Chapter 20- Molecular Techniques
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Restriction Enzymes
Gel electrophoresis
RFLP
STRs
Southern blots
Sequencing
Recombinant DNA
• Genomic libraries
• PCR
• RNA analysis
– northerns
– cDNAs
– microarrays
• Gene therapy and
genetically modified
organisms
Restriction Endonucleases
• Isolated from bacteria
• Defense mechanism for
bacteria
• Recognize specific
sequences
• Named after organism
they were isolated from
I. Background
Nucleic acids
• Notice their sugar
component (deoxyribose)
• Notice their base (a purine
or a pyrimidine)
• Notice their phosphate
groups.
• In a basic buffer, the
hydrogen atoms dissociate
and the phosphate groups
become negatively charged.
They act like an acid.
3
Gel Electrophoresis
• Place DNA/RNA in
basic buffer
• Negative charges
predominate
• Apply electric current
• Migration toward anode
• Separation according to
size
TECHNIQUE
Mixture of
DNA molecules of
different
sizes
– Cathode
Power
source
Anode
+
Gel
1
–
Power
source
+
Longer
molecules
2
RESULTS
Shorter
molecules
Use of restriction enzymes and gel electrophoresis for
identifying gene mutations
Normal -globin allele
175 bp
DdeI
Sickle-cell
allele
Large fragment
201 bp
DdeI
Normal
allele
DdeI
DdeI
Large
fragment
Sickle-cell mutant -globin allele
376 bp
DdeI
201 bp
175 bp
Large fragment
376 bp
DdeI
DdeI
(a) DdeI restriction sites in normal and
sickle-cell alleles of -globin gene
(b) Electrophoresis of restriction fragments
from normal and sickle-cell alleles
• Restriction fragment length polymorphisms
(RFLP)
Restriction Fragment Length Polymorphism Analysis
• Emanate from SNP- single
nucleotide polymorphisms
• RFLP
• “rif-lip” vs. R-F-L-P
• Restriction Enzymes
• Used to detect alleles and mutations.
• Huntington’s Disease- trinucleotide
repeats make the RFLP
longer….how would this migrate on
the gel?
DNA
Fingerprinting
• Short tandem repeats
(STRs)
• DNA sequences
repeated in a row
• Varies in number
from individual to
individual
• 13 standard STRS
• Guilty? Innocent?
“check CODIS”
• STR sites- short
tandem repeats
• 13 predetermined sites
• Number of repeats for
an individual can be
entered
• 4 nucleotide repeats at
13 sites
• COmbined DNA
Index System
Fig. 20-24
(a) This photo shows Earl
Washington just before
his release in 2001,
after 17 years in prison.
Source of
sample
STR
marker 1
STR
marker 2
STR
marker 3
Semen on victim
17, 19
13, 16
12, 12
Earl Washington
16, 18
14, 15
11, 12
Kenneth Tinsley
17, 19
13, 16
12, 12
(b) These and other STR data exonerated Washington and
led Tinsley to plead guilty to the murder.
Transfers and blots
• Dr. Southern -1973
• Analyze different genes
• Need probe
3 C C G A TT G A A T C G 5
Southern Blot
Short term
Quick viewing
of DNA
fragments
Long term storage
Blot can be reused
(re-hybridized to
other probes) and
stored easily
Image from:http://openlearn.open.ac.uk/file.php/2645/S377_1_007i.jpg
Animation:
http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html
1
Fig. 20-11
TECHNIQUE
DNA + restriction enzyme
Restriction
fragments
I
II
III
Heavy
weight
Nitrocellulose
membrane (blot)
Gel
Sponge
I Normal II Sickle-cell III Heterozygote
-globin allele
allele
2 Gel electrophoresis
1 Preparation of restriction fragments
Paper
towels
Alkaline
solution
3 DNA transfer (blotting)
Radioactively labeled
probe for -globin gene
I
II III
Probe base-pairs
with fragments
Fragment from
sickle-cell
-globin allele
Nitrocellulose blot
Fragment from
normal -globin
allele
4 Hybridization with radioactive probe
I
II III
Film
over
blot
5 Probe detection
Of course, in order to design a probe, we
need to know the sequence
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Genome sequence
www.nlm.nih
Genbank
J. Craig Venter
Fig. 20-12
TECHNIQUE
DNA
(template strand)
Primer
DNA
polymerase
DNA (template
strand)
Deoxyribonucleotides
dATP
ddATP
dCTP
ddCTP
dTTP
ddTTP
dGTP
ddGTP
Labeled strands
Shortest
Direction
of movement
of strands
Longest
Longest labeled strand
Detector
Laser
RESULTS
Shortest labeled strand
Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand
Dideoxyribonucleotides
(fluorescently tagged)
Recombinant DNA techniques
• Cut DNA with endonuclease
• Isolate DNA band from
electrophoresis gel
• Isolate plasmid from bacteria
• Cut with same endonuclease
• Mix human DNA with plasmid
and ligate
• Transform recombinant plasmid
back into bacteria
• Ampicillin plates
• lacZ operon
Restriction site
DNA
1
5
3
3
5
Restriction enzyme
cuts sugar-phosphate
backbones.
Sticky end
2
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
One possible combination
3
DNA ligase
seals strands.
Recombinant DNA molecule
Fig. 20-4-4
Hummingbird
cell
TECHNIQUE
Bacterial cell
lacZ gene
Restriction
site
ampR gene
Sticky
ends
Bacterial
plasmid
Gene of interest
Hummingbird
DNA fragments
Nonrecombinant
plasmid
Recombinant plasmids
Bacteria carrying
plasmids
RESULTS
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying recombinant
plasmid with disrupted lacZ gene
One of many
bacterial
clones
Fig. 20-2b
Recombinant
bacterium
3 Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Protein expressed
by gene of interest
Gene of
Interest
Copies of gene
Protein harvested
4 Basic research and
Basic
research
on gene
Gene for pest
resistance inserted
into plants
various applications
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth hormone treats stunted
growth
Recombinant DNA allows us to make
human proteins
• Growth
Hormone
• EPO
Cloned genes can be stored in genomic
libraries
Foreign genome
cut up with
restriction
enzyme
Large insert
Large plasmid with many genes
or
BAC
clone
Recombinant
phage DNA
Bacterial
clones
(a) Plasmid library
Recombinant
plasmids
(b) Phage library
Phage
clones
(c) A library of bacterial artificial
chromosome (BAC) clones
These libraries could be transferred to nylon
membrane and analyzed for genes
TECHNIQUE
Radioactively
labeled probe
molecules
Multiwell plates
holding library
clones
Probe
DNA
Gene of
interest
Single-stranded
DNA from cell
Film
•
Nylon membrane
Location of Nylon
DNA with the membrane
complementary
sequence
PCR- polymerase chain reaction
• Kary Mullis-Nobel Prize in
Chemistry 1993
• Taq polymerase (Thermus
aquaticus)
• Nucleotides
• Primers
• Denaturation
• Annealing
• Extension
• 20-40 cycles
• MANY uses
5
TECHNIQUE
3
Target
sequence
3
Genomic DNA
1 Denaturation
5
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
Studying Transcription- 1st half of gene
expression
• Isolate mRNA
• Transcribed genes
– cDNA libraries
– Northern blots
– microarrays
cDNA
• Complimentary DNA
• Reverse transcriptase
– Howard Temin and
David Baltimore
– Nobel Prize in
Physiology and
Medicine (1975)
DNA in
nucleus
mRNAs in
cytoplasm
Reverse
mRNA transcriptase
Poly-A tail
DNA Primer
strand
Degraded
mRNA
• Typical PCR follows
DNA
polymerase
cDNA
Microarrays
• Genome-wide expression study
• Glass slide with thousands of
single-stranded DNA fragments
• a.k.a.- gene chip/ DNA chip
• Thousands of genes on one chip
• Libraries for entire genome of an
organism
• Isolate mRNA
• Make and label cDNA
• Allow hybridization
• Which genes are transcribed?
Large groups? When? Which
tissues?
Using Microarrays
TECHNIQUE
1 Isolate mRNA.
2 Make cDNA by reverse
transcription, using
fluorescently labeled
nucleotides.
3 Apply the cDNA mixture to a
microarray, a different gene in
each spot. The cDNA hybridizes
with any complementary DNA on
the microarray.
4 Rinse off excess cDNA; scan
microarray for fluorescence.
Each fluorescent spot represents a
gene expressed in the tissue sample.
Tissue sample
mRNA molecules
Labeled cDNA molecules
(single strands) DNA fragments
representing
specific genes
DNA microarray
DNA microarray
with 2,400
human genes
Combining techniques for gene expression
• Isolate RNA from
different tissues
• RNA from different
stages of development
• Personalized medicine
TECHNIQUE
1 cDNA synthesis
2 PCR amplification
mRNAs
cDNAs
Primers
-globin
gene
3 Gel electrophoresis
RESULTS
Embryonic stages
1 2 3 4 5
6
Determined vs. Differentiated
• All nucleated cells have the same
genomic DNA
• Totipotent- new organism PLUS
extraembryonic membranes
• Pluripotent-new organism
• Mulitpotent- hemocytoblastmany lineages
• unipotent stem cells –
spermatogonia/oogonia
• Can we use a differentiated
cell to create a new
organism?.....depends
•
The cell that is capable of developing into any cell
type, including extraembryonic tissue (e.g., a
zygote)
•
not fixed as to developmental potentialities ;
especially : capable of differentiating into one of
many cell types; not capable of differentiating into
extra-embryonic tissue (e.g., blastomere)
•
A cell that possesses the ability to differentiate into
various but limited number of cell types, especially
into cells of a closely related family of cells. (e.g.,
hemocytoblast)
•
The cell that has the ability to self-renew but gives
rise to only one type of cell or tissue. (e.g.,
gametes).
Plants and some animals have regeneration
EXPERIMENT
RESULTS
Transverse
section of
carrot root
2-mg
fragments
Fragments were Single
Embryonic
Plantlet was
cultured in nu- cells
plant developed cultured on
trient medium; free in
from a cultured agar medium.
stirring caused suspension single cell.
Later it was
single cells to began to
planted
shear off into
divide.
in soil.
the liquid.
A single
somatic
carrot cell
developed
into a mature
carrot plant.
Fig. 20-17
EXPERIMENT
Frog egg cell Frog tadpole
Frog embryo
UV
Less differentiated cell
Fully differentiated
(intestinal) cell
Donor
nucleus
transplanted
Donor
nucleus
transplanted
Enucleated
egg cell
Egg with donor nucleus
activated to begin
development
RESULTS
Most develop
into tadpoles
Most stop developing
before tadpole stage
Cell signaling systems and
successful cloning with
animals
• Cytoplasmic signals can regulate
gene expression
• Dolly- from sheep udder cells
• 1997
• Dolly looked like her “nuclear
mother” not egg donor
• Reproductive cloning to generate
animals with desirable traits- but
doesn’t ensure it.
• Environmental influences and
other phenomena
TECHNIQUE
Mammary
cell donor
Egg cell
donor
2
1
Egg cell
from ovary
3 Cells fused
Cultured
mammary cells 3
4 Grown in
Nucleus
removed
Nucleus from
mammary cell
culture
Early embryo
5 Implanted
in uterus
of a third
sheep
Surrogate
mother
6 Embryonic
development
RESULTS
Lamb (“Dolly”)
genetically identical to
mammary cell donor
CC- Carbon Copy the Cat and her mother.
Genetically ModifiedCloned
gene
Organisms
• Transgenic
• Plasmid or
virus for
transferring
new gene
into genome
• Where does
it insert?
1
Insert RNA version of normal allele
into retrovirus.
Viral RNA
2
Retrovirus
capsid
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
3
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
4
Inject engineered
cells into patient.
Bone
marrow
Fig. 20-25
TECHNIQUE
Agrobacterium tumefaciens
Ti
plasmid
Site where
restriction
enzyme cuts
T DNA
DNA with
the gene
of interest
RESULTS
Recombinant
Ti plasmid
Plant with new trait
Southern Blot:
DNA hybridization probes… detect specific
DNA sequences
Northern Blot:
gene expression… detect specific RNA
sequences (mRNA’s)
Western Blot:
Protein immunoblot… detect presence of
specific proteins