Download Chapter 17. Application of Recombinant DNA Technology in

Chapter 17.
Application of Recombinant
DNA Technology in Genetics
The DNA Toolbox
• In recombinant DNA, nucleotide sequences from two
different sources, often two species, are combined in vitro into
the same DNA molecule.
• Methods for making recombinant DNA are central to genetic
engineering, the direct manipulation of genes for practical
purposes.
• DNA technology has revolutionized biotechnology, the
manipulation of organisms or their genetic components to
make useful products.
• To work directly with specific genes, scientists prepare genesized pieces of DNA in identical copies, a process called DNA
cloning.
DNA Cloning and Its Applications
• Most methods for cloning pieces of DNA in the laboratory
share general features, such as the use of bacteria and their
plasmids.
• Plasmids are small circular DNA molecules that replicate
separately from the bacterial chromosome.
• Cloned genes are useful for making copies of a particular
gene and producing a protein product.
• Gene cloning involves using bacteria to make multiple
copies of a gene.
• Foreign DNA is inserted into a plasmid, and the
recombinant plasmid is inserted into a bacterial cell.
• Reproduction in the bacterial cell results in cloning of the
plasmid including the foreign DNA.
• This results in the production of multiple copies of a
single gene.
DNA Cloning
Bacterium
1 Gene inserted into
Cell containing gene
of interest
plasmid
Bacterial
Plasmid
chromosome
Recombinant
DNA (plasmid)
Gene of
interest
2
2 Plasmid put into
bacterial cell
Recombinant
bacterium
DNA of
chromosome
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
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Protein harvested
4 Basic research and
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
Using Restriction Enzymes to Make
Recombinant DNA
• Bacterial restriction enzymes cut DNA molecules at specific
DNA sequences called restriction sites.
• A restriction enzyme usually makes many cuts, yielding
restriction fragments.
• The most useful restriction enzymes cut DNA in a staggered way,
producing fragments with “sticky ends” that bond with
complementary sticky ends of other fragments.
• DNA ligase is an enzyme that seals the bonds between
restriction fragments.
Restriction Enzymes
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Action of Restriction Enzymes
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Use of Restriction Enzymes
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Cloning a Eukaryotic Gene in a Bacterial
Plasmid
• In gene cloning, the original plasmid is called a cloning
vector.
• A cloning vector is a DNA molecule that can carry
foreign DNA into a host cell and replicate there.
Plasmid Vector
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Construction of Recombinant DNA
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Transformation
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Hummingbird
cell
TECHNIQUE
Bacterial cell
lacZ gene
ampR gene Bacterial
Restriction
site
Sticky
ends
Gene of interest
Hummingbird
DNA fragments
plasmid
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
Selection of Recombinant DNA
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Storing Cloned Genes in DNA Libraries
• A genomic library that is made using bacteria is the
collection of recombinant vector clones produced by
cloning DNA fragments from an entire genome.
• A genomic library that is made using bacteriophages is
stored as a collection of phage clones.
Foreign genome
cut up with
restriction
enzyme
or
Recombinant
phage DNA
Bacterial
clones
(a) Plasmid library
Recombinant
plasmids
Phage
clones
(b) Phage library
BAC
• A bacterial artificial chromosome (BAC) is a large plasmid
that has been trimmed down and can carry a large DNA
insert.
• BACs are another type of vector used in DNA library
construction.
Large plasmid
Large insert
with many genes
BAC
clone
(c) A library of bacterial artificial
chromosome (BAC) clones
YAC
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How big does a genomic library have to
be to have a 95% or 99% chance of
containing all the sequences in a
genome?
• The number of clones required to contain a genome
depends on several factors: the average size of the cloned
inserts, the size of the genome to be cloned, and the level
of probability desired.
• The number of clones in a library can be calculated as
N = ln(1-P)/ln(1-f)
N: the number of required clones
P: the probability of recovering a given sequence
f: the fraction of the genome in each clone
Human Genomic Library
• We wish to prepare a human genomic library large
enough to have 99% chance of containing all the
sequences in the genome.
• If we construct the library using a plasmid vector with
an average insert size of 5kb, more than 2.4 million
clones would be required for a 99% probability of
recovering any given sequence from the genome.
• If the library is constructed in a YAC vector with an
average insert size of 1Mb, then the library would only
need to contain about 14,000 YACs.
Question
• An ampicillin-resistant, tetracycline-resistant plasmid,
pBR322, is cleaved with PstI, which cleaves within the
ampicillin-resistant gene. The cut plasmid is ligated with
PstI-digested Drosophila DNA to prepare a genomic library,
and the mixture is used to transform E. coli K12.
1. Which antibiotics should be added to the medium to
select cells that have incorporated a plasmid?
2. What growth pattern should be selected to obtain
plasmids containing Drosophila inserts?
3. How can you explain the presence of colonies that are
resistant to both antibiotics?
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cDNA library
• A complementary DNA (cDNA) library is made by
cloning DNA made in vitro by reverse transcription of all
the mRNA produced by a particular cell.
• A cDNA library represents only part of the genome —
only the subset of genes transcribed into mRNA in the
original cells.
DNA in
nucleus
mRNAs in
cytoplasm
mRNA
Reverse
transcriptase
Poly-A tail
DNA Primer
strand
Degraded
mRNA
DNA
polymerase
cDNA
cDNA Synthesis
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Screening a Library for Clones Carrying a
Gene of Interest
• A clone carrying the gene of interest can be identified
with a nucleic acid probe having a sequence
complementary to the gene.
• This process is called nucleic acid hybridization.
• A probe can be synthesized that is complementary to
the gene of interest.
• For example, if the desired gene is
5¢
… G G C T A A C T T AG C …
– Then we would synthesize this probe
3¢ C C G A T T G A A T C G 5¢
3¢
Radioactive
DNA probe
Single-stranded
DNA
Mix with singlestranded DNA from
genomic library
Base pairing
indicates the
gene of interest
· The DNA probe can be used to screen a large number of
clones simultaneously for the gene of interest.
· Once identified, the clone carrying the gene of interest
can be cultured.
Hybridization is Used to Identify Similar
DNA Sequences
• Prepare library
– Distribute libraries clones on
petri dish
– Transfer clones to nitrocellulose
disk
• Prepare probe
– Previous cloned DNA
– PCR fragment
– Oligonucleotide
• Screen library
– Expose probe to clones on
nitrocellulose
– Determine location of matching
clone by autoradiography or
fluorescence
Library Screening
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TECHNIQUE
Radioactively
labeled probe
molecules
Multiwell plates
holding library
clones
Probe
DNA
Gene of
interest
Single-stranded
DNA from cell
Film
•
Nylon membrane
Nylon
Location of
membrane
DNA with the
complementary
sequence
Amplifying DNA in Vitro: The Polymerase
Chain Reaction (PCR)
• The polymerase chain reaction, PCR, can produce many
copies of a specific target segment of DNA.
• Kary Mullis (Chemistry 1993: invention of the polymerase
chain reaction)
• A three-step cycle — heating, cooling, and replication —
brings about a chain reaction that produces an
exponentially growing population of identical DNA
molecules.
Amplifying DNA in Vitro: The
Polymerase Chain Reaction (PCR)
• The polymerase chain reaction, PCR, can produce many copies of a
specific target segment of DNA.
• A three-step cycle — heating (denaturation), cooling (annealing),
and replication (extension)—brings about a chain reaction that
produces an exponentially growing population of identical DNA
molecules.
Kary Mullis (1944-present): 1993 Nobel Prize in Chemistry
TECHNIQUE
Genomic DNA
1
2
5¢
3¢
3¢
Target
sequence
5¢
Denaturation 5¢
3¢
3¢
5¢
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
PCR Process
5¢
TECHNIQUE
3¢
Target
sequence
Genomic DNA
3¢
5¢
1 Denaturation
5¢
3¢
3¢
5¢
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
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Cycle 1
yields 2 molecules
Genomic
DNA
3¢
5¢
1 Heat to
3¢
5¢
5¢
3¢
separate
DNA strands
3¢
5¢
5¢
2 Cool to allow
primers to form
hydrogen bonds
with ends of
target sequences
Target
sequence
3¢
5¢
3 DNA
polymerase adds
nucleotides
to the 3¢ end
of each primer
5¢
5¢
3¢
5¢
3¢
Primer
5¢
New DNA
3¢
Cycle 2
yields 4 molecules
Cycle 3
yields 8 molecules
Gel Electrophoresis
• One indirect method of rapidly analyzing and comparing
genomes is gel electrophoresis.
• This technique uses a gel as a molecular sieve to
separate nucleic acids or proteins by size.
• A current is applied that causes charged molecules to
move through the gel.
• Molecules are sorted into “bands” by their size.
TECHNIQUE
Power
source
Mixture of DNA molecules
of different sizes
Anode +
– Cathode
Gel
1
Power
source
–
+
Longer
molecules
2
Shorter
molecules
Mixture of DNA
fragments of
different sizes
Power
source
Longer
(slower)
molecules
Gel
Shorter
(faster)
molecules
Completed gel
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Gel Electrophoresis and Hybridization to
Map DNA Fragments
• Southern blot
– Cut whole genomic DNA with restriction enzyme
– Separate DNA fragments by electrophoresis
– Blot fragmented DNA to a filter
– Hybridize to DNA probe
– Observe matched bands by autoradiography or
fluorescence
Southern Blotting
• A technique called Southern blotting combines gel
electrophoresis of DNA fragments with nucleic acid
hybridization.
• Specific DNA fragments can be identified by Southern
blotting, using labeled probes that hybridize to the DNA
immobilized on a “blot” of gel .
Stain with
Ethidium bromide
Fig.
9.15
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Radioactively labeled
probe for b-globin gene
I
II
III
Probe base-pairs
with fragments
Fragment from
sickle-cell
b-globin allele
Nitrocellulose blot
Fragment from
normal b-globin
allele
4 Hybridization with radioactive probe
I
II
III
Film
over
blot
5 Probe detection
Blot is
removed
washed, and
exposed to
X-ray film
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Use of RFLPs for Mapping
– Single nucleotide polymorphism (SNP) is a
variation at one base pair within a coding or
noncoding sequence
– Restriction fragment length polymorphism
(RFLP) is a variation in the size of DNA
fragments due to a SNP that alters a restriction
site
• In restriction fragment analysis, DNA fragments
produced by restriction enzyme digestion of a DNA
molecule are sorted by gel electrophoresis.
• Restriction fragment analysis is useful for comparing
two different DNA molecules, such as two alleles for a
gene.
• The procedure is also used to prepare pure samples of
individual fragments.
Normal b-globin allele
175 bp
DdeI
201 bp
DdeI
Normal Sickle-cell
allele
allele
Large fragment
DdeI
DdeI
Large
fragment
Sickle-cell mutant b-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 b-globin gene
(b) Electrophoresis of restriction fragments
from normal and sickle-cell alleles
Restriction
enzymes added
DNA sample 1
DNA sample 2
w
Cut
z
x
Cut
Cut
y
y
Longer
fragments
z
x
Shorter
fragments
w
y
y
STR site 1
STR site 2
Crime scene DNA
Number of short tandem
repeats match
Suspect’s DNA
Number of short tandem
repeats do not match
Crime scene
DNA
Suspect’s
DNA
(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.
Question
DNA samples were extracted from chimpanzee hair and used
as templates for PCR. The primers used in our study flank
highly polymorphic sites in human DNA that result from
variable numbers of tandem nucleotide repeats. Several
offspring and their putative parents were tested to determine
whether the putative parents are real parents of the offspring.
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Question
• The following partial restriction map shows a
recombinant plasmid, pBIO220, formed by cloning a
piece of Drosophila DNA (striped segment), including
the rosy gene, into the vector pBR322, which also
contains the penicillin-resistant gene. The vector part
of the plasmid contains only the two E recognition
sequences shown and no A or B sequences. The gel
shows several restriction digests of pBIO220.
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1. Use the stained gel pattern to deduce where
restriction-enzyme recognition sequences are located
in the cloned fragment.
2. A PCR-amplified copy of the entire 2000-bp rosy
gene was used to probe a Southern blot of the same
gel. Use the Southern blot results to deduce the
location of rosy in the cloned fragment. Redraw the
map showing the location of the rosy gene.
Question
• The gel shows the pattern of bands of fragments produced
with several restriction enzymes. The enzymes used are
identified above and below the gel.
• One of the six restriction maps shown below is consistent
with the pattern of bands shown in the gel.
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1. From your analysis of the pattern of bands on the gel,
select the correct map.
2. In a Southern blot prepared from this gel, the
highlighted bands (pink) hybridized with the pep
gene. Where is the pep gene located?
DNA Sequencing
• Relatively short DNA fragments can be sequenced by the
dideoxy chain termination method.
• Modified nucleotides called dideoxyribonucleotides
(ddNTP) attach to synthesized DNA strands of different
lengths.
• Each type of ddNTP is tagged with a distinct fluorescent
label that identifies the nucleotide at the end of each
DNA fragment.
• The DNA sequence can be read from the resulting
spectrogram.
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General Principals of Sanger Sequencing
Method
Fig. 9.17
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Automated DNA sequencing
TECHNIQUE
DNA
(template strand)
Primer
DNA
polymerase
Deoxyribonucleotides
Dideoxyribonucleotides
(fluorescently tagged)
dATP
ddATP
dCTP
ddCTP
dTTP
ddTTP
dGTP
ddGTP
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TECHNIQUE
DNA (template
strand)
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
Automated DNA Sequencing
• Output from an
automated DNA
sequencing reaction
– Each lane displays the
sequence obtained
from a separate DNA
sample and primer.
Sequencing Results
• Computer reads of the sequence complementary to the
template strand from right to left (5’ à 3’ direction).
Machine generates complementary strand. Ambiguities are
recorded as an “N” and can sometimes be resolved by a
technician.
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Positional Cloning: Use of Polymorphic DNA
Markers to Clone Genes via Linkage
A pedigree of the royal family descended from Queen Victoria
In which hemophilia A is segregating
Positional Cloning: Step 1
• Find extended families in which disease is segregating.
• Use panel of polymorphic markers spaced at 10 cM
intervals across all chromosomes.
– 300 markers total
• Determine genotype for all individuals in families for
each DNA marker.
• Look for linkage between a marker and disease
phenotype.
• Once region of
chromosome is
identified, a high
resolution mapping is
performed with
additional markers to
narrow down region
where gene may lie.
Positional cloning: Step 2
(Identifying Candidate Genes)
• Once region of chromosome has been narrowed down
by linkage analysis to 1000 kb or less, all genes within
are identified.
• Candidate genes
– Usually about 17 genes per 1000 kb fragment
– Identify coding regions
• Computational analysis to identify conserved
sequences between species
• Computational analysis to identify exon-like
sequences by looking for codon usage, ORFs, and
splice sites
• Appearance on one or more EST clones derived from
cDNA
Computational Analysis of Genomic
Sequences to Identify Candidate Genes
Gene Expression Patterns Can Pinpoint
Candidate Genes
• Look in public database of EST sequences representing
certain tissues.
• Northern blot
– RNA transcripts in the cells of a particular tissue
(e.g., with disease) separated by electrophoresis and
probed with candidate gene sequence
Northern Blot Example of SRY
Positional cloning: Step 3
• Find the gene responsible for the phenotype.
– Expression patterns
• RNA expression assayed by Northern blot or PCR
amplification of cDNA with primers specific to
candidate transcript
• Look for misexpression (no expression,
underexpression, overexpression)
– Sequence differences
• Missense mutations identified by sequencing
coding region of candidate gene from normal
and abnormal individuals
– Transgenic modification of phenotype
• Insert the mutant gene into a model organism.
Transgenic Analysis Can Prove Candidate
Gene
Example: Positional Cloning of Cystic
Fibrosis Gene
• Linkage analysis places CF on chromosome 7
Northern blot analysis reveals only one of candidate
genes is expressed in lungs and pancreas.
-Every CF patient has a mutated allele of the CFTR gene
on both chromosome 7 homologs.
-Location and number of mutations indicated under
diagram of chromosome
-CFTR is a membrane protein.
-TMD-1 and TMD-2 are transmembrane domains.
Proving CFTR is the Right Gene
• Phenotype eliminates gene function.
– Cannot use transgenic technology.
• Instead perform CFTR gene “knockout” in mouse to
examine phenotype without CFTR gene.
– Targeted mutagenesis
• Introduce mutant CFTR into mouse embryonic
cells in culture.
• Rare double recombinant events with
homologous wild-type CFTR gene are selected
for.
• Mutant cell is introduced into normal mouse
embryos where they incorporate into germ line.
• Knockout mouse created.