burner flame until it glows red hot.
3. Remove the lid from the E. coli OP50 culture plate with your free hand. Do not
place the lid on the lab bench; hold the lid face down just above the culture plate
to help to prevent contaminants from falling on the plate or lid.
4. Stab the inoculating loop into a clear area of the E. coli OP50 culture plate several times to cool it.
5. Use the loop tip to scrape a visible cell mass from a bacterial colony on the E. coli
OP50 culture plate. Do not gouge the agar. Replace the lid on the E. coli OP50 culture plate.
6. Lift the lid of the new LB agar plate just enough to perform streaking as described
below. The object is to serially dilute the bacteria with each successive streak, so
that individual cells are separated in at least one of the streaks. Do not place the
lid on the lab bench; replace the plate lid after each streak.
i. Streak 1: Glide the loop tip back and forth across the surface of the LB agar to
make a streak across the top quarter of the plate. Avoid gouging the agar.
ii. Streak 2: Reflame the inoculating loop and cool it by stabbing it into the agar
away from the first (primary) streak. Draw the loop tip through the end of the
primary streak and, without lifting the loop, make a zigzag streak across one
quarter of the agar surface.
iii. Streak 3: Reflame the loop and cool it in the agar. Draw the loop tip once
through the end of the previous streak and make another zigzag streak in the
adjacent quarter.
iv. Streak 4: Reflame the loop and cool it. Draw the tip once through the end of
the previous streak and make a final zigzag streak in the remaining quarter of
the plate.
v. Reflame the loop and allow it to cool before placing it on the lab bench.
7. Label the bottom of three LB/amp plates with your group number, the date, and
the appropriate E. coli RNAi feeding strain (“dpy-11 RNAi,” “bli-1 RNAi,” or “unc22 RNAi”).
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SCRAPE cell mass
from bacterial
colony
FLAME and
cool loop
STREAK 1
STREAK 3
FLAME and
cool loop
STREAK 2
FLAME and
cool loop
STREAK 4
8. Follow the procedures outlined in Steps 2–6 to streak each feeding strain onto a
separate LB/amp plate.
9. Place all four plates upside down in a 37°C incubator. Incubate them for 15–20 h,
until optimal growth of well-formed colonies is achieved. At this point, colonies
should range from 0.5 to 3 mm in diameter.
10. Take time for responsible cleanup.
37°C
Plates are inverted to prevent condensation that
collects on the lids from
falling onto the agar, causing the colonies to run
together.
i. Segregate unwanted bacterial cultures into a “bio bag” or heavy-duty trash
bag for proper disposal.
ii. Wipe the lab bench with soapy water and 10% bleach or disinfectant at the
end of the lab.
iii. Wash your hands before leaving the lab.
◗ II. Grow E. coli Overnight Cultures
REAGENTS, SUPPLIES, & EQUIPMENT
For each group
Bunsen burner
4 Culture tubes (15 mL) (sterile)
E. coli OP50 culture plate from Part I
E. coli RNAi feeding strain cultures (dpy-11,
bli-1, and unc-22) on LB/amp plates
from Part I
Inoculating loop (optional)
LB/amp broth (10 mL)
LB broth (5 mL)
Micropipette tips (10–100 µL, sterile)
Permanent marker (red)
Pipette aid or bulb
2 Pipettes (5 mL) (sterile)
Test tube rack
To share
“Bio bag” or heavy-duty trash bag
Bleach (10%) <!> or disinfectant (e.g., Lysol)
Shaking water bath or incubator set at 37°C
See Cautions Appendix for appropriate handling of materials marked with <!>.
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If working in a team, one
partner should handle the
pipette and the other
should handle the tubes
and caps.
1. Use a red permanent marker to label a sterile 15-mL culture tube with your group
number, the date, and “OP50.”
2. Use a 5-mL pipette to sterilely transfer 2 mL of LB broth into the culture tube as
follows:
i. Make sure that the culture tube cap is unscrewed to the “loose” position.
ii. Attach a pipette aid or bulb to a 5-mL pipette. Briefly flame the pipette cylinder.
iii. Remove the cap of the bottle containing LB broth using the little finger of
your hand holding the pipette bulb. Flame the mouth of the LB bottle.
Pipette flaming can be eliminated if individually
wrapped pipettes are used.
iv. Use the pipette to withdraw 2 mL of LB. Reflame the mouth of the bottle and
replace the cap.
v. Remove the cap of the labeled culture tube from Step 1. Expel the LB into the
tube, reflame, and replace the cap.
3. Use a sterile micropipette tip to scrape a visible cell mass from a selected colony
on your E. coli OP50 culture plate (from Part I) and drop it tip-first into the culture tube. Reflame and replace the tube cap in the loose position. Alternatively,
use an inoculating loop as follows:
i. Sterilize the loop in the Bunsen burner flame until it glows red hot. Cool the
loop by stabbing it several times into a clear area near the edge of your E. coli
OP50 culture plate (from Part I).
ii. Use the loop to scrape a visible cell mass from a selected colony on your E. coli
OP50 culture plate. Immerse the cell mass in the LB broth and agitate the
loop to dislodge the cell mass.
Loop flaming can be eliminated if an individually
wrapped, sterile plastic
loop is used.
iii. Replace the tube cap in the loose position. Reflame the loop before setting it
on the lab bench.
4. Label three sterile 15-mL culture tubes with your group number, the date, and one of
the E. coli RNAi feeding strains (“dpy-11 RNAi,” “bli-1 RNAi,” or “unc-22 RNAi”).
5. Use a 5-mL pipette to sterilely transfer 2 mL of LB/amp broth into each of the
three labeled culture tubes as described in Step 2.
6. Use a sterile pipette tip (or a flamed and cooled inoculating loop) to transfer a single colony of RNAi feeding bacteria from each of the dpy-11, bli-1, and unc-22
LB/amp plates (from Part I) into the appropriate culture tubes as described in
Step 3.
7. Incubate the tubes for 12–24 h in a 37°C shaking water bath (with continuous
shaking) or for 24–48 h in a 37°C incubator (without shaking). To allow air flow,
do not seal the tube lids during incubation.
8. Take time for responsible cleanup.
37°C
i. Segregate unwanted bacterial cultures and tubes, pipettes, and micropipette
tips that have come into contact with cultures into a “bio bag” or heavy-duty
trash bag for proper disposal.
ii. Wipe the lab bench with soapy water and 10% bleach or disinfectant at the
end of lab.
iii. Wash your hands before leaving the lab.
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◗ III. Seed NGM-Lite and NGM-Lite/Amp + IPTG Plates with E. coli
REAGENTS, SUPPLIES, & EQUIPMENT
For each group
Bunsen burner
E. coli OP50 overnight culture from Part II
E. coli RNAi feeding strain (dpy-11, bli-1, and
unc-22) overnight cultures from Part II
3 NGM-lite + ampicillin <!> + isopropyl-β-Dthiogalactopyranoside <!> (NGM-lite/amp
+ IPTG) plates
4 NGM-lite plates
Permanent markers (black and red)
Pipette aid or bulb
4 Pipettes (5 mL)
Test tube rack
To share
“Bio bag” or heavy-duty trash bag
Bleach (10%) <!> or disinfectant (e.g., Lysol)
See Cautions Appendix for appropriate handling of materials marked with <!>.
1. Label the bottom of four NGM-lite plates with your group number and the date
with a black marker. Use a red marker to label the bottom of the plates “OP50.”
Seed the plates in a clean
area to avoid contamination.
2. Use a 5-mL pipette to sterilely seed each of the NGM-lite plates with OP50 as follows:
i. Attach the pipette aid or bulb to the 5-mL pipette. Briefly flame the pipette
cylinder.
ii. Remove the cap from the OP50 overnight culture (from Part II) using the little finger of your hand holding the pipette bulb. Flame the mouth of the
OP50 overnight culture.
iii. Use the pipette to withdraw 1 mL of overnight culture. Reflame and replace
cap.
iv. Add one to two drops of overnight culture to the center of the surface of each
NGM-lite plate. The drops should occupy most of the plate surface but should
not touch the edge of the dish.
Avoiding the edges of the
dish ensures that the
worms will remain in the
center of the plate.
3. Use a red marker to label the bottom of three NGM-lite/amp + IPTG plates with
your group number, the date, and one of the E. coli RNAi feeding strains (“dpy11 RNAi,” “bli-1 RNAi,” or “unc-22 RNAi”).
4. Use a 5-mL pipette to sterilely transfer one to two drops of each feeding strain
overnight culture to the appropriate NGM-lite/amp + IPTG plate as described in
Step 2.
5. Grow the seeded plates face-up for 24–36 h at room temperature.
The bacterial lawn should
be confluent and dry before
any worms are added.
6. Take time for responsible cleanup.
i. Segregate unwanted bacterial cultures and tubes, pipettes, and micropipette
tips that have come into contact with cultures into a “bio bag” or heavy-duty
trash bag for proper disposal.
ii. Wipe the lab bench with soapy water and 10% bleach or disinfectant.
iii. Wash your hands before leaving the lab.
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OVERVIEW OF EXPERIMENTAL METHODS IN STAGE B: CULTURING C. ELEGANS
I. CHUNK WILD-TYPE C. ELEGANS
OBSERVE
worms
PICK UP
chunk
DIP
implement
in ethanol
TRANSFER
chunk to
new plate
IGNITE
ethanol
VERIFY
worm
transfer
II. PICK INDIVIDUAL C. ELEGANS
FLAME
end of
pick
TRANSFER
worms to
new plate
ATTACH
glob of
bacteria
VERIFY
worm transfer
and health
PICK
large
worms
CUT
chunk with
worms
INCUBATE
48 h at room
temperature
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STAGE B: CULTURING C. ELEGANS
◗ I. Chunk Wild-Type C. elegans
REAGENTS, SUPPLIES, & EQUIPMENT
For each group
Binocular dissecting microscope
Bunsen burner
Ethanol (95%) <!> in a 50- or 100-mL beaker
Metal spatula or forceps
OP50-seeded NGM-lite plate from Part III of
Stage A
Permanent marker (black)
Sterilization prevents crosscontamination with different C. elegans strains and
non-OP50 bacteria.
CAUTION! Be extremely
careful to avoid igniting the
ethanol in the beaker. Do
not panic if the ethanol is
accidentally ignited. Cover
the beaker with a glass
Petri dish lid or other nonflammable cover to cut off
oxygen and rapidly extinguish the fire.
To feed worm strains, it is
easier to transfer a chunk of
worm-filled agar from a
well-grown plate to a new
plate rather than to pick
individual worms.
Wild-type worms on NGM-lite plate
To share
“Bio bag” or heavy-duty trash bag
Bleach (10%) <!> or disinfectant (e.g., Lysol)
See Cautions Appendix for appropriate handling of materials marked with <!>.
1. Obtain a fresh OP50-seeded NGM-lite plate (from Part III of Stage A) and a plate
with wild-type worms.
2. Examine both plates under the dissecting microscope for signs of bacterial or
mold contamination—any growth of a different color or morphology (shape)
from the OP50 lawn. Obtain a new plate if you detect any contamination.
3. Use a black permanent marker to label the bottom of the fresh OP50-seeded
NGM-lite plate with your group number, the date, and “wild type.”
4. Use your dissecting microscope to identify a region of the plate of wild-type
worms that is densely populated with worms and eggs.
5. Sterilize a metal spatula or forceps by dipping the end of the implement into the
beaker of ethanol and then briefly passing it through a Bunsen burner flame to
ignite the ethanol. Allow the ethanol to burn off away from the Bunsen flame; the
implement will become too hot if left in the flame.
6. Use a sterilized spatula or forceps to cut a 1-cm (~3/8-in) square chunk of agar
from the worm- and egg-dense region of the plate identified in Step 4.
7. Carefully remove the piece of agar with worms from the wild-type plate and place
it upside down on the lawn of the fresh OP50-seeded NGM-lite plate.
Placing the agar piece upside
down makes it easier for the
worms to crawl into the new
bacterial food source.
8. Examine the new plate under the microscope to verify that you have successfully
chunked the worms. Within a few minutes, worms should crawl from the agar
chunk and be visible in the bacterial lawn.
9. Store the new plate lid-side down for ~48 h at room temperature before continuing with Part II of Stage B (or Parts I or II of Stage C). Choose a place where the
plate will not be disturbed.
10. Take time for responsible cleanup.
i. Segregate any bacterial cultures that need to be discarded into a “bio bag” or
heavy-duty trash bag for proper disposal.
ii. Wipe the lab bench with soapy water and 10% bleach or disinfectant.
iii. Wash your hands before leaving the lab.
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562 / Chapter 4
◗ II. Pick Individual C. elegans
Repeat this procedure until you can efficiently pick worms to a new plate. Once you
feel accomplished, try to pick several worms at once.
REAGENTS, SUPPLIES, & EQUIPMENT
For each group
To share
Binocular dissecting microscope
Bunsen burner
Forceps
OP50-seeded NGM-lite plate from Part III of
Stage A
Permanent marker (black)
Wild-type worms on NGM-lite plate from
Part I
Worm pick
“Bio bag” or heavy-duty trash bag
Bleach (10%) <!> or disinfectant (e.g., Lysol)
See Cautions Appendix for appropriate handling of materials marked with <!>.
1. Examine the plate of wild-type worms you chunked in Part I under the dissecting
microscope. Confirm that no contaminants have grown since chunking. Obtain a
new plate of worms if necessary.
2. Use a black permanent marker to label the bottom of the fresh OP50-seeded
NGM-lite plate (from Part III of Stage A) with your group number, the date, and
“wild type.”
3. Examine a worm pick. The flattened end of the platinum wire should be bent at
roughly a 45° angle. Adjust the pick with the forceps if necessary. You may need
to adjust your pick from time to time during the course of this exercise.
4. Hold the worm pick like a pencil and sterilize the tip in a Bunsen burner flame
until it glows red hot.
5. Attach a glob of bacteria to the worm pick by wiping the flat head across the lawn
of bacteria on the fresh OP50-seeded NGM-lite plate, as shown to the left. The
bacteria will act like double-stick tape when you pick worms.
6. Open the lid of your plate of worms and identify a large worm.
Bacteria from a plate that
has aged 2–3 wk serve as a
better source of sticky bacteria.
7. Gently tap the top of the worm with the glob of bacteria on the bottom of the flattened pick. The glob of bacteria will attach the worm to the pick.
8. To transfer the worm, gently wipe the bottom of the pick in the lawn of the fresh
OP50-seeded plate. Make sure to avoid using too much force or you may tear the
agar surface and possibly crush the worm.
9. Examine the new plate under the microscope to confirm that the worm has survived picking. If it is visibly damaged or fails to move within several minutes,
transfer another worm.
10. For most experiments, it is important to use only one stage of worms. To avoid
later confusion, “burn” any embryos or other smaller larval stages that are accidentally transferred to the new plate by heating the worm pick in a Bunsen burner flame until it glows red hot and then immediately touching the flattened end
to any unwanted worm. (Alternatively, carefully pick each unwanted worm and
flame it in the Bunsen burner.)
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Culturing and Observing C. elegans / 563
11. Store the plate at room temperature. Choose a place where the plate will not be
disturbed.
12. Take time for responsible cleanup.
i. Segregate any bacterial cultures that need to be discarded into a “bio bag” or
heavy-duty trash bag for proper disposal.
ii. Wipe the lab bench with soapy water and 10% bleach or disinfectant.
iii. Wash your hands before leaving the lab.
STAGE C: OBSERVING WILD-TYPE AND MUTANT C. ELEGANS
◗ I. Observe the C. elegans Life Cycle
REAGENTS, SUPPLIES, & EQUIPMENT
For each group
Wild-type worms on NGM-lite plate from
Part I or II of Stage B
Binocular dissecting microscope
1. Obtain a plate with wild-type worms.
2. Observe the worms under a dissecting microscope. Note any physical (morphological) differences among the worms.
3. Note any differences in behavior, paying particular attention to how they move on
the plate.
4. Lift the plate several centimeters (~1 in) above the microscope stage and drop it.
Note any changes in worm movement. You may need to tap the plate several times
to induce movement.
Adult
Embryonic
development (14 h)
L4 larva (~10 h)
L1 larva (~12 h)
L3 larva (~8 h)
L2 larva (~7 h)
5. Study the diagram of the C. elegans life cycle above and attempt to identify an
example of each stage of the worm life cycle on the plate.
i. An adult hermaphrodite is a large worm with embryos inside. (The wild-type
strain used in this experiment produces few if any adult males.)
ii. The embryo is a small, oval object.
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564 / Chapter 4
iii. An L1 larva has recently hatched and is the smallest of the four larval stages.
iv. L2 and L3 larvae are larger than L1 worms but not as large as an adult. Examine
worms of different sizes to familiarize yourself with these larval stages.
v. The final juvenile stage, an L4 larva, is almost as large as an adult hermaphrodite. The lack of internal embryos is one marker that distinguishes an L4
larva from an adult. A clear, crescent-shaped patch near the center of the
body is another characteristic of an L4 larva. The egg-laying structure, called
the vulva, will develop in this patch when the L4 molts into an adult.
◗ II. Observe C. elegans Mutants
REAGENTS, SUPPLIES, & EQUIPMENT
All RNAi experiments in
subsequent labs begin
with identifying L4 hermaphrodites, so it is important to become proficient at identifying them.
For each group
Binocular dissecting microscope
Mutant worms on NGM-lite plates (dpy-11,
rol-6, bli-1, and unc-22)
Wild-type worms on NGM-lite plate from
Part I or II of Stage B
1. Obtain plates with mutant and wild-type worms.
2. Observe the worms under a dissecting microscope. Note any physical (morphological) differences among the wild-type and mutant worms. Record your observations and make sketches as needed.
3. Note any differences in behavior, paying particular attention to how the wild-type
and mutant worms move on the plate. Gently tap the plates on the microscope stage
to induce movement. Record your observations and make sketches as needed.
RESULTS AND DISCUSSION
1. How many stages of C. elegans development were you able to identify? Describe
each stage.
2. Why is it necessary for C. elegans to pass through several larval stages and how is
this type of development different from humans?
3. How does a hermaphrodite produce offspring without mating?
4. What physical (morphological) differences did you observe in the mutant
worms? What differences in behavior or movement did you notice? Did your
classmates identify the same characteristics of the mutant C. elegans?
5. Based on each mutant phenotype that you observed, what do you think would be
the function of the protein produced by the wild-type gene?
6. The mutant bli-1 and dpy-11 strains contain mutations that affect the cuticle, the
outer layer of the worm that is secreted by the epidermal (skin) cells. These two
very different phenotypes show how the nature of the mutation in a strain (the
genotype) affects the phenotype.
i. bli-1 encodes a collagen. What is a collagen? How can mutations in a collagen
affect the cuticle?
ii. dpy-11 encodes an enzyme. What do enzymes do? How can mutations in
enzymes affect the cuticle?
683-692_SubjIndex_Genome Science 10/3/12 3:25 PM Page 683
Index
A
ABA (American Breeders Association), 163
ABI (Applied Biosystems, Inc.), 34, 40
Abi-Rached, Laurent, 196–197
Activator (Ac), 334
Adenomatous polyposis coli (APC) gene, 187
Aegilops cylindrica (grass), 314
Affymetrix, 45, 191
Age-Related Eye Disease Study (AREDS), 354
Age-related macular degeneration (AMD),
354, 538
Agriculture and evolution, 208–210
Agrobacterium-mediated gene transfer,
348–351
Ahringer, Julie, 538
Altman, Sidney, 541
Alu, 161–162
Alu insertion
about, 231–233
amplifying DNA by PCR, 238
analyzing PCR products by gel electrophoresis, 238–239
answers to questions, 299–304
experimental methods overview, 235
further reading, 233
isolating DNA from cheek cells, 236–237
isolating DNA from hair sheaths, 237–238
planning and preparation instructions,
271–281
planning and preparation overview, 234
recipes for reagents and stock solutions,
283–291
results and discussion, 243–250
using BLAST to find DNA sequences,
239–241
using BLAST to identify additional alleles,
241–242
using Map Viewer, 242–243
Ambros, Victor, 529
Ambrose, Stanley, 201
AMD (age-related macular degeneration),
354, 538
American Breeders Association (ABA), 163
Amino acids, 11–14, 19–20, 351, 544–545
Anderson, Stephen, 34
Annotating genomic DNA
about, 49–50
answers to questions, 125–135
further reading, 50
planning and preparation instructions,
113–124
planning and preparation overview, 51
Stage A: annotating genes in a synthetic
contig
adding EST evidence to the project,
61–62
browsing the gene models, 61, 66
developing alternatively spliced models for the fourth gene, 65–66
developing alternatively spliced models for the third gene, 62–65
DNA Subway project creation, 52
editing the ends of a nonspliced gene,
56–58
editing the ends of a spliced gene, 59
examining and fixing two gene models, 62
examining exon/intron borders, 55–56
fixing splice sites, 60–61
identifying and masking repeats, 52
merging exons and editing the ends of
a spliced gene, 59–60
naming a gene and inserting a start
codon, 53–55
predicting genes in a DNA sequence,
52–53
searching databases for biological evidence, 56
Stage B: annotating a chromosome
sequence, 66–71
Antennapedia, 514
Antisense RNA, 523–524, 525
Antisense strand of DNA, 19
APC (adenomatous polyposis coli) gene, 187
Apollo, 31, 53–55, 56, 114–115
Apoptosis, 521–523
Applied Biosystems, Inc. (ABI), 34, 40
Arabidopsis, 331
Arabidopsis
detecting a transposon lab (see
Transposon detection in
Arabidopsis)
detecting epigenetic DNA methylation lab
(see Epigenetic DNA methylation
detection in Arabidopsis)
Arabidopsis Gibberellin-Insensitive (GAI) gene,
346
AREDS (Age-Related Eye Disease Study), 354
Argonaute family, 527–529, 531, 533, 534–535
Augustus, 52–53, 119
Autism spectrum disorder (ASD), 189
Avery, Oswald, 7
B
Bacillus thuringiensis (Bt), 350–351
BACs (bacterial artificial chromosomes),
43–44, 328–329
Bacteriophages, 7
Baltimore, David, 548, 549
Baross, John, 548
Bartel, David, 527, 546
Basic Local Alignment Search Tool (BLAST), 29
Bat Cave, New Mexico, 324
Bates, Lynn, 352
Baulcombe, David, 525, 526, 532
BCR (breakpoint cluster region), 186
Beachell, Henry, 346
Beadle, George, 6, 172, 323
Beal, William James, 340
Bent, Andrew, 349
Benz, Bruce, 326
Bernal, John Desmond, 545
β-Carotene, 353–354
Bevasiranib, 538
Biofortified foods, 353, 354
Biofuels and plant biomass, 356–357
Bioinformatics, 10
Biological Laboratory, Cold Spring Harbor, 163
Bithorax1 mutant, 514
Bitter-taste ability investigation
about, 251–253
amplifying DNA by PCR, 258
analyzing PCR products by gel electrophoresis, 259–260
answers to questions, 304–309
digesting PCR products with HaeIII, 259
experimental methods overview, 255
further reading, 253
683
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684 / Index
Bitter-taste ability investigation (continued)
isolating DNA from cheek cells, 256–257
isolating DNA from hair sheaths, 257–258
multiple sequence alignment and evolution of TAS2R38 gene, 263–266
planning and preparation instructions,
271–281
planning and preparation overview, 254
recipes for reagents and stock solutions,
283–291
results and discussion, 266–269
using BLAST to find DNA sequences,
260–262
using Map Viewer for TAS2R38 gene
locating, 262–263
Blackburn, Elizabeth, 549, 550
Blakeslee, Albert, 252
BLAST (Basic Local Alignment Search Tool),
29, 123
BLAT (BLAST-like Alignment Tool), 29
Borlaug, Norman, 345, 346
Borrelia burgdorferi, 549
Botstein, David, 175, 176p
Boveri, Theodor, 3
Brachypodium distachyon (grass), 357
Brachystola, 3
Branson, Herman, 10
BRE (B-recognition element), 23
Breakpoint cluster region (BCR), 186
B-recognition element (BRE), 23
Brenner, Sydney, 11, 520, 523, 529
Bridges, Calvin, 4
Britten, Roy, 159
Brown, Pat, 44
Bruce, A.B., 343
Bt (Bacillus thuringiensis), 350–351
Buckler, Edward, 355
Buck v. Bell, 168
Burbank, Luther, 163
Burkitt’s lymphoma, 186
Burnham, Charles, 323p
Burr, Benjamin, 353
Bush, Vannevar, 311
C
C (colored), 334
Caenorhabditis elegans (worm)
culturing and observing lab (see Culturing
and observing C.elegans)
development in, 519–521
number of genes in, 158
Calgene, 523
Calrose76, 347
Cancer
cell death processes role in, 522
miRNAs involvement in, 539–540
pharmacogenetics and, 191
structural variation and genetic disease
and, 186–190
Cantor, Charles, 175, 176p
Capecchi, Mario, 515, 516, 523
Carotenoids in plants, 354
Carrington, James, 533
Casal, Caspar, 352
Caskey, Thomas, 177, 178p
Cavalli-Sforza, Luigi Luca, 208, 209
CCAAT box, 23–24
CCD (charge-coupled device) camera, 38
cDNA (complementary DNA) libraries, 30
Cech, Thomas, 541, 546, 547
ced (cell death abnormal), 521–522
Celera, 36, 37
Cellulosic ethanol, 356–357
centiMorgan (cM), 173
CF (cystic fibrosis), 182
Chalcone synthase (CHS), 524
Chandler, Peter, 347
Chaplin, George, 204, 205
Chargaff, Erwin, 10
Charge-coupled device (CCD) camera, 38
Chase, Martha, 7
Chilton, Mary-Dell, 348, 349p
Chromosome detection using bioinformatics
about, 73–74
answers to questions, 136–139
comparing human and chimpanzee
genomes, 76
comparing human and chimpanzee
genomes by syntenic dotplot,
77–79
comparing locations of homologous genes
on human and chimpanzee chromosomes, 76–77
determining the origin of the centromere
in human chromosome 2, 79–80
extending the analysis of the evolution of
human chromosome 2, 80
further reading, 74
planning and preparation instructions,
113–124
planning and preparation overview, 75
Chromosomes
detecting using bioinformatics lab (see
Chromosome detection using
bioinformatics)
heredity and, 3–4
human disease and, 171–173, 174
human genome structure and, 156–157
transposons content lab (see Transposon
content in grasses)
Chronic myeloid leukemia (CML), 186
CHS (chalcone synthase), 524
Cigna, 191
CIMMYT (International Maize and Wheat
Improvement Center), 346
Climate change and human evolution,
199–200
Clough, Steven, 349
Clustal W, 109–111, 122
cM (centiMorgan), 173
CML (chronic myeloid leukemia), 186
CMV (cytomegalovirus), 524
CNVs (copy-number variations), 186, 189
CODIS (Combined DNA Index System),
177–178, 179
CoGe comparative genomics tool, 116
Colon cancer, 187, 188
Colored (C), 334
Comparing diversity in eukaryotes lab. See
Eukaryotes diversity comparison
Complementary DNA (cDNA) libraries, 30
Copper Cross, 342
Copy-number variations (CNVs), 186, 189
Corey, Robert, 10
Corn. See Maize (corn)
Correns, Carl, 3
Cosuppression, 524–525
CPY450s (cytochrome P450 mono-oxidases),
191
Creighton, Harriet, 5, 334
Crick, Francis, 8, 544
Croce, Carlo, 539
Crossing-over, genetic, 5
Culturing and observing C.elegans
about, 551–553
answers to questions, 659–660
further reading, 553
objectives, 551
planning and preparation instructions,
627–639
planning and preparation overview, 554
recipes for reagents and stock solutions,
641–658
results and discussion, 564
Stage A: culturing E. coli, 554, 555–560
Stage B: culturing C. elegans, 561–563
Stage C: observing wild-type and mutant
C. elegans, 563–564
C-value paradox, 331
Cycle sequencing, 34–37
Cyclic array sequencer, 39–40
Cystic fibrosis (CF), 182
Cytochrome P450 mono-oxidases (CPY450s),
191
Cytomegalovirus (CMV), 524
D
D1S80 VNTR, 177, 178
Darwin, Charles, 2, 340
Davenport, Charles, 163
Davis, Mark, 538
Davis, Ronald, 175
Dawkins, Richard, 532
ddNTPs (dideoxynucleotide triphosphates),
33
Demic diffusion model, 209
Denisovan, 196–198
Deoxynucleotide triphosphates (dNTPs), 33
Deoxyribonucleic acid. See DNA
Department of Agriculture, 312
Detecting a lost chromosome using bioinformatics lab. See Chromosome detection using bioinformatics
683-692_SubjIndex_Genome Science 10/3/12 3:25 PM Page 685
Index / 685
Detecting a transposon in Arabidopsis lab. See
Transposon detection in Arabidopsis
Detecting a transposon in corn lab. See
Transposon detection in corn
Detecting epigenetic DNA methylation in
Arabidopsis lab. See Epigenetic
DNA methylation detection in
Arabidopsis
Detecting genetically modified foods by PCR
lab. See Genetically modified foods
detection by PCR
Determining the transposon content in grasses lab. See Transposon content in
grasses
De Vries, Hugo, 3
Dicer, 538
Dicer and Slicer identification, 527–529, 531,
534
Dideoxy DNA sequencing, 33–34
Dideoxynucleotide triphosphates (ddNTPs), 33
Ding, Shou Wei, 533, 534
Discovering evidence for pseudogene function lab. See Pseudogene function
Disease
chromosomal and molecular basis of,
171–173, 174
identifying genes behind complex genetic
disorders, 183–186
identifying genes behind simple genetic
disorders, 180–183
Displacement theory of human origin, 192–193
Dissociation (Ds), 334
DMD (Duchenne muscular dystrophy), 182
DMs (double minutes), 186
DNA (deoxyribonuleic acid)
annotating genomic DNA lab (see
Annotating genomic DNA)
automated sequencing, 34–37
composition of, 8, 9f
dideoxy sequencing, 33–34
directionality, 19
discovery that it is genetic material, 5–8
identifying and classifying living things
using barcodes lab (see DNA barcodes use)
implications of availability of personal
DNA information, 155–156
linkage mapping lab (see Linkage mapping a mutation)
methylation detection lab (see Epigenetic
DNA methylation detection in
Arabidopsis)
microarrays, 44–45
molecular clock concept, 193
next-generation sequencing, 37–41
patterns in DNA sequences, 15–18
polymorphisms and human identity
CODIS and, 177–178, 179
DNA fingerprinting requirements,
178–190
forensic DNA fingerprinting, 176
microsatellites use, 177
VNTR polymorphisms, 176–177
repetitive, 159–162
structure of, 8, 10
transposons, 336
DNA barcodes use
about, 425–427
answers to questions, 501–505
bioinformatics methods
performing a phylogenetic analysis,
442–444
submitting DNA barcodes to
GenBank, 444
uploading and assembling the DNA
sequences in DNA Subway, 439–441
using BLAST to find DNA sequences
in databases, 437–438
using BLAST to identify your species,
441–442
experimental methods
amplifying DNA by PCR, 435–436
analyzing PCR products by gel electrophoresis, 436–437
collecting, documenting, and identifying specimens, 431
isolating DNA from plant or animal
tissue, 432–435
overview, 429–431
further reading, 427
objectives, 425
planning and preparation instructions,
463–474
planning and preparation overview, 428
recipes for reagents and stock solutions,
475–484
results and discussion, 445
DNA Plant Technology, Inc., 524
DNA Subway, 50, 116–118
dNTPs (deoxynucleotide triphosphates), 33
Doebley, John, 324, 326, 338
Domestication syndrome, 317–318, 325
Donis-Keller, Helen, 175, 176p
Double minutes (DMs), 186
Double-stranded RNA (dsRNA), 526
Dougherty, William, 532
Dozy, Andrea-Marie, 173
DPE (downstream promoter element), 23
Drosha, 531
Drosophila melanogaster (fruit fly), 4, 175,
512–514
Drug response, 191
Ds (Dissociation), 334
dsRNA (double-stranded RNA), 526
Duchenne muscular dystrophy (DMD), 182
Duckweed (Spirodela polyrhiza), 357
Dugdale, Richard, 164
E
East, Edward, 342, 343
E. coli feeding strains used to induce RNAi
and knock down genes
about, 565–567
answers to questions, 660–662
bioinformatics methods, 572–573
experimental methods overview, 569
further reading, 567
inducing RNAi by feeding, 570–571
objectives, 565
observing and scoring phenotypes, 571
planning and preparation instructions,
627–639
planning and preparation overview, 568
recipes for reagents and stock solutions,
641–658
transferring wild-type C. elegans to OP50seeded NGM-lite plates, 570
Eichelbaum, Michel, 191
Einkorn, 314
Emerson, Rollins, 323
Emmer, 314
Enattah, Nabil, 207
ENCODE (Encyclopedia of DNA Elements),
158
Enhancers, 24
Epigenetic DNA methylation detection in
Arabidopsis
about, 447–449
answers to questions, 505–510
bioinformatics methods
identify the FWA amino acid sequence
and function, 460–461
using BLAST to find DNA sequences
in databases, 457–459
using Map Viewer to determine the
chromosome location of the FWA
gene, 459–460
experimental methods
amplifying DNA by PCR, 455–456
analyzing PCR products by gel electrophoresis, 456–457
digesting DNA with McrBC, 454–455
isolating DNA, 453–454
overview, 451
plant Arabidopsis seeds, 452
further reading, 449
objectives, 447
planning and preparation instructions,
463–474
planning and preparation overview,
450
recipes for reagents and stock solutions,
475–484
results and discussion, 461–462
ERO (Eugenics Record Office), 163, 170
Estabrook, Arthur, 164, 165p, 169
Ethanol, 356
EU (European Union), 351
Eugenics
discrediting of, 169–171
Mendelian genetics and, 162–165
social engineering and, 166–168
Eugenics Record Office (ERO), 163, 170
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686 / Index
Eugenics: The Science of Human Improvement
by Better Breeding (Davenport), 163
Eukaryotes diversity comparison
about, 81–82
aligning and comparing the human and
chimpanzee adh1 gene sequences,
85
aligning and comparing the maize and
teosinte adh1 gene sequences, 85
aligning and comparing two maize adh1
gene sequences, 84–85
answers to questions, 140–142
extending the analysis of adh1 gene
sequences, 85
further reading, 82
planning and preparation instructions,
113–124
planning and preparation overview, 83
Sequence Server project setup, 84
Eukaryotic core promoter, 23–24
European Union (EU), 351
Evans, Martin, 516
Evolution
bitter-taste ability demonstration lab (see
Bitter-taste ability investigation)
climate change and population bottlenecks, 199–200
concept of a human population and,
202–203
DNA molecular clock, 193
ethnicity and, 203–204
evidence of recent selection in the
genome, 205–208
fossil records and, 192–193
genetic diversity and population bottlenecks, 199–202
genetic legacies of Neandertal and
Denisovan, 195–199
human metabolism adaptations, 204–205
information obtainable from DNA,
194–195
mitochondrial DNA polymorphisms lab
(see Mitochondrial DNA polymorphisms)
natural selection as explained by Darwin, 2
spread of agriculture and, 208–210
Exons and introns, 24–27
Exonucleases, 547
“Experiments in Plant Hybridization”
(Mendel), 2, 8
Eyeless gene, 518
F
Fearon, Eric, 187
Fedoroff, Nina, 336
Ferris, James, 545
Fertile Crescent, 313
FGenesH, 52–53, 119
Fiers, Walter, 34
Finger Prints (Galton), 176
Fire, Andy, 525, 526, 527
Fischer, Eugen, 171
“Fitter Family” contests, 163
FlavrSavr tomato, 523–524
Fly. See Drosophila melanogaster (fruit fly)
Fodor, Steven, 45
Fogarty, John E., 311
Fomivirsen (Vitravene), 524
Food and Agriculture Organization, UN, 352
Ford Foundation, 346
Forensic DNA fingerprinting, 176, 178–190
Fossil records and evolution, 192–193
454 Life Sciences, 39, 41
FOXP2 transcription factor, 198
Fraley, Robert, 349
Franklin, Rosalind, 10
Fromm, Michael, 349, 350
Fuller, Dorian, 318
G
GAI (Arabidopsis Gibberellin-Insensitive) gene,
346
GAI (gibberellic acid insensitive). See
Identifying GAI gene family members in plants
Galton, Francis, 162, 176
Garrod, Archibald, 172, 173p
Gasohol, 356
GBrowse, 120
Gehring, Walter, 514, 515, 518
Gene Boy, 108–109, 118
GeneChip, 45, 46
Gene targeting, 515
Genetically modified (GM) food
detecting GM foods lab (see Genetically
modified foods detection by PCR)
development of enriched rice and corn,
353–356
FlavrSavr tomato, 523–524
Green Revolution and, 345–347, 348
plants and, 348–351
Genetically modified foods detection by PCR
about, 411–412
answers to questions, 499–501
bioinformatics methods
using BLAST to find DNA sequences
in databases, 419–421
using BLAST to identify transgenes
driven by the 35S promoter,
421–422
experimental methods
amplifying DNA by PCR, 417–418
analyzing PCR products by gel electrophoresis, 418–419
isolating DNA from soybean and food
product, 415–417
overview, 414
plant soybean seeds, 415
further reading, 412
objectives, 411
planning and preparation instructions,
463–474
planning and preparation overview, 413
recipes for reagents and stock solutions,
475–484
results and discussion, 422–424
Genetic code
automated DNA sequencing, 34–37
chromosome detection techniques lab (see
Chromosome detection using
bioinformatics)
codons and, 12–14
dideoxy DNA sequencing, 33–34
DNA directionality, 19
DNA microarrays, 44–45
eukaryotes diversity comparison lab (see
Eukaryotes diversity comparison)
exons and introns, 24–27
finding genes in genomic sequence, 27–29
gene annotation, 29–32
gene annotation lab (see Annotating
genomic DNA)
genome assembly strategies, 41–44
heredity and, 3–4, 11–14
identifying members of a gene family lab
(see Identifying GAI gene family
members in plants)
minimal and synthetic genomes, 46–48
next-generation DNA sequencing, 37–41
patterns in DNA sequences, 15–18
pseudogene function lab (see Pseudogene
function)
reading frames, 19–21
RNA code translation into amino acids,
11–12
RNA splicing mechanism, 26f
3′- and 5′-untranslated regions and promoters
protein-binding sites, 21
sequence elements in the core promoter, 23–24
transcription and translation, 22
transposons investigations (see
Transposon content in grasses;
Transposon detection in
Arabidopsis; Transposon detection
in corn)
Genetic code labs planning and preparation
about, 113–114
Apollo annotation editor, 114–115
BLAST sequence search tool, 123
Clustal W and muscle sequence alignment
tools, 122
CoGe comparative genomics tool, 116
DNA Subway, 116–118
Gene Boy, 118
gene prediction tools, 119
genome browsers Map Viewer and
GBrowse, 120
InterProScan, 120
Jalview multiple sequence alignment and
phylogenetic tree editor, 120–121
Phylip phylogenetic tree tool, 121
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RepeatMasker, 121–122
Sequence Server, 123
TARGeT, 123
TargetScan, 123–124
tRNAscan-SE, 124
Genetics and Evolution (Morgan), 170
GENEWIZ DNA sequencing services,
277–281
Genome Analyzer, 40
Genome as information
DNA as information (see DNA)
genetic code (see Genetic code)
heredity and (see Heredity)
laboratory preparation and planning,
113–124
meaning of “genome,” 1
Genome function
antisense RNA, 523–524, 525
cell death mutants, 521–523
cosuppression of genes, 524–525
culturing and observing lab (see Culturing
and observing C.elegans)
development in C. elegans and, 519–521
Dicer and Slicer identification, 527–529
foundational biochemical interactions,
544
functional genomics screens, 537
homeotic genes in Drosophila, 512–514
Hox genes in the mouse, 514–518
laboratory preparation and planning,
627–639
miRNAs regulation of genes, 529–531
prebiotic evolution, 544–546
recipes for reagents and stock solutions,
641–658
RNA and (see RNA)
RNAi and knock down genes lab (see E.
coli feeding strains used to induce
RNAi and knock down genes)
study of model organisms, 511–512
transposon silencing, 534–535
Genome-wide association studies (GWAS),
184–186
Genomics, 10
Genotype, 3
Germany and eugenics, 171
gibberellic acid insensitive (GAI). See
Identifying GAI gene family members in plants
Gibberellin, 346–347
Gilbert, Walter, 11
Gleevec, 190
Glyphosate (Roundup), 350
Glyphosate-resistant crops, 350
Goddard, Henry, 167–168
Goff, Stephen, 344
Golden rice, 353–356
Grass (Aegilops cylindrica), 314
Grass (Brachypodium distachyon), 357
Green Revolution, 345–347, 348
Greider, Carol, 549, 550
Griffith, Fred, 6
Gros, François, 11
Guo, Susan, 525
GWAS (genome-wide association studies),
184–186
H
Haldane, J.B.S., 1
Hall, Benjamin, 11
Hall, Timothy, 349
Hammer, Karl, 317
Hannon, Greg, 527
HapMap (International Haplotype Map)
Project, 184
Harberd, Nicholas, 346, 347p
Hardy, Godfrey, 170
Hardy–Weinberg equilibrium, 245–246
Harjes, Carlos, 355
HC-Pro protein, 533
HD (Huntington’s disease), 182
Hereditary Genius (Galton), 162
Heredity
chromosome behavior and, 3–4
composition of DNA, 8, 9f
discovery that DNA is genetic material, 5–8
evolution by natural selection, 2
genetic code, 11–14
inheritance studies by Mendel, 2–3
proof of genetic crossing-over, 5
structure of DNA, 8, 10
studies by Morgan, 511–512
Herrick, James, 172
Hershey, Alfred, 7
Heterosis, 342–345
Heun, Manfred, 314
Hi-Bred Corn Company, 342
Hidden Markov model (HMM), 27–28
Hiei, Yukoh, 350
Hierarchical cloning, 42–44
Hill, J. Lister, 311
HIV (human immunodeficiency virus), 1
HLAs (human leucocyte antigens), 196–198
HMM (hidden Markov model), 27–28
Hoagland, Mahlon, 12
Holley, Robert, 12
Holmes, Oliver Wendell Jr., 169
Homeotic genes in Drosophila, 512–514
Homogenously staining region (HSR), 186
Homology, 29
Hood, Leroy, 34, 175
Hopscotch, 338
Horsh, Robert, 349
Horvitz, Robert, 522, 523
How to Make a Eugenical Family Study
(Davenport), 163
Hox genes in the mouse, 514–518
HSR (homogenously staining region), 186
Hsu, Tao-Chiuh, 171, 172
Human genome
alternative gene splicing, 158
bitter-taste ability demonstration lab (see
Bitter-taste ability investigation)
chromosomal and molecular basis of
human disease, 171–173, 174
chromosomes, 156–157
density of genetic information, 156
discrediting of eugenics, 169–171
DNA polymorphisms and human identity
CODIS and, 177–178, 179
DNA fingerprinting requirements,
178–190
forensic DNA fingerprinting, 176
microsatellites use, 177
VNTR polymorphisms, 176–177
eugenics and Mendelian genetics, 162–165
eugenic social engineering, 166–168
evolution and (see Evolution)
gene insertion lab (see Alu insertion)
identifying genes behind complex genetic
disorders, 183–186
identifying genes behind simple genetic
disorders, 180–183
implications of availability of personal
DNA information, 155–156
laboratory preparation and planning,
271–281
mapping, 173–176
noncoding regions, 158–159
nucleotides, 157
pharmacogenomics and pharmacogenetics, 190–191
recipes for reagents and stock solutions,
283–291
reference human genome size, 157–158
repetitive DNA, 159–162
structural variation and genetic disease,
186–190
structure of, 156–158
therapeutic proteins, 173
Human Genome Project, 36, 519
Human Genome Sciences, 36
Human identity and DNA polymorphisms
CODIS and, 177–178, 179
DNA fingerprinting requirements,
178–190
forensic DNA fingerprinting, 176
microsatellites use, 177
VNTR polymorphisms, 176–177
Human immunodeficiency virus (HIV), 1
Human leucocyte antigens (HLAs), 196–198
Hunkapiller, Mike, 34, 35p, 36
Hunt, John, 172, 173
Huntington’s disease (HD), 182
Hurwitz, Jerard, 11
Hybrid plants, 321, 323, 341, 342–343. See
also Maize (corn)
I
Identifying GAI gene family members in
plants
about, 95–96
answers to questions, 145–149
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Identifying GAI gene family members in
plants (continued)
determining how GAI proteins from different plant species are related,
98–99
extending the analysis of GAI sequences,
100–101
further reading, 96
identifying the cause of the GAI semidwarf phenotype, 99–100
next wave of the green revolution, 100
planning and preparation instructions,
113–124
planning and preparation overview, 97
Illumina Genome Analyzer, 40
Immigration Restriction Act (1924), 167
India, 345, 346
Ingram, Veron, 172, 173
Innocence Project, 180
Inr box, 23
Insect-resistant crops, 350
International Haplotype Map (HapMap)
Project, 184
International Health Research Act (1960), 311p
International Maize and Wheat Improvement
Center (CIMMYT), 346
International Rice Research Institute (IRRI),
346
InterProScan, 120
IR8 rice hybrid, 346, 347
Iressa, 191
Itano, Harvey, 172
J
Jablonski, Nina, 204, 205
Jacob, François, 11
Jalview, 120–121
Janssens, Frans Alfons, 5
J. Craig Venter Institute, 46
Jeffreys, Alec, 176, 177p
Jennings, Walter, 346
Johannsen, Wilhelm, 3
Jones, Donald, 342
Jorgensen, Rich, 524
Joshua-Tor, Leemor, 528
Joyce, Gerald, 546
Jukes, The (Dugdale), 164, 165p
Jumping genes, 161
K
Kaiser Wilhelm Institute of Anthropology,
Human Genetics, and Eugenics, 171
Kallikaks, The (Goddard), 167–168
Kan, Yuet Wai, 173
Kemp, John, 349
Kemphues, Ken, 525
Kennedy, John F., 311p
Khorana, Har Gobind, 12, 46
Klein, Theodore, 349p, 350
Knockout genes, 515–518
Kohne, David, 159
Konishi, Saeko, 319
Koornneef, Maarten, 95
Kuahuqiao, China, 315, 316f, 317
Kwashiorkor (protein deficiency), 352
L
L1, 161–162
Laboratory planning and preparation
amplifying DNA by PCR, 274–275,
465–467, 633–634
analyzing DNA by gel electrophoresis,
275–277, 468–470, 634–636
culturing bacteria, 627–628
culturing C. elegans, 629–633
digesting PCR products with restriction
enzymes, 275, 467–468
genetic code labs (see Genetic code labs
planning and preparation)
isolating DNA, 465
isolating DNA from cheek cells, 271–273
isolating DNA from hair sheaths, 273–274
isolating worm DNA, 633
obtaining seeds and specimens, 463–464
recombination, 636
sequencing PCR products, 277–281,
470–474
transforming bacteria, 636–639
Lactose tolerance, 206–208
Lalueza-Fox, Carles, 198
Lasker, Mary, 311
Laughlin, Harry, 167, 171
Lee, Yoontae, 531
Let-7, 529
Leven, Albert, 172
Lewis, Edward, 514
Li, Changbao, 320
lin-4 and lin-14 mutants, 529
Lincoln, Tracey, 546
LINEs (long interspersed elements), 336
Linkage mapping a mutation
about, 393–395
answers to questions, 494–499
bioinformatics methods
digest CAPS markers electronically, 405
using BLAST to find DNA sequences
in databases, 403–405
using Map Viewer, 406–407
experimental methods
amplifying DNA by PCR, 400–401
analyzing CAPS markers by gel electrophoresis, 402
digesting PCR products to produce
CAPS markers, 401
isolating DNA, 398–400
overview, 397
planting Arabidopsis seeds, 398
further reading, 395
objectives, 393
planning and preparation instructions,
463–474
planning and preparation overview, 396
recipes for reagents and stock solutions,
475–484
results and discussion, 407–410
LOD (logarithm of the odds), 184
LOH (loss of heterozygosity), 187
Londo, Jason, 315
Long interspersed elements (LINEs), 336
Loomis, W. Farnsworth, 203–204
Loss of heterozygosity (LOH), 187
Lowe, Scott, 538
Lucito, Robert, 189
Lutein, 354
Lyme disease, 549
M
MacLeod, Collin, 7
MacNeish, Richard, 326
Maize (corn)
corn hybrids advantages, 342
detecting a transposon lab (see
Transposon detection in corn)
development of enriched rice and corn,
353–356
evidence of first appearance, 326–327
genetic diversity of, 324
genetics investigation lab (see Eukaryotes
diversity comparison)
heterosis and modern corn development,
339–343
hierarchical shotgun sequencing of the
maize genome, 330
importance to civilization, 321
insights from maize genome sequence,
328–331
investigating the genetics of lab (see
Transposon detection in corn)
key elements of maize domestication syndrome, 325
modern corn development, 339–343
quality protein maize, 351–353
scenario supporting cultivation start, 328
teosinte’s connection to, 322–324
transposition mechanism and, 338–339
use as popcorn, 324
whole-genome comparisons between
maize and other grains, 331–334
Maize Genome Sequencing Project, 328
Mangelsdorf, Paul, 324
Mapping the human genome
alleles and RFLPs, 175
common language of DNA markers,
175–176
crossover and linkage, 175
genetic distance and, 173, 175
Map Viewer, 76, 120
Marker-assisted selection, 356
Martienssen, Rob, 534
Matsuoka, Makoto, 347
McCarty, Maclyn, 7
McClintock, Barbara, 5, 88, 323p, 334–335,
337, 338
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Mello, Craig, 526, 527, 534
Mendel, Gregor, 2–3, 164
Mendelian genetics, 2–3, 162–165
Menozzi, Paolo, 208
Mer/L2 symbiosis, 162
Mertz, Edwin, 352
Meselson, Matthew, 11
Mesoamericans, 351–352
Methionine, 12
Mexico, 345
Miescher, Friedrich, 8
Miller, Stanley, 544
Miniature Ping (mPing), 337–338
miRNAs (microRNAs), 529–531, 539–540
MITE (miniature inverted-repeat transposable element), 338
Mitochondrial DNA (mtDNA), 193, 194, 196
Mitochondrial DNA polymorphisms
about, 211–213
amplifying DNA by PCR, 218
analyzing PCR products by gel electrophoresis, 219–220
answers to questions, 293–299
examining genes and chromosome structure, 222
experimental methods overview, 215
further reading, 213
isolating DNA from cheek cells, 216–217
isolating DNA from hair sheaths, 217–218
planning and preparation instructions,
271–281
planning and preparation overview, 214
recipes for reagents and stock solutions,
283–291
results and discussion, 223–229
using BLAST to find DNA sequences,
220–222
using MITOMAP to explore role of mutations, 222–223
MITOMAP, 222–223
Mittenthal, Jay, 548
Mizutani, Satoshi, 548, 549
Moazed, Danesh, 534
Molecular clock, 193
Monaco, Anthony, 198
Monsanto, 349
Montmorillonite clay, 545–546
Morgan, Thomas Hunt, 1, 4, 5, 170, 173,
511–512
Mouse, 514–518
mPing (miniature Ping), 337–338
mtDNA (mitochondrial DNA), 193, 194,
196
Mt. Toba, Sumatra, 201
Mu (Mutator), 338
Müller, Hermann, 4, 170, 512
Mullis, Kary, 34, 35p
Multiregional theory of human origin, 192
Muscle, 122
Mutagenesis, 512
Mycoplasma capricolom, 47–48
Mycoplasma genitalium, 47
Mycoplasma mycoides, 47
N
Nakamura, Yusuke, 176, 177p
National Cancer Institute, 311p, 312
National Eye Institute, 354
National Institute of Food and Agriculture,
312
National Institutes of Health (NIH), 311
National Plant Genome Initiative, 328
National Science Foundation, 328
Neandertal, 195–199, 201, 227–228
Negative eugenics, 164
Neomycin (neo), 515–516
Neufeld, Peter, 180
Neurospora, 6
Nevado de Toluca, 329, 331
Newman, Horatio Hackett, 170
NIH (National Institutes of Health), 311
Nirenberg, Marshall, 12
Nixtamalization of corn, 352
Noncoding regions, 158–159
Nucelotides, 157
Nuclein, 8
Nüsslein-Volhard, Christiane, 512
O
Office of Scientific Research and
Development (OSRD), 311
Olfactory receptors (ORs), 205
Oligonucleotide arrays, 45
Olson, Maynard, 175, 176p
Oncogenesis, 186–190
1000 Genomes Project, 155
Online Mendelian Inheritance in Man
(OMIM), 157–158
Opaque2 mutation, 353
Orange corn, 353–356
ORF (open reading frame), 20
Orgel, Leslie, 544
On the Origin of Species (Darwin), 2
ORs (olfactory receptors), 205
Oryza sp. (rice). See Rice cultivation
OSRD (Office of Scientific Research and
Development), 311
Ötzi the Iceman, 209, 210, 227
Özkan, Hakan, 314
P
Pääbo, Svante, 196
Pakistan, 345, 346
Papaya ringspot virus (PRSV), 524–525
Parham, Peter, 196–197
Pauling, Linus, 10, 172
Pax6 gene, 518
PCR (polymerase chain reaction), 34–36
Pearl, Raymond, 170
Pederson, Irene, 533
Pellagra, 352
Penrose, Lionel, 170
PG (polygalacturonase), 523
Pharmacogenetics, 190–191
Pharmacogenomics, 190–191
Phenotype, 3
Philippines, 346
PHYLIP, 30, 121
Piazza, Alberto, 208
Pioneer Hi-Bred, 342, 345
Piperno, Dolores, 326
piRNAs (piwi-interacting RNAs), 534
PIWI domain, 528
Planning and preparation for labs. See Laboratory planning and preparation
Plant genomes
biofuels and plant biomass, 356–357
challenges in plant research, 312
detecting epigenetic DNA methylation lab
(see Epigenetic DNA methylation
detection in Arabidopsis)
detecting GM foods lab (see Genetically
modified foods detection by PCR)
detecting transposons labs (see
Transposon content in grasses;
Transposon detection in
Arabidopsis; Transposon detection
in corn)
development of enriched rice and corn,
353–356
domestication of maize (see Maize [corn])
domestication syndrome, 317–318, 325
focus on public health research in U.S.,
311–312
gene silencing studies on, 524–525
genetic modification of plants, 348–351
Green Revolution and, 345–347, 348
heterosis and hybrids, 342–343
heterosis and modern corn development,
339–343
hierarchical shotgun sequencing of the
maize genome, 330
identifying and classifying living things
lab (see DNA barcodes use)
identifying members of a gene family lab
(see Identifying GAI gene family
members in plants)
laboratory preparation and planning,
463–474
linkage mapping a mutation lab (see
Linkage mapping a mutation)
modern corn development, 339–343
molecular genetic basis of heterosis,
343–345
molecular mechanisms of transposition,
335–339
move to increase research on plants, 312
origins of wheat and rice cultivation,
313–317
quality protein maize, 351–353
recipes for reagents and stock solutions,
475–484
shattering QTLs, 318–321
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Plant genomes (continued)
transposition during development, 334–335
whole-genome comparisons between
maize and other grains, 331–334
Plasterk, Ronald, 534
Polonies, 37, 39
Poly(A) tail, 22
Polygalacturonase (PG), 523
Polymerase chain reaction (PCR), 34–36
Polymerase II (Pol II), 22
Popcorn, 323–324
Positional cloning, 180–183
Positive eugenics, 162–163
Potrykus, Ingo, 354
Prebiotic evolution, 544–546
Protein deficiency (kwashiorkor), 352
Protein p53, 522, 538
PRSV (papaya ringspot virus), 524–525
Pseudogene function
about, 103–104
answers to questions, 149–153
extending the analysis of pseudogene
sequences, 111
further reading, 104
planning and preparation overview, 105
using Gene Boy to determine the proteincoding capacity of the PTENP1
pseudogene, 108–109
using Map Viewer to determine the relationship between PTEN and
PTENP1, 107–108
using Map Viewer to locate the PTEN
gene and PTENP1, 106
using TargetScan and Clustal W to identify a potential function for the
PTENP1 pseudogene, 109–111
Pseudogenes, 161
PV92 genotype, 241–242, 243–244
Q
Qing, Ji, 540
QTLs (quantitative trait loci), 318–321,
355–356
Quality protein maize (QPM), 351–353
R
Rainbow papaya, 524
Ramakrishnan, Venkatraman, 543
Reading frames, 19–21
Recipes for reagents and stock solutions
amplifying DNA by PCR, 286–287,
478–481, 650–651
analyzing DNA by gel electrophoresis,
287–290, 482–484, 651–654
culturing bacteria, 642–645
culturing C. elegans, 645–648
digesting PCR products with restriction
enzymes, 481
isolating DNA, 476–477
isolating DNA from cheek cells, 284–285
isolating DNA from hair sheaths, 285–286
isolating worm DNA, 649–650
preparing plasmid minipreps, 656–658
sequencing PCR products, 290–291
transforming bacteria, 654–656
Reduced height-1 genes (Rht-B1 and Rht-D1),
346
Reid, Robert, 338
RepeatMasker, 121–122
Retinoblastoma, 187, 188
Retrotransposons, 331, 336
RFLPs (restriction-fragment-length polymorphisms), 175
Rhoades, Marcus, 323p
Rhombomers, 515
Ribonucleic acid. See RNA
Ribozymes, 541
Rice cultivation
evolution of rice, 314–317
Green Revolution and, 345–347
origins of, 313–317
QTLs for shattering, 319–321
transposition mechanism and, 337–338
Riis, Jacob, 166
RISC (RNA-induced silencing complex), 527
RITS (RNA-induced initiation of transcriptional gene silencing), 534
RNA (ribonucleic acid)
amino acids and, 12, 13f, 14
codons and, 12, 13f, 14
complexity of, 540–541
composition of, 9f
enzymes, 541–543
protein world and, 548
RNA code and amino acids, 11–12
RNA world and RNA protocells, 544–546
RNA world to a DNA world, 548–550
self-replicating RNA and RNA stability,
546–547
silencing mechanism, 525–527
splicing mechanism, 26f
transposons, 336
RNAi (RNA interference)
constructing a feeding vector lab (see
RNAi feeding vector construction)
examining the RNAi mechanism lab (see
RNAi mechanism examination)
inducing RNAi and knock down genes lab
(see E. coli feeding strains used to
induce RNAi and knock down
genes)
pathways, 531–532
as a research tool, 536–537
RNA silencing and, 526
role in silencing viral and transposon
DNA, 532–534
as therapeutic, 538–540
RNAi feeding vector construction
about, 591–594
answers to questions, 666–673
further reading, 594
objectives, 591
planning and preparation instructions,
627–639
planning and preparation overview,
595–597
recipes for reagents and stock solutions,
641–658
results and discussion, 615, 619
Stage A: identify a disease-causing gene
and design PCR primers in silico,
597–599
Stage B: clone gene-specific DNA, 600,
601–604
Stage C: transformation of E. coli, 606,
607–611
Stage D: vector analysis, 612, 613–615
Stage E: isolate plasmid DNA, 616,
617–618
Stage F: create RNAi feeding strain, 620,
621–626
RNAi mechanism examination
about, 575–576
answers to questions, 662–666
bioinformatics methods, 584–587
experimental methods
amplifying DNA by PCR, 582–583
analyzing PCR products by gel electrophoresis, 583–584
induce RNAi by feeding, 580–581
isolating DNA from C. elegans,
581–582
overview, 579
transfer wild-type and dpy-13 C. elegans to OP50-seeded NGM-lite
plates, 580
further reading, 577
objectives, 575
planning and preparation instructions,
627–639
planning and preparation overview, 578
recipes for reagents and stock solutions,
641–658
results and discussion, 588–589
RNA-induced silencing complex (RISC), 527
RNase III family, 527
RNase P, 541
Roberts, Richard, 24
Rocheford, Torbert, 354
Rockefeller Foundation, 345, 346
Rogers, Stephen, 349
Römpler, Holger, 198
Rothberg, Jonathan, 41
Roundup (glyphosate), 350
Rous sarcoma virus (RSV), 548
Rowley, Janet, 186
RSV (Rous sarcoma virus), 548
Russell, Wilbert, 342
Ruvkun, Gary, 529
S
Sang, Tao, 320
Sanger, Fred, 33, 34
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Scaffolds, 44
Schaal, Barbara, 315
Scheck, Barry, 180
Schell, Jeff, 349
Schulman, Alan, 338
Science, The Endless Frontier (Bush), 311
Sebat, Jonathan, 189
Segregating pseudogenes, 206
Selfish Gene, The (Dawkins), 532
SELF PRUNING (SP), 345
Semi-dwarf varieties of plants, 345, 347
Sense strand of DNA, 19
Sequence Server, 123
Sequence-tagged site (STS), 176
Seven daughters of Eve, 209, 210
SFT (SINGLE FLOWER TRUSS), 344
Sharp, Philip, 24
Shattering QTLs, 318–321
Short interfering RNAs (siRNAs), 527
Short tandem repeats (STRs), 177
shRNAs (short hairpin RNAs), 538
Shull, George, 169, 340, 341, 342, 343
Sickle cell disease, 172–173, 174
Simon, Mel, 43
SINGLE FLOWER TRUSS (SFT), 344
Single-nucleotide polymorphisms (SNPs),
155
Single-stranded RNA (ssRNA), 526
siRNAs (short interfering RNAs), 527
Skin pigmentation and evolution, 203–204
Skolnick, Mark, 175
Small nuclear RNAs (snRNAs), 24–25, 541
Small nucleolar RNAs (snoRNAs), 543
Small temporal RNA (stRNA), 529
Smith, C. Earle, 324
Smith, Hamilton, 46
Smith, Lloyd M., 34
Smithies, Oliver, 515, 516
SNAP, 52–53, 119
snoRNAs (small nucleolar RNAs), 543
SNPs (single-nucleotide polymorphisms), 155
snRNAs (small nuclear RNAs), 24–25, 541
SOLiD system, 40–41
Sorangium, cellulosum, 549
Sorghum, 331, 332, 334
Southern Dent corn variety, 339–340
Soybean, 331, 332
SP (SELF PRUNING), 345
Spiegelman, Sol, 11
Spirodela polyrhiza (duckweed), 357
ssRNA (single-stranded RNA), 526
Stark, Benjamin, 541
Start codon, 20
Station for Experimental Evolution, 163
Steitz, Thomas, 543
Sterilization laws, 167–168, 171
Stern, Curt, 5
Stevens, Nettie, 3
Stop codon, 20
Streptomyces, 549
stRNA (small temporal RNA), 529
STRs (short tandem repeats), 177
STS (sequence-tagged site), 176
Stuitje, Antoine, 524
Sturtevant, Alfred, 4, 5
Sulston, John, 519, 520, 521, 523
Supreme Court, U.S., 168
Sutton, Walter, 3
Sykes, Brian, 209
Syntenic dotplot, 77–79
Synteny, 158
Szostak, Jack, 545, 546, 549, 550
T
Tamoxifen, 191
Tanno, Ken-ichi, 318
Tarceva, 191
TARGeT (tree analysis of related genes and
transposons), 100, 123
TargetScan, 109–111, 123–124
TAS2R38 gene, 262–266
TATA box, 23
Tatum, Edward, 6, 172
TBP (TATA-binding protein), 23
Telomere, 549
Temin, Howard, 548, 549
Teosinte
connection to maize, 322–324
discovery of popcorn from, 324
origin of, 321
Terminal inverted repeats (TIRs), 336
Tetrahymena, 541
TFs (transcription factors), 22
Therapeutic proteins, 173, 174
TIGR (The Institute for Genomic Research), 36
TIRs (terminal inverted repeats), 336
Tishkoff, Sarah, 207, 208
Tjio, Joe Him, 172
Toluca volcano, 329, 331
Toluqueño, Palomero, 329
Torrent, Ion, 41
Torroni, Antonio, 209
Tourist, 338
Trait Book (Davenport), 163
Transcription and translation, 21–22
Transcription factors (TFs), 22
Transposition, 335–339
Transposon content in grasses
about, 87–89
answers to questions, 142–145
comparing the transposable element content in brachypodium, rice, and
corn, 92–93
extending the analysis of transposons using
other tools in DNA Subway, 94
further reading, 89
identifying transposable elements in the
adh1 region of the brachypodium
genome, 91–92
identifying transposable elements in the
adh1 region of the corn genome, 92
identifying transposable elements in the
adh1 region of the rice genome, 92
planning and preparation instructions,
113–124
planning and preparation overview, 90
Transposon detection in Arabidopsis
about, 375–376
answers to questions, 489–494
bioinformatics methods
determining the insertion site of the
Ds transposon, 387–388
identify the CLF amino acid sequence,
385–386
using BLAST to determine the function of the CLF protein, 388
using BLAST to find DNA sequences
in databases, 383–385
using Map Viewer to determine the
chromosomal location of the CLF
gene, 386–387
experimental methods
amplifying DNA by PCR, 382
analyzing PCR products by gel electrophoresis, 382–383
isolating DNA, 380–381
overview, 379
plant seeds, 380
further reading, 376–377
objectives, 375
planning and preparation instructions,
463–474
planning and preparation overview, 378
recipes for reagents and stock solutions,
475–484
results and discussion, 388–391
Transposon detection in corn
about, 359–360
answers to questions, 485–489
bioinformatics methods
determining the insertion site of the
Ds transposon, 370–371
identify the BZ amino acid sequence,
369–370
using BLAST to determine the function of the BZ protein, 371–372
using BLAST to find DNA sequences
in databases, 367–369
using Map Viewer to determine the
chromosomal location of the BZ
gene, 370
experimental methods
amplifying DNA by PCR, 365–366
analyzing PCR products by gel electrophoresis, 366–367
isolating DNA from maize, 364–365
overview, 363
plant maize seed, 364
further reading, 361
objectives, 359
planning and preparation instructions,
463–474
planning and preparation overview, 362
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692 / Index
Transposon detection in corn (continued)
recipes for reagents and stock solutions,
475–484
results and discussion, 372–374
Transposons, 161–162. See also Transposon
content in grasses; Transposon
detection in Arabidopsis;
Transposon detection in corn
Trichuris (worm), 316, 317
Triticum sp. (wheat). See Wheat cultivation
tRNAscan-SE, 124
Troyer, David and Chester, 339p
Troyer Reid hybrid corn, 339p
Tryptophan, 12
Tschermak-Seysenegg, Erich von, 3
Tumor-inducing (Ti) plasmid vector, 348
Tumor-suppressor genes, 187
Turcatti, Gerardo, 40
Tuschl, Tom, 527, 534
23andMe, 155
U
Untranslated regions (UTRs), 22
Urey, Harold, 544
Using an Alu insertion polymorphism to
study human populations lab. See
Alu insertion
Using a single-nucleotide polymorphism to
predict bitter-taste ability lab. See
Bitter-taste ability investigation
Using DNA barcodes to identify and classify
living things lab. See DNA barcodes use
Using E. coli feeding strains to induce RNAi
and knock down genes lab. See E.
coli feeding strains used to induce
RNAi and knock down genes
Using mitochondrial DNA polymorphisms in
evolutionary biology lab. See
Mitochondrial DNA polymorphisms
UTRs (untranslated regions), 22
V
Vance, Vicki, 533
Van Montagu, Marc, 349
Variable-number tandem repeats (VNTRs),
160, 176–177
VEGF (vascular endothelial growth factor),
538
Venter, J. Craig, 36–37, 46
Vicens, Quentin, 547
Vielle-Calzada, Jean-Philippe, 329
Viroids, 547
Vitamin A deficiency, 353–356
Vitravene (fomivirsen), 524
VNTRs (variable-number tandem repeats),
160, 176–177
Vogel, Friedrich, 190
Vogelstein, Bert, 187
Volcanic eruptions and plant cultivation, 331
Von Hansemann, David, 186
W
Walbot, Virginia, 338, 349
Wallace, Douglas, 204
Wallace, Henry, 342, 345
Wallace, Margaret, 337
Warner, Mark, 169
Watson, James, 8, 10, 11, 39–40, 156
Weinberg, Wilhelm, 170
Weismann, August, 164, 165p
Weiss, Samuel, 11
Wessler, Susan, 337, 338
Wheat cultivation
Green Revolution and, 345–347
origins of, 313–317
White, Ray, 176, 177p
White-Harrison, Susan, 525
WHO (World Health Organization), 352
Whole-genome shotgun, 41–42
Wieschaus, Eric, 512
Wigler, Michael, 189
Wilkins, Maurice, 10
Willcox, George, 318
Wilson, Allan, 194
Wilson, Edmund, 3, 175
Winkler, Hans, 1
Woese, Carl, 544
World Health Organization (WHO), 352
World’s Fair Corn, 340
Worm (Trichuris), 316, 317
Wu, Louisa, 533
Y
Yellow Dent corn hybrid, 340
Yerkes, Robert, 165
Yonath, Ada, 543
Yu, Jun, 344
Z
Zamecnik, Paul, 11, 12, 523
Zamir, Dani, 344
Zapalote Chico, 338
Zeaxanthin, 354
Zech, Lore, 186
Zein genes, 352–353
Zong, Cheng, 315