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CHAPTER 14
Recombinant plasmid
enters E. coli
A
B
C
● Figure 14-5
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Isolating a Human Gene by DNA Cloning.
The term “cloning” is somewhat confusing
because it is used in three ways. First, cloning means making identical copies of a DNA molecule.
Second, cloning means isolating a particular gene from the rest of the DNA. Third, human cloning
means the creation of a genetically identical copy of a human being, human cell, or human tissue.
Although human clones are naturally produced in the form of identical twins, laboratory-produced
human cloning is controversial. Artificial therapeutic cloning involves cloning cells from an adult
for use in medicine. Artificial reproductive cloning involves making cloned humans and is illegal
in many countries.
Although the exact method of cloning differs from gene to gene, we will use the cloning of
human Factor VIII as an example of a general cloning strategy. The first step in cloning human
Factor VIII is the construction of a genomic library. The construction of a genomic library em-
ploys the use of a plasmid vector (or cloning vector). A plasmid vector is a circular DNA mole-
cule that can infect and replicate inside a bacterium. Plasmid DNA can be combined with human
DNA to form a recombinant plasmid. A major disadvantage of plasmid vectors is that they accept
only small DNA fragments. Consequently, other vectors that accept long DNA fragments have been
designed which include cosmid vectors based on the bacteriophage
; BACs (bacterial artificial
chromosomes) based on the F-factor plasmids; PACs (P1 artificial chromosomes) based on the
P1 bacteriophage; and YACs (yeast artificial chromosomes).
Figure 14-5A Construction of a genomic library:
• Human DNA is cut with an RE into DNA fragments. A plasmid DNA vector is also cut with the
same RE and the two types of DNA are mixed together.
• Human DNA fragments will be inserted into the plasmid DNA and sealed with DNA ligase. This
forms a recombinant plasmid, that is, DNA from two different sources has been “recombined.”
• pBR322 is a widely used versatile plasmid that contains two antibiotic resistance genes (ampi-
cillin and tetracycline). The tetracycline gene has a BAMH1 RE site that can be used to insert
human DNA fragments into the pBR322 plasmid. This results in bacteria that will be resistant to
only ampicillin and will not grow in the presence of tetracycline.
• The recombinant plasmids are mixed with Escherichia coli bacteria and plated on Petri plates
forming bacterial colonies which constitute a genomic library. Plasmids enter bacteria by conju-
gation (Gram-), transduction (most Gram
by using bacteriophages), or transformation
(using temperature changes or CaCl
2
).
Figure 14-5B Screening the library:
• Bacteria colonies are transferred to filter paper, lysed, and the DNA is denatured to single strands
under alkaline conditions.
• Because a portion of the amino acid sequence of Factor VIII protein was already known, the DNA
sequence that codes for these amino acids can be deduced. A DNA probe was made based on this
deduced DNA sequence. The radiolabeled DNA probe hybridizes to the DNA strand of comple-
mentary nucleotide sequence, that is, the Factor VIII gene.
• The filter paper is exposed to photographic film to identify the radiolabeled DNA.
Figure 14-5C Amplification:
• The corresponding bacterial colony is plucked from the Petri dish and introduced to a nutrient
broth culture overnight.
• The recombinant plasmid DNA is separated from the bacterial DNA such that you now have mil-
lions of copies of the recombinant plasmid containing the Factor VIII gene.
• Hence, you have cloned the Factor VIII gene.
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MOLECULAR BIOLOGY TECHNIQUES
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CHAPTER 14
mRNA with poly (A) tail
Reverse transcriptase
4dNTP, Magnesium
RE sticky ends
DNA ligase
Recombinant plasmid
enters
E. coli
● Figure 14-6
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MOLECULAR BIOLOGY TECHNIQUES
Construction of a cDNA Library.
A major disadvantage of a genomic library is that most of
the DNA fragments that are recombined with the plasmid vector are repetitive DNA sequences,
introns (noncoding regions of the gene), or spacer DNA, not DNA sequences that code for pro-
teins. This means that the library you have to screen through to find your gene of interest (e.g.,
Factor VIII) is very large and therefore disadvantageous. The question is: Is there another way?
And the answer is: Yes, through the construction of a complementary DNA (cDNA) library. Please
note that the main difference between a genomic library and a cDNA library is that a genomic
library uses genomic (chromosomal) DNA, whereas a cDNA library uses DNA copied from mRNA.
DNA copied from mRNA is called cDNA. The advantage of a cDNA library should already be
apparent. In a cDNA library, you only have to screen through DNA sequences that code for pro-
teins because the DNA used to construct the library is copied from mRNA. Because the liver syn-
thesizes large amounts of Factor VIII, it would be a good candidate organ to use in the construc-
tion of the cDNA library. The enzyme that is critical in the formation of a cDNA library is called
reverse transcriptase. Reverse transcriptase produces DNA from an RNA template.
• All the mRNA from the liver is isolated. mRNA can be isolated by using the fact that eukaryotic
mRNA has a poly A tail at the 3
end. A chromatograph column with oligo dT tails attached to a
resin can be used to bind the poly A tails of the mRNA. The mRNA is then eluted off the column.
• The mRNA is hybridized with a poly (T) primer which acts as a primer for reverse transcriptase.
• Reverse transcriptase copies the mRNA into a cDNA chain, thereby forming a DNA/mRNA
hybrid. Reverse transcriptase needs 4dNTP and magnesium for its activity.
• Alkaline conditions are used to selectively degrade the mRNA.
• DNA polymerase copies the single strand of DNA into double-stranded DNA which uses in this
case the 3
end of the single-stranded DNA which can fold back on itself and form a few chance
base pairings.
• A DNA nuclease cleaves the hairpin loop thus forming a double-stranded cDNA copy of the orig-
inal mRNA.
• All the double-stranded cDNAs formed can be inserted into a plasmid vector to form a cDNA
library. The cDNA has blunt ends which need to be converted to sticky ends by RE treatment to
facilitate ligation with the plasmid.
• The cDNA library can then be screened and amplified as indicated in Figure 14-5B and C. RE
restriction enzyme; dNTP
deoxynucleotide triphosphate.
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CHAPTER 14
A
A
B
● Figure 14-7
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