Plasmids

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Plasmids
Small circular autonomously replicating
extrachromosomal DNA
Modified plasmids, called cloning vectors Are
used by molecular biologists to isolate Large
quantities of a given DNA fragment Plasmids used
for cloning share three properties Unique
restriction site Antibiotic resistance Origin
of replication
Bacterial genome (5000kb)
Plasmid DNA (3kb)
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Plasmid elements
Origin of replication This is a DNA element that
allows the plasmid to be replicated and
duplicated in bacteria. Each time the bacterium
divides, the plasmid also needs to divide and go
with the daughter cells. If a plasmid cannot
replicate in bacteria, then it will be lost.
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Plasmid elements
Antibiotic resistance This allows for the
presence of the plasmid to be selectively
maintained in a given strain of bacteria
Lab bacterial strains are sensitive to
antibiotics. When grown on plates with
antibiotics, they die. The presence of a plasmid
with the antibiotics resistance gene allows
these lab strains to grow on plates with the
antibiotic. You are therefore selecting for
bacterial colonies with the Plasmid
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Plasmid elements
Unique restriction sites For cloning the plasmid
needs too be linearized. Most cloning vectors
have unique restriction sites. If the plasmid
contains more than one site for a given
restriction enzyme, this results in
fragmentation of the plasmid
Why does this matter?
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pUC18
pUC18 is a commonly used plasmid pUC plasmid
University of California
Plasmid replicon copy No pBR322 pMB1 15 pUC18
pMB1 500 pACYC p15A 10 pSC101 pSC101 5
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Why are plasmids important?
Most genes are present as two copies in the
entire genome. Plasmids allow us to obtain 1000s
of copies of a gene in a pure form
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How is a specific gene isolated (CLONED)?
Its like going to the library and looking for a
specific book. It involves screening through a
genomic library. A genomic library is a large
collection of plasmids containing pieces of DNA
from a specific species. The set of cloned
fragments is so comprehensive that virtually the
entire genome is represented in the
library. The fragments that make up the library
are initially generated by digesting genomic DNA
(e.g. human) with a restriction enzyme- say
EcoRI The EcoRI sites are randomly distributed
in the genome- fragments of varying lengths will
be generated. Some fragments will contain one
gene, others two genes or cut genes in half.
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The library is random!
Each fragment is cloned into the plasmid, each
plasmid is put (transformed) into E.coli
C
D
A
B
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Fragments,bookmark title
The library is not bookmarked or even titled and
is in fragments! There is no organization to
the library. It is simply a populations of
cloned fragments representing the entire
genome. The equivalent of this would be if you
went to the University Library to find all the
books in a large heap, the books had no title,
and in addition instead of entire books you often
found parts of books. How do you use such a
library? How do you find the book you are
interested in.
Lets work our way through this problem with a
simple example Organism has EIGHT genes in its
genome
A
B
C
D
E
F
G
H
EcoRI
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Genomic library
If we wanted to study gene C- Create a
restriction map of gene C Determine it
sequence Study proteinC What do we need to do
We need to initially clone the gene and make many
copies of gene C Creating a genomic library
provides a means of obtaining many copies of gene
C To generate a genomic library Total genomic
DNA is isolated from the species of interest The
DNA is cut with EcoRI
A
B
C
D
E
F
G
H
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Genomic library
These genomic DNA fragments are mixed with a
plasmid that has been linearized at a single
EcoRI site (say pUC18)
Both the plasmid and genomic DNA have been cut
with EcoRI, they have complementary sticky ends
G A A T T C C T T A A G
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Recombinant plasmid
This process where foreign DNA is joined to
plasmid DNA is called ligation It results in
recombinant plasmid (foreign DNAplasmid) Each
plasmid has one foreign EcoRI fragment Each
foreign fragment is still present as only one
copy! This is not useful.
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Transformation
The entire collection of these plasmids bearing
genomic DNA inserts is called a Genomic
Library! These plasmids are added back into
bacteria by a process called transformation
The bacteria are selected for the presence of the
Plasmid by growth on media containing antibiotics
Each colony of E. coli will harbor one plasmid
with one piece of genomic DNA We at this point
dont know which colony has which piece of
foreign DNA
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How are genomic libraries used?
If we are interested in studying gene C, you need
the plasmid containing gene C Having a genomic
library means you have gene C, but where is it?
Which colony on the Petri dish contains gene
C? Genomic libraries are much more complex than
the one described for our hypothetical 8 gene
organism You need to identify one recombinant
plasmid out of 100,000s present in a
library. Identifying and isolating a specific
plasmid is called screening a library. This
requires a probe A probe is a sequence
complementary to part of the sequence one wishes
to pull out You radiolabel the probe and once
labeled the probe is used to identify the plasmid
containing E. coli colony How do we get the
probe?
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PROBES
Probes are obtained in a number of ways
RNA as a source The probe for hemoglobin can be
obtained from mRNA of immature red blood
cells. The major transcript of these cells is
from the hemoglobin gene. So isolating RNA from
these cells, we can obtain a relatively pure
probe for the hemoglobin gene Protein If you
have a purified protein, the amino acid sequence
can be determined. From the amino acid sequence,
using the genetic code a corresponding DNA
sequence can be synthesized and this small DNA
piece can be used as a probe Homology Probes
from conserved genes-Many genes are conserved
from one species to another Chimpanzee and human
DNA are 97 identical. If you know the sequence
of a gene in chimps, then you will be able to
know the sequence for the gene in humans! The
histone genes are highly conserved across phyla.
Histone proteins have three Amino acid
differences between humans and peas Histone genes
have been isolated in yeast, they can serve as
probes for screening a Human genomic library-
cloning by phone The computer databases
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How is the probe used to screen a genomic library?
The E.coli containing the genomic inserts are
plated onto a series of petridishes. The media on
the petridishes contain ampicillin so only E.coli
with the plasmid containing the ampicillin
resistance gene will grow and form colonies
Colonies are imprinted onto A paper filter
Filter
The bacteria on the filter are lysed The DNA in
the bacteria (plasmid and chromosomal) is
denatured (converted from ds to ss) Then you
take the filter and to it add radiolabeled probe
(small part of Gene C). The probe will bind the
single stranded plasmid that has a complementary
sequence. The bacterial colony containing the
plasmid with gene C will bind the probe.
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Autoradiography
Filter autoradiogram
Now you have the complete GeneC!!!
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Isolate the plasmid
To isolate the gene C fragment, we grow up a
large population of E. coli containing the
plasmid with the gene C insert. A simple
procedure allows us to isolate the plasmid (which
is smaller than Chromosomal DNA) Once we have
purified the plasmid we have 1000s of copies of
Gene C in a plasmid We can take the plasmid and
cut it with EcoRI. When the digest is run on an
agarose gel, we get two bands- one corresponding
to the plasmid and one to the insert. The DNA
present in the band corresponding to the insert
can be isolated from the gel PURE GENE C!!!!!
Marker
EcoRI
Uncut
Gene C
plasmid
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The genomic library and a specific probe enabled
us to achieve two goals Out of the billions of
base pairs in a large genome, we have been able
to identify a few 1000 base pairs that correspond
to a specific gene of interest. In addition we
were able to isolate this sequence on a
specifically engineered plasmid That allows us to
make large quantities of this rare
sequence. Genomic libraries are described in
terms of average fragment size and the number of
plasmids that must be screened to have the entire
genome represented To have a good probability
(gt99) of identifying a given DNA sequence (gene)
present in the collection of plasmids (library).
The number of plasmids (colonies) that must be
screened is a function of the size of the genome
of the species from which the Library was
constructed.
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Genomic libraries
Species Genome size average plasmids
insert size screened E. Coli 5000kb 16
kb 1300 Drosophila 150,000kb 16
kb 46,000 Human 3000,000kb 16 kb gt100,000
The larger the genome, the more difficult the
task of isolating a plasmid with a given gene
from a library At present, genomic libraries
exist for a large number of organisms
including Yeast, C.elegans, Drosophila,
Zebrafish, Xenopus, Chickens, Mouse, Humans etc
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cDNA
Often we have RNA rather than DNA as the starting
material For instance in the case of the human
hemoglobin gene, we started with globin mRNA RNA
is difficult to work with. In contrast to DNA,
RNA breaks down and degrades very easily. There
are no restriction enzymes that cut RNA at
specific sites. RNA cannot be cloned. It cannot
be inserted into a plasmid and amplified since
all Plasmids are DNA. The enzyme reverse
transcriptase has proven very useful to molecular
biologists. This enzyme catalyzes the synthesis
of DNA from a RNA template. It is normally found
in a large class of viruses. The genome of these
viruses is RNA!! These viruses are called
retroviruses.They infect eukaryotic cells and use
these cells to grow/replicate Retroviruses carry
an RNA genome. Interestingly they will integrate
into the DNA of the host. For RNA to integrate
into DNA, first the RNA has to be converted to
DNA Remember the central dogma of molecular
biology Information flows from DNA to RNA to
protein! DNA----gtRNA----gtprotein Reverse
Transcriptase reverses this dogma (partially)
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cDNA synthesis
Protein coat
RNA genome
Reverse transcriptase
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cDNA/splicing
So from globin mRNA, a complementary DNA molecule
can be created using reverse Transcriptase. This
complementary DNA is called cDNA. This DNA can
now be inserted into a plasmid and cloned. What
is the relationship between a cDNA clone and a
genomic clone?
Splicing In eukaryotes, the coding sequences are
interrupted by introns
Primary transcript
Splicing
Genomic clones represent the organization of the
DNA in the nucleus!
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Genomic clones represent the organization of the
DNA in the nucleus! cDNA represents the
organization of mRNA sequences after the gene has
been transcribed, processed and exported to the
cytoplasm. cDNA clones contain the sequence of
nucleotides that code for the protein! cDNA
clones do not contain the sequence of the
promoter of the gene or the intron. cDNA
libraries can be made. They are analogous to
genomic libraries discussed earlier The starting
material is different. Organism, tissue or cell
type. First mRNA is isolated, then this is
converted to to dsDNA using reverse
transcriptase These DNA fragments are inserted
into plasmids, transformed into E. coli to
produce a cDNA library.
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Properties of genomic and cDNA libraries
Genomic library cDNA library Source Nucleii
cytoplasmic RNA (any cell) (specific cell
type) Frequency Depends on copy Depends on
abundance of a gene number, gene stability of
RNA in library size, genome size Use Studies
on gene Studies directed organization
towards coding regions structure
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Southern blotting
Rapid method of identifying specific DNA
fragments in a large mixture of fragments
plasmid
EcoRI
Insert GeneC
Marker
EcoRI
Uncut
How do you determine which band corresponds to
insert and which to the plasmid
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Southern blotting allows us to answer this
question
This method is very similar to the method we used
to screen a genomic library
TTTTTTT AAAAAAA
The probe AAAAAAA specifically hybridizes with
the insert (geneC) A probe with this specific
sequence is generated and made radioactive The
plasmid is cut with restriction enzyme and run on
an agarose gel The DNA is transferred to a
membrane Incubate the filter with the
radio-labeled probe
Marker
EcoRI
Uncut
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What about a genome?
What if Gene C was in a large genome. Could we
identify the fragment by this method?
EcoRI
GeneC
Transfer to membrane
Hybridize with Probe
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Mapping deletions.
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You can build a genomic restriction map using
this method.
If we digest the DNA with HindIII instead of
EcoRI what will happen?
E
E
E
E
GeneC
H
H
H
H
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Rapid low resolution analysis of hemoglobin gene
MstII
0.2kb
1.1kb
Exon1
Exon2
0.3kb deletion
Marker
WT
Del
1.1 0.2
1
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Northern blot
This is a rapid method that allows you to
determine the cell type in which a specific gene
is active (MAKING RNA) and being transcribed.
Brain
Bone
Blood
Embryo
Lung
liver
These tissues differ because each is transcribing
a unique Subset of genes. Each tissue contains a
unique and distinct mRNA population
Presence of RNA is a reflection of gene activity
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Microarrays
These are glorified northern blots These are a
recent development Allows us to examine gene
expression of all of the genes in the
genome! They are a reverse northern blot
Each spot is DNA for one defined gene Each gene
DNA is spotted in a grid So extensive that they
cover the entire genome.
Make total RNA from normal and mutant cell Label
each total RNA differently Add labeled RNA from
normal and mutant to array and let
hybridize Measure label and determine change
WT
Mut
1
2
Ratio of WT/mut
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