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UNIT 5
Recombinant DNA
Technology
Objectives
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Define Recombinant DNA technology
Outline steps involved in creating recombinant DNA
Define type II restriction enzymes and their use in recombinant DNA
technology
Explain restriction mapping and construct restriction maps
State the properties of cloning vectors and reproduce genetic map of
common cloning vectors
Outline the steps involved in cloning prokaryotic DNA into a plasmid
cloning vector
Outline the steps involved in cloning eukaryotic DNA into a plasmid
cloning vector
Understand the methods of transformation and the steps involved in
the selection of transformants
Explain the concept of gene libraries and methods of screening a gene
library
Recombinant DNA technology
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a body of techniques for cutting and splicing
together different pieces of DNA.
When segments of foreign DNA are transferred
into another cell or organism, the protein for
which they code may be produced along with
substances coded for by the native genetic
material of the cell or organism.
These cells become "factories" for the
production of the protein coded for by the
inserted DNA
Restriction Enzymes
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The major tools of recombinant DNA technology are
restriction enzymes (nucleases)
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first discovered in the late 1960s
They work by cutting up the foreign DNA, a process called
restriction.
Most restriction enzymes are very specific
recognizing short, specific nucleotide sequences in DNA
molecules and cutting at specific points within these
sequences.
There are hundreds of restriction enzymes and more than
150 different recognition sequences.
Restriction enzymes
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Nucleases are further described by addition of
the prefix "endo" or "exo" to the name:
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endonuclease applies to sequence specific nucleases
that break nucleic acid chains somewhere in the
interior, rather than at the ends, of the molecule.
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exonucleases function by removing nucleotides from
the ends of the molecule
Types of Restriction Enzymes
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Type I Recognise specific sequences·but then track along DNA
(~1000-5000 bases) before cutting one of the strands and
releasing a number of nucleotides (~75) where the cut is made.
A second molecule of the endonuclease is required to cut the
2nd strand of the DNA
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Type II Recognise a specific target sequence in DNA, and then
break the DNA (both strands), within the recognition site
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Type III Intermediate properties between type I and type II.
Break both DNA strands at a defined distance from a
recognition site
Restriction Endonucleases
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The first endonucleases discovered was from Escherichia coli
EcoRI
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A restriction endonuclease functions by "scanning" the length of
a DNA molecule.
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Once it encounters its particular specific sequence (recognition
site), it will bond to the DNA molecule and makes one cut in
each of the two sugar-phosphate backbones of the double helix.
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Once the cuts have been made, the DNA molecule will break
into fragments.
Restriction Endonucleases
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The positions of these two cuts, both in relation to each other,
and to the recognition sequence itself, are determined by the
identity of the restriction endonuclease
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The length of the recognition site for different enzymes can be
four, five, six, eight or more nucleotide pairs long.
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Restriction endonucleases that cleave recognition sites of four
and six nucleotide pairs are used for most of the protocols for
molecular cloning since these restriction sites are common in
DNA.
Restriction Endonucleases
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Different endonucleases yield different sets of cuts, but
one endonuclease will always cut a particular base
sequence the same way.
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Some restriction endonucleases digest DNA leaving 5′phosphate extensions(protruding ends, sticky ends)
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Some leave 3′-hydroxyl extensions
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Some cut the backbone of both strands within a recognition
site to produce blunt-ended (flush-ended) DNA molecules.
Use of Restriction Enzymes in
recombinant DNA Technology
How Restriction Enzymes are
named
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generally have names that reflect their origin—
The first letter of the name (capitalized ) comes from
the genus of the bacteria
 the second two letters (lower case) come from the
species
 Roman numerals following the nuclease names
indicate the order in which the enzymes were
isolated from that strain of bacteria.
 For example EcoRI comes from Escherichia coli
RY13
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Type II Restriction Enzymes
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Many Type II restriction endonucleases recognise
PALINDROMIC sequences
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Symmetrical sequences which read in the same order of
nucleotide bases on each strand of DNA (always read 5' to 3')
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For example, EcoRI recognises the sequence
5'-G A A T T C-3'
3'-C T T A A G-5 '
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The high specificity for their recognition site means that
DNA (target sequence and cloning vector) will always
be cut reproducibly into defined fragments (important
for molecular cloning)
Enzyme
Organism from which derived
Target sequence (cut at *) 5' -3'
Bam HI
Bacillus amyloliquefaciens
G* G A T C C
Bgl II
Bacillus globigii
A* G A T C T
Eco RI
Escherichia coli RY 13
G* A A T T C
Eco RII
Escherichia coli R245
* C C A/T G G
Hae III
Haemophilus aegyptius
GG*CC
Hind III
Haemophilus inflenzae Rd
A* A G C T T
Hpa I
Haemophilus parainflenzae
G T T * AA C
Kpn I
Klebsiella pneumoniae
G GTAC * C
Pst I
Providencia stuartii
CTGCA*G
Sma I
Serratia marcescens
CCC*GGG
SstI
Streptomyces stanford
GAGCT*C
Sal I
Streptomyces albus G
G*TCGAC
Taq I
Thermophilus aquaticus
T*CGA
Xma I
Xanthamonas malvacearum
C*CCGGG
Note: Only one strand of the target DNA is shown
Frequency of cutting
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Because of their restriction site specificity, the
restriction endonucleases cut DNA into
fragments whose average length is determined
by
the number of base pairs in the restriction site
 and to a lesser extent by the ratio of bases in the
DNA.
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For DNA that has equal amounts of all four
bases, each base has a probabilty of 1/4 at any
particular position in the DNA sense strand.
Frequency of cutting
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Because of their restriction site specificity, the
restriction endonucleases cut DNA into
fragments whose average length is determined
by the number of base pairs in the restriction
site (and to a lesser extent by the ratio of bases
in the DNA).
For DNA that has equal amounts of all four
bases, each base has a probability of 1/4 at any
particular position in the DNA strand.
Frequency of cutting
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For a restriction site of 4 base pairs, the
probability of random occurrence of that
sequence is (1/4)(1/4)(1/4)(1/4) = 1/256.
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For 6 base pairs, the probability is 1/4,096, and
for 8 base pairs it is 1/65,536.
Frequency of cutting
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Thus, a restriction endonuclease with a 6 base
pair restriction site would generate fragments
whose average length is 4,096 base pairs.
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Such fragments are large enough to contain a
complete gene (provided that the are no cut sites
within the gene for the restriction endonuclease
that is used).
Frequency of cutting
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Effect of base composition:
For DNA whose base composition differs from
50% GT it is necessary to calculate the probability of
a site as the product of the probabilities of each of
its components.
 For example, if a DNA is 66.7% GC (2/3 of its base
pairs are GC) and one assumes random orientation
of the base pairs, A and T will each have
probabilities of 1/6 and G and C will have
probabilities of 1/3 each.
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Frequency of cutting
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Effect of base composition cont’d. :
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Thus the probability of GAATTC would be:
(1/3)(1/6)(1/6)(1/6)(1/6)1/3) = 1/11,664
as opposed to 1/4096 when all four bases are
present in equal amounts.
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Thus, the average fragment length generated by Eco
RI would be longer in a DNA with a higher GC
content.
Steps in creating recombinant
DNA
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The DNA (insert, cloned DNA, target DNA,
foreign DNA) from a donor organism is
extracted
Then DNA is enzymatically cleaved (cut,
digested) and joined (ligated) to another DNA
entity (cloning vector) to form a new
recombined DNA molecule (cloning vectorinsert DNA construct, DNA construct).
Steps in creating recombinant
DNA
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The cloning vector-insert DNA construct is
transferred into and maintained within a host
cell, a process called transformation.
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The host cell that takes up the DNA construct
(transformed cells) are identified (by screening)
and selected (separated, isolated) from those
that do not.
Transformation
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Many cells lack the ability to take up DNA from their
surroundings and are hence not competent
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The cell membranes of such cells have to be made
porous to make them competent.
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Competence can be achieved by treatment of cells with
low temperature and calcium chloride.
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Heat shock treatment during transformation closes these
pores.
Transformation
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Transformation is an inefficient process (1 cell in 1000).
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Furthermore a few cells will be transformed by
recircularized plasmid DNA
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others by nonplasmid DNA
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while a few by plasmid-insert DNA construct.
Desirable features of cells used in
Transformation exercises
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To ensure that the plasmid-insert DNA
construct is perpetuated in its original form the
E. coli cells used:
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must lack REs
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must also be incapable of homologous
recombination.
Screening of Transformants
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After transformation, it is necessary to
identify the cells that contain the plasmidcloned DNA constructs
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This procedure is called screening
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The screening method used will depend on
the plasmid system used in the
transformation process.
Screening when a pBR322 system is
used
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In a pBR322 system in which the target sequence was
inserted into the BamH1 site, the identification is
accomplished in two steps:
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First the cells from the transformation mixture are
plated onto medium that contains ampicillin.
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Only those cells that contain either the pBR322 or
pBR322-cloned DNA construct (both of which have an
intact ampicillin resistance gene) can grow under these
conditions.
pBR322
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The nontransformed cells are sensitive to
ampicillin.
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The BamH1 gene of pBR322 is within the
tetracycline resistance gene, so insertion of
DNA into this gene disrupts the coding
sequence and tetracycline resistance is lost.
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Cells transformed with the pBR322-cloned
DNA construct are resistant to ampicillin but
sensitive to tetracycline.
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Cells transformed with the recircularized plasmid
are resistant to both ampicillin and tetracycline
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Hence cells that grow on ampicillin containing
medium are transferred to a tetracycline
containing medium.
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Each location of inoculating cells on a
tetracycline-agar plate corresponds to the site of a
colony on an original ampicillin-agar plate.
Screening when a pUC 19 vector
system is used in transformation
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Belongs to the pUC series of plasmids.
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They are all based on pBR322 from which 40% of the
DNA has been deleted, including the tetracycline gene.
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All their cloning sites are also clustered into one site
called a multiple cloning site (MCS).
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pUC vector contain the pBR322 ampicillin resisitance
gene to allow selection for bacteria that have received a
copy of the vector.
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The MCS lies within the DNA sequence coding
for the amino terminal portion of the enzyme βgalactosidase.
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The bacteria used with the pUC vectors carry a
gene fragment that encodes the carboxyl portion
of β-galactosidase.
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The two polypeptides by themselves have no
activity.
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When clones containing the plasmid are plated
on medium containing the galactoside,
X- gal, colonies with non-recombinant pUC
plasmid will turn blue.
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β-galactosidase cleaves the X-gal.
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This cleavage releases galactose plus an indigo dye
that stains the bacterial colony blue.
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IPTG is also added to the growth medium to
induce the expression of the lacZ gene
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Recombinant transformants will produce white
colonies on this medium since the galactosidase
gene is disrupted by the insert.
Defining a Gene Library
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A gene library is a collection of host cells
that contain all of the genomic DNA of the
source organism.
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A gene library is also called a clone bank, or a
gene bank.
Creating a gene Library
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The source organism’s genomic DNA is
digested (cut) into clonable elements and
inserted into host cells.
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Conditions of the digestion reaction are set to
give a partial, not a complete, digestion
Screening a Gene Library
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After a library is created, the clone (s) (cell lines)
with the target sequence must be identified.
Three popular methods of identification are used:
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Colony hybridization with a labeled DNA probe
followed by screening for the probe label
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immunological screening for the protein product
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screening for protein activity.
Screening by Colony Hybridization
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Genomic DNA libraries are often screened by
plating out cells from each cell line on to a
master plate
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Samples of each colony are then transferred to
a solid matrix such as a nitrocellulose.
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The colonies from the master plate that
correspond to samples containing hybridized
DNA are then isolated and cultured.
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The cells on the membrane are lysed and the target
DNA is denatured by alkali treatment
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The single strands of DNA are then irreversibly bound
to the matrix , this process is often carried out at a
high temperature.
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Then, the DNA probe, which is labeled with either a
radiosotope or another tagging system, is incubated
with the bound DNA sample.
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If the sequence of nucleotides in the DNA probe is
complementary to a nucleotide sequence in the
genome of the cell, then base pairing (i.e.,
hybridization) occurs
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The hybridization can be detected by
autoradiography or other visualization
procedures, depending on the nature of the
probe label.
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If the nucleotide sequence of the probe does not
base pair (bind) with a DNA sequence in the
sample, then no hybridization occurs and the
assay gives a negative result.
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General probes range in length from 100 to more
than 1,000 bp, although both larger and smaller
probes can be used.
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Stable binding requires a greater than 80% match
within a segment of 50 bases.
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There are at least two possible sources of probes for
screening a genomic library:
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First, cloned DNA from a closely related organism can be
used (a heterologous probe).
Second, a probe can be produced by chemical synthesis.
Probe detection
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Isotopic detection of hyridization is done when the
probe is labelled with radioactive isotope.
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The nitrocellulose membrane with bound DNA
hybridized to probe is overlaid with X-Ray film
(autoradiography). Darkened areas on the film indicate
positions of the DNA probe hybrid.
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For nonisotopic detection of hybridization, biotin (or
similar nonradioactive label) can be attached to one of
the four deoxyribonucleotides that is incorporated into
the probe.
Isotopic detection
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When a probe with this kind of tag (label) hybridizes to
the sample DNA, detection is based on the binding of
an intermediary compound (e.g., streptavidin) that
carries an appropriate enzyme.
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Depending on the assay system, the enzyme can be
used for the formation of either a chromogenic
(colored) molecule that can be visualized directly or a
chemiluminescent response that can be detected by
autoradiography
Nonisotopic detection
Analysing hybridization results
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Because most libraries are created from partial
digestions, a number of colonies (clones) may give a
positive response to the probe.
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The next task is to determine which clone, if any,
contains the complete sequence of the target gene.
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Preliminary analyses that use the results of gel
electrophoresis and restriction endonuclease
mapping reveal the length of each insert and identify
those inserts that are the same and those that share
overlapping sequences.
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If an insert in any one of the clones is large
enough to include the full gene, then the
complete gene can be recognized after DNA
sequencing
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(because it will have start and stop codons and a
contiguous set of nucleotides that code for the
target protein).
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If the search for an intact gene fails, then
another library can be created with a different
restriction endonuclease and screened with
either the original probe or probes derived from
the first library.
Screening by Immunological Assay
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Used if a DNA probe is not available
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If a cloned DNA sequence is transcribed and translated,
the presence of the protein, or even part of it, can be
determined by an immunological assay.
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First use a primary antibody that is raised against the
protein. Then wash with a secondary antibody.
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In many assay systems, the secondary antibody has an
enzyme, such as alkaline phosphatase, attached to it.
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After washing the matrix, a colorless substrate is
added.
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If the secondary antibody has bound to the
primary antibody, the colorless substrate is
hydrolyzed by the attached enzyme and
produces a colored compound that accumulates
at the site of the reaction.
Cloning DNA Sequences That Encode Eukaryotic
Protein in prokaryotic cells
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Prokaryotic organisms do not have the ability to
express eukaryotic genes since they do not have
the machinery to remove introns.
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If Eukaryotic genes are to be expressed in
prokaryotic hosts their introns must first be
removed.
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To achieve this cDNA is made and used for the
transformation of prokaryotes
cDNA Synthesis
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cDNA synthesis is preceded by extraction of
mRNA from the eukaryotic cells
The poly (A) tail of the mRNA can be used to
separate the mRNA fraction of a tissue from the
ribosomal and transfer RNAs.
mRNA extraction
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Extracted cellular eukaryotic RNA is passed
through a column packed with cellulose beads to
which is bound short chains of thymidine
residues, each about 15 nucleotides long (oligodT15).
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The poly (A) tails of the messenger RNA
molecules bind by base pairing to the oligo-dT
chains.
cDNA Synthesis
The process is divided into two steps:
1. First Strand Synthesis
 After the mRNA fraction is purified the following are
added:
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i. short (unbound) sequences of oligo-dT molecules
ii. the enzyme reverse transcriptase
iii. the four deoxyribonucleotides (dATP, dTTP, dGTP,
dCTP).
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The oligo-dT molecules base pair with the poly (A) tail
regions and provide an available 3’ hydroxyl group to
prime the synthesis of a DNA strand.
2. Second Strand Synthesis
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The second DNA strand is synthesized by the
addition of the Klenow fragment of E. coli DNA
polymerase I:
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The polymerase uses the first DNA strand as a
template and adds deoxyribonucleotides to the
growing strand, starting from the end of the
hairpin loop.
After the reaction is complete, the sample is treated
with:
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the enzyme Rnase H, which degrades the mRNA
molecules and
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S1 nuclease, which opens the hairpin loops and
degrades single-stranded complementary DNA
(cDNA) copies of the more prevalent mRNAs in the
original sample.