Download Fig. 20.14a

Agenda 12/12/11
• Collect dye electrophoresis worksheet
• Go over paper plasmid activity – do a mock digest and
electrophoresis
– look at one student’s recombinant plasmid and restriction map – pick an
enzyme to cut and talk about different sized fragments you would get
• A few more slides on gene cloning/biotech
• Intro 6B restriction enzyme lab – go over digest procedures (20 min)
• Will check Ch. 20 notes tomorrow
Homework –
Ch. 21 Notes and self-quiz due Thurs. 12/16 – use my slides to focus
on what you need to know
DNA Scissors part 2 restriction map due Wednesday – do on own, then
check key on website
Read and do prelab 6B and be prepared to do day 1 (digest) tomorrow
– 4th will run into lunch!!!! 5th period should come in 15 minutes
early, same thing for day 2
Bridging the paper plasmid activity
• What if we put whole eukaryotic gene into
a prokaryote?
– What can’t they do? (see next slide)
Continuing
on gene
cloning…
• Complementary
DNA(cDNA) is
DNA made in
vitro using
mRNA as a
template and the
enzyme reverse
transcriptase.
-impt. since
bacteria can’t do
Fig. 20.5
mRNA
Copyright
© 2002 Pearson Education, Inc., publishing as Benjamin Cummings
processing
Cloned genes are stored in DNA
libraries
• A complete set of recombinant plasmid
clones, each carrying copies of a particular
segment from the initial genome, forms a
genomic library.
– The library can be saved and used as a source
of other genes or for gene mapping.
• cDNA libraries also exist
– Limitations are every cell only expressed certain
mRNA’s at a time, so won’t get every gene
• In addition to plasmids, certain
bacteriophages are also common cloning
vectors for making libraries.
– The recombinant phage
DNA is packaged in a
capsid in vitro and
allowed to infect a
bacterial cell.
– Infected bacteria
produce new phage
particles, each with
the foreign DNA.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
The polymerase chain reaction
(PCR) clones DNA entirely in vitro
• DNA cloning is the best method for preparing
large quantities of a particular gene or other
DNA sequence.
• When the source of DNA is scanty or impure,
the polymerase chain reaction (PCR) is
quicker and more selective.
• This technique can quickly amplify any piece
of DNA without using cells.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• The DNA is
incubated in a
test tube with
special DNA
polymerase, a
supply of
nucleotides,
and short
pieces of
singlestranded DNA
as a primer.
Fig. 20.7
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• PCR can make billions of copies of a targeted
DNA segment in a few hours.
– This is faster than cloning via recombinant bacteria.
• In PCR, a three-step cycle: heating, cooling, and
replication, brings about a chain reaction that
produces an exponentially growing population of
DNA molecules.
– The key to easy PCR automation was the discovery of
an unusual DNA polymerase, isolated from bacteria
living in hot springs, which can withstand the heat
needed to separate the DNA strands at the start of
each cycle.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Devised in 1985, PCR has had a major
impact on biological research and
technology.
• PCR has amplified DNA from a variety of
sources:
– fragments of ancient DNA from a 40,000-yearold frozen wooly mammoth,
– DNA from tiny amount of blood or semen found
at the scenes of violent crimes,
– DNA from single embryonic cells for rapid
prenatal diagnosis of genetic disorders,
– DNA of viral genes from cells infected with
difficult-to-detect viruses such as HIV.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Intro Restriction Digest Lab
Electrophoresis (aka DNA Fingerprinting)
Real World Applications
• Crime scene
• Human relatedness
• Paternity
• Animal relatedness
• Anthropology studies
• Disease-causing organisms
• Food identification
• Human remains
• Monitoring transplants
Restriction enzymes
• Type 2 restriction endonucleases are used in
this lab. (most common)
• Cut DNA at a specific sequence.
• Enzymes used
– HindIII
– EcoRI
– PstI
Why are there restriction
enzymes?
• Evolved by
bacteria to protect
against viral
infection
• Over 3000 known
enzymes
Bacteriophage Lambda DNA
• Show picture p. 31 of BioRad Instruction
Manual
– Shows where HindIII cuts
– Similar maps exist for other restriction enzymes,
including the additional ones we will use - what
were they?
– Differs from our restriction digest of the paper
plasmid
• If plasmid cut once, how many fragments?
• If linear phage DNA cut once, how many fragments?
Day 1 – Restriction Digest
Day 2 - Electrophoresis
Day 3 – Analysis of Gel
The DNA Digestion Reaction
Restriction Buffer provides optimal
conditions
• NaCI provides the correct ionic
strength
• Tris-HCI provides the proper pH
• Mg2+ is an enzyme co-factor
DNA Digestion Temperature
Why incubate at 37°C?
• Body temperature is optimal for these and
most other enzymes
What happens if the temperature is too hot or
cool?
• Too hot = enzyme may be denatured (killed)
• Too cool = enzyme activity lowered, requiring
longer digestion time
Lab Procedure day 1
• Each group of 4 students sets up 4 tubes.
• Handout and go through Quick Guide
procedures together
• Make hypotheses about each tube
bs.kaist.ac.kr
Things to remember :
• Set up Digests on ICE!
• Concentrations are important (More is not
better)
– THESE ARE TINY AMOUNTS – BE SURE YOUR TIP IS IN
BOTTOM AND YOU GET IT ALL – NO AIR BUBBLES!!!
• Add reagents in Correct order and use fresh
tip for buffer and enzymes.
– DNA, then buffer, then enzymes are always last.
• Reason for Enzymes being last: Enzymes are
sensitive to conditions outside of their normal
range. Strong buffer solutions can effect the
• Keep it clean and don’t digest too long
– Contaminations of DNases can be
devastating.
Lane 8 has little to no
DNase activity
Lane 1 has a large
amount of DNase activity
biosyn.com
Agenda 12/13/11
• Run restriction digest – digest for 30 minutes
• While waiting for digest, go over electrophoresis
procedures and prelab (last 20-30 minutes of class once
all digests in water bath)
• Leave Ch. 20 notes out to be checked during period
Homework –
Ch. 21 Notes and self-quiz due Thurs. 12/16 – use my
slides to focus on what you need to know
DNA Scissors worksheet due Wed – check answers with
key on website and be sure you understand!
Things to remember :
• Set up Digests on ICE!
• Concentrations are important (More is not
better)
– THESE ARE TINY AMOUNTS – BE SURE YOUR TIP IS IN
BOTTOM AND YOU GET IT ALL – NO AIR BUBBLES!!!
• Add reagents in Correct order and use fresh
tip for buffer and enzymes.
– DNA, then buffer, then enzymes are always last.
• Reason for Enzymes being last: Enzymes are
sensitive to conditions outside of their normal
range. Strong buffer solutions can effect the
efficiency of the digest
• Keep it clean and don’t digest too long
– Contaminations of DNases can be
devastating.
Lane 8 has little to none
DNase activity
Lane 1 has a large
amount of DNase activity
biosyn.com
Look at Quick Guide for Day 2
• Switch off who does pipetting for each
tube – other person can be uncapping etc.
• Will do overnight staining
Making a standard curve
QuickTime™ and a
decompressor
are needed to see this picture.
• Measure to leading
edge of each band
in the marker/
ladder
Standard Curve
QuickTime™ and a
decompressor
are needed to see this picture.
• Graph of lambda
HindIII marker (base
pair vs. Distance
migrated)
• Using semi log graph
paper
Using graph to find size of
Unknowns
QuickTime™ and a
decompressor
are needed to see this picture.
Agenda 12/14/11
• Electrophoresis of digest from yesterday and staining
• I check DNA Scissors worksheet & prelab while runs
• While running, you work on Analysis 1-2 and Questions
1-6,8 in lab manual
Homework –
Ch. 21 Notes and self-quiz due tomorrow – use my slides
to focus on what you need to know
Finish analysis questions (except for the couple you can’t
do yet)
Lab procedure day 2
• Add loading dye to each of your 4 tubes,
load marker and contents of 4 tubes and
run gel.
• 1 group per gel
• Run at 100V for approx. 30 min
• Stain the gel
Agenda 12/15/11
• Next 4 slides – possible errors and modified lab abstract
format
• 25 min - Analyze electrophoresis lab data – put plastic wrap
on white paper and measure – Add a Table 6.3 to bottom of
p. 73 for the PstI results and note the estimated base pair
length of the uncut DNA in the L tube
• I check Ch. 21 while you measure, graph and fill in tables
• Give actual bp data from p. 56 of BioRad manual and briefly
go over analysis questions (15 min)
• Start Ch. 20 slides and 21 highlights, slides 33-71 (15 min)
Homework –
Finish 6B analysis to be checked tomorrow
Write abstract for lab 6 (modified format) – Transformation and
Restriction Digest – due tomorrow I suggest unless you
want to work on it over break
Things that can go wrong
• Impeded digestion due to incorrect set
up. (too much or too little buffer etc.)
• Star digest activity
– Under non-standard reaction
conditions, some restriction enzymes
are capable of cleaving sequences
which are similar but not identical to
their defined recognition sequence.
This altered specificity has been
termed star activity"
Star digestion example
• Examples:
– EcoRI is supposed
to only cut
GAATTC but,
under extreme
conditions, it might
possibly cut
CAATTC also.
QuickTime™ and a
decompressor
are needed to see this picture.
http://www.fermentas.com/en/support/technical-reference/restriction-enzymes/star-activity
Star digests
• Things that can cause Star digests
– Too much glycerol in reaction. (More is not
better)
– Incorrect buffer or buffer concentration
– Extended digest times. Don’t leave them over
night
Lab 6 Abstract
• Paragraph 1- Transformation – hypoth.,
results, lab error
• Paragraph 2- Digest & Electrophoresis –
hypoth, results, lab error
• Paragraph 3 – Relate both to steps in
Gene Cloning/Biotech Curriculum
• References
• Attach – Semilog graph
• Can turn in tomorrow or when back
• Comparisons among whole sets of genes
and their interactions is the field of
genomics.
• One indirect method of rapidly analyzing and
comparing genomes is gel electrophoresis.
– Gel electrophoresis separates macromolecules nucleic acids or proteins - on the basis of their
rate of movement through a gel in an electrical
field.
• Rate of movement depends on size, electrical charge,
and other physical properties of the macromolecules.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• For linear DNA molecules, separation
depends mainly on size (length of
fragment) with longer fragments migrating
less along the gel.
Fig. 20.8
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
1. Restriction fragment analysis detects DNA
differences that affect restriction sites
• Restriction fragment analysis indirectly
detects certain differences in DNA nucleotide
sequences.
– After treating long DNA molecules with a restriction
enzyme, the fragments can be separated by size via gel
electrophoresis.
– This produces a series of bands that are characteristic of
the starting molecule and that restriction enzyme.
– The separated fragments can be recovered
undamaged from gels, providing pure samples of
individual fragments.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• We can use restriction fragment analysis to
compare two different DNA molecules
representing, for example, different alleles.
– Because the two alleles must differ slightly in
DNA sequence, they may differ in one or more
restriction sites.
– If they do differ in restriction sites, each will
produce different-sized fragments when
digested by the same restriction enzyme.
– In gel electrophoresis, the restriction fragments
from the two alleles will produce different band
patterns, allowing us to distinguish the two
alleles.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Restriction fragment analysis is sensitive enough
to distinguish between two alleles of a gene that
differ by only 1 base pair in a restriction site.
HERE WE ARE RUNNING
PURIFIED SEGMENT OF DNA
WITH GENE OF INTEREST
Fig. 20.9
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Gel electrophoresis combined with nucleic acid
hybridization allows analyses to be conducted on
the whole genome, not just cloned and purified
genes.
• Although electrophoresis will yield too many
bands to distinguish individually, we can use
nucleic acid hybridization with a specific probe to
label discrete bands that derive from our gene of
interest.
• The radioactive label on the single-stranded
probe can be detected by autoradiography,
identifying the fragments that we are interested
in.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• We can tie together several molecular techniques
to compare DNA samples from three individuals.
– We start by adding the restriction enzyme to each of
the three samples to produce restriction fragments.
– We then separate the fragments by gel
electrophoresis.
– Southern blotting (Southern hybridization) allows us
to transfer the DNA fragments from the gel to a sheet
of nitrocellulose paper, still separated by size.
• This also denatures the DNA fragments.
– Bathing this sheet in a solution containing our probe
allows the probe to attach by base-pairing (hybridize)
to the DNA sequence of interest and we can visualize
bands containing the label with autoradiography.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• For our three individuals, the results of these steps show that individual
III has a different restriction pattern than individuals I or II. Here we are
running all DNA but probe identifies the fragments that have to do with
our gene of interest.
Fig. 20.10
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Southern blotting can be used to examine
differences in noncoding DNA as well.
• Differences in DNA sequence on
homologous chromosomes that produce
different restriction fragment patterns are
scattered abundantly throughout genomes,
including the human genome.
• These restriction fragment length
polymorphisms (RFLPs) can serve as a
genetic marker for a particular location
(locus) in the genome.
– A given RFLP marker frequently occurs in
numerous variants in a population.
• RFLPs are detected and analyzed by
Southern blotting, frequently using the
entire genome as the DNA starting
material.
– These techniques will detect RFLPs in
noncoding or coding DNA.
• Because RFLP markers are inherited in a
Mendelian fashion, they can serve as
genetic markers for making linkage maps.
– The frequency with which two RFPL markers or a RFLP marker and a certain allele for a
gene - are inherited together is a measure of
the closeness of the two loci on a
DNA MICROASSAYS
• Automation has allowed scientists to detect
and measure the expression of thousands
of genes at one time using DNA
microarray assays.
– Tiny amounts of a large number of singlestranded DNA fragments representing different
genes are fixed on a glass slide in a tightly
spaced array (grid).
– The fragments are tested for hybridization with
various samples of fluorescently-labeled cDNA
molecules.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Fig. 20.14a
• Spots where any of the cDNA hybridizes
fluoresce with an intensity indicating the
relative amount of the mRNA that was in
the tissue.
Fig. 20.14b
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Ultimately, information from microarray
assays should provide us a grander view:
how ensembles of genes interact to form a
living organism.
– It already has confirmed the relationship
between expression of genes for
photosynthetic enzymes and tissue function in
leaves versus roots of the plant Arabidopsis.
– In other cases, DNA microarray assays are
being used to compare cancerous versus
noncancerous tissues.
• This may lead to new diagnostic techniques and
biochemically targeted treatments, as well as a fuller
understanding of cancer.
• Perhaps the most interesting genes
discovered in genome sequencing and
expression studies are those whose
function is completely mysterious.
• One way to determine their function is to
disable the gene and hope that the
consequences provide clues to the gene’s
normal function.
– Using in vitro mutagenesis, specific changes
are introduced into a cloned gene, altering or
destroying its function.
– When the mutated gene is returned to the cell,
it may be possible to determine the function of
the normal gene by examining the phenotype
of the mutant.
• In nonmammalian organisms, a simpler
and faster method, RNA interference
(RNAi), has been applied to silence the
expression of selected genes.
– This method uses synthetic double-stranded
RNA molecules matching the sequences of a
particular gene to trigger breakdown of the
gene’s mRNA.
– The mechanism underlying RNAi is still
unknown.
– Scientists have only recently achieved some
success in using the method to silence genes
in mammalian cells.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• The next step after mapping and sequencing
genomes is proteomics, the systematic study of
full protein sets (proteomes) encoded by
genomes.
– Difficult due to vast number
• Genomic and proteomics are giving
biologists an increasingly global perspective
on the study of life.
• Advances in bioinformatics, the application
of computer science and mathematics to
genetic and other biological information, will
play a crucial role in dealing with the
enormous mass of data.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• These analyses will provide understanding of the
spectrum of genetic variation in humans.
– Because we are all probably descended from a small
population living in Africa 150,000 to 200,000 years ago,
the amount of DNA variation in humans is small.
– Most of our diversity is in the form of single nucleotide
polymorphisms (SNPs), single base-pair variations.
• In humans, SNPs occur about once in 1,000 bases, meaning
that any two humans are 99.9% identical.
– The locations of the human SNP sites will provide useful
markers for studying human evolution and for identifying
disease genes and genes that influence our
susceptibility to diseases, toxins or drugs.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Ch. 21 Highlights – Genes and
Development
Differential gene expression leads to different cell
types in multicellular organisms
• Zygote undergoes transformation through 3
interrelated cell processes
1) Cell division – mitosis increases # of cells
2) Cell differentiation – cells become specialized in
structure and function
3) Morphogenesis – organization of cells into tissues
and organs
What are the terms that describe an embryo going
through these stages? (Remember Ch. 47)
Fig. 21.2
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
– cell lineage, a type of fate map.
• A fate map traces the development of an embryo.
CHAPTER 21
THE GENETIC BASIS OF
DEVELOPMENT
Section B: Differential Gene Expression
1. Different types of cells in an organism have the same DNA
2. Different cell types make different proteins, usually as a result of
transcriptional regulation
3. Transcriptional regulation is directed by maternal molecules in the
cytoplasm and signals from other cells
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Differing Gene Expression
• The differences between cells in a
multicellular organism come almost entirely
from differences in gene expression, not
differences in the cell’s genomes.
• These differences arise during development,
as regulatory mechanisms turn specific
genes off and on.
• An important question that emerges is
whether genes are irreversibly inactivated
during differentiation.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
1. Different types of cell in an
organism have the same DNA
• Much evidence supports the conclusion that
nearly all the cells of an organism have
genomic equivalence - that is, they all have
the same genes.
• An important question that emerges is
whether genes are irreversibly inactivated
during differentiation.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
In many plants, whole new organisms can develop from differentiated somatic cells.
During the 1950s, F.C. Steward and his students found that differentiated root cells
removed from the root could grow into normal adult plants when placed in a medium
culture.
•These cloning experiments produced genetically identical individuals, popularly called
clones.
In plants, at least, cell can remain totipotent.
•Plant cloning is now used extensively in agriculture.
Animal Cloning Much Harder
• In tadpoles, the ability of
the transplanted nucleus to
support normal
development is inversely
related to the donor’s age.
• 1997 when Ian Wilmut
and his colleagues
cloned an adult sheep
• One, “Dolly,” of
several hundred
implanted embryos
completed normal
development.
– Improper
methylation in
many cloned
embryos interferes
with normal
development.
Stem Cells
• Stem cells that can
differentiate into
multiple cell types are
multipotent or, more
often, pluripotent.
• Embryonic vs. adult
– embryonic stem cells
are “immortal” because
of the presence of
telomerase that allows
these cells to divide
indefinitely.
• Under the right
conditions, cultured
stem cells derived
from either source can
differentiate into
specialized cells.
• Beyond the study of differentiation, stem cell
research has enormous potential in medicine.
• The ultimate aim is to supply cells for the repair of
damaged or diseased organs.
– For example, providing insulin-producing pancreatic
cells to diabetics or certain brain cells to individuals
with Parkinson’s disease could cure these diseases.
• At present, embryonic cells are more promising
than adult cells for these applications.
• However, because embryonic cells are derived
from human embryos, their use raises ethical and
political issues.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
More Ch. 21
• What controls differentiation
and morphogenesis?
1) Cytoplasmic
determinants – maternal
substances in egg that are
distributed unevenly in early
cells of embryo
2) Cell-cell signals result
from molecules, such as
growth factors, that are
produced by one cell to
influence neighboring cells –
called induction.
– Causes cells to differentiate
Determination – series of events that lead to
observable differentiation of a cell. Differentiation is
caused by cell-cell signals and is irreversible. When
cell is irreversible committed to its final fate, it is said
to be “determined.”
Fig. 21.9
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
More Ch. 21
Pattern formation – sets up the body plan as a
result of cytoplasmic determinants and
inductive signals.
– Determines head/tail, left/right, back/front
– Uneven distribution of substances called
morphogens plays a role in establishing these
axes.
• Bicoid mRNA- a morphogen
– Concentrated at extreme
anterior end
More Ch. 21
• Homeotic genes – master regulatory genes that
determine the location and organization of body
parts, esp. appendages of each segment
Hox genes = one class of Homeotic genes (include 180
nucleotide sequence called homeobox)
– Highly conserved in evolution
- changes in these genes and the genes that regulate
them can have a profound effect on morphology,
leading to evolutionary change
ex: variable expression of a Hox gene in a fish fin bud
and a chicken leg bud leads to longer skeletal
extension in chicken leg compared to fish fin
• In fact, the
vertebrate genes
homologous to the
homeotic genes of
fruit flies have even
kept their
chromosomal
arrangement.
Fig. 21.15
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Apoptosis (Cell suicide) and Normal
Development
• A built-in cell suicide mechanism is
essential to development in all animals.
– Similarities between the apoptosis genes in
mammals and nematodes indicate that the
basic mechanism evolved early in animal
evolution.
– The timely activation of apoptosis proteins in
some cells functions during normal
development and growth in both embryos and
adults.
• It is part of the normal development of the
nervous system, normal operation of the
immune system, and for normal morphogenesis
ofPearson
human
hands
and
feet.
Copyright © 2002
Education, Inc.,
publishing as
Benjamin
Cummings
• Problems with the cell suicide mechanism
may have health consequences, ranging
from minor to serious.
– Failure of normal cell death during
morphogenesis of the hands and feet can result
in webbed fingers and toes.
– Researchers are also investigating the
possibility that certain degenerative diseases of
the nervous system result from inappropriate
activation of the apoptosis genes.
– Others are investigating the possibility that some
cancers result from a failure of cell suicide which
normally occurs if the cell has suffered
irreparable damage, especially DNA damage.
Agenda 12/16/11
• Finish slides from yesterday (if we don’t finish, you need to finish on
your own over break and write down any questions you have for first
day back)
• Collect lab reports that are done
• Check that 6B Analysis is done in lab manual
Homework –
Write abstract for lab 6 – Transformation and Restriction Digest - if not
done
Review Manual - Molecular Genetics (Ch. 11) and Lab 6 & 7 due Tues
1/3
Molecular Genetics/Biotech Test Thurs. 1/5
Will also cover Ch. 22-25 before finals so could get started on those.