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113
MOLECULAR BIOLOGY TECHNIQUES
In the previous discussion of cloning the Factor VIII gene using
either a genomic library or cDNA library, we saw that the amplification step employed culturing bac-
teria in a nutrient broth overnight. There is another method to amplify DNA called polymerase chain
reaction (PCR). PCR is based on repeated cycles of replication using specially designed primers,
DNA polymerase, dATP, dGTP, dCTP, and dTTP. To design the primers, some knowledge of the DNA
sequence to be amplified is necessary. After 20–40 repeated cycles of the PCR reaction, millions of
copies of the desired DNA can be obtained within a few hours. Many PCR methods have been devel-
oped over the years as indicated below:
1. Reverse transcription PCR (RT-PCR). RT-PCR uses mRNA as the starting material and an ini-
tial reverse transcriptase step to produce cDNA. Eukaryotic genes contain introns (noncoding
sequence) that interrupt the exons (coding sequence) and therefore will not be expressed in bac-
teria because bacteria do not have a splicing mechanism. So, to get a copy of a gene without
introns, we can start with mRNA (the introns are already spliced out) and use RT-PCR to con-
vert the mRNA into an intron-free DNA copy of the gene.
2. Differential display PCR. Differential display PCR is a type of RT-PCR that is used to compare
mRNA populations from two different cell lines, tissues, or different times in embryological
development to identify differentially expressed genes. This technique uses an oligo-dT primer
as the first primer (because eukaryotic mRNAs have a poly A tail) and a mixture of random
primers as the second primer. The PCR will produce cDNAs corresponding to all the mRNAs.
The cDNAs are separated by gel electrophoresis and a series of DNA bands corresponded to all
the mRNAs is displayed. The DNA bands from the different cell lines, tissues, and different times
in embryological development can be compared.
3. Real-time PCR. Real-time PCR is a type of quantitative PCR that uses a fluorescent dye whose
fluorescence increases when it binds to DNA. Because each cycle of PCR makes more DNA, the
dye binds to the new DNA and the fluorescence increases. Real-time PCR uses a fluorescence-
detecting thermocycler machine to amplify DNA sequences and simultaneously measure the
amount of DNA produced. Most dyes do not recognize any specific DNA sequence, so these dyes
just measure total DNA. However, sophisticated fluorescent probes can be constructed that rec-
ognize only a specific DNA sequence and therefore can measure a specific PCR product.
Figure 14-7A The PCR reaction:
• A region of double-stranded DNA to be amplified is shown undergoing the PCR reaction. Each
cycle of the PCR reaction begins with 94
C heat treatment to separate the double-stranded DNA
into single strands (denature). Because the temperature rises to 94
C, it is necessary to use Taq
polymerase which is a thermostable DNA polymerase isolated from the thermophilic bacterium
Thermus aquaticus. This bacterium lives in hot springs and hydrothermal vents.
• The DNA primers hybridize to the single-stranded DNA at 50–68
C.
• The DNA primers act as the primer for DNA synthesis at 72
C by DNA (Taq) polymerase, dATP,
dGTP, dCTP, and dTTP.
• Of the DNA put into the original reaction, only the DNA sequence bracketed by the two primers
is amplified because there are no primers attached anywhere else.
• This is repeated for 25–30 cycles to produce millions of copies of the original region of DNA.
Figure 14-7B PCR and viral detection: It is good news when a virus has been isolated and its
DNA (or RNA) sequence determined because it allows one to design primers to be used in PCR.
PCR is one of the most sensitive methods to detect viral infection at very early stages because of
the ability of PCR to amplify DNA that is present in even very minute amounts.
• A blood sample from a suspect patient is taken and cells removed by centrifugation. If even a
trace amount of virus is present in the serum, its DNA can be isolated and amplified by PCR.
• PCR will produce enough DNA so that it can be detected by gel electrophoresis.
• Note that if you want to detect an RNA virus (e.g., human immunodeficiency virus [HIV]), you
must first use reverse transcriptase to convert the RNA into cDNA and then amplify by PCR.
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114
CHAPTER 14
Implanted
Pseudo-pregnant ewe
Transgenic sheep
with human
Factor IX gene
Birth
Isolate of colony
containing human
Factor IX gene
Human Factor
IX gene
colony
A
B
A
● Figure 14-8
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MOLECULAR BIOLOGY TECHNIQUES
Producing a Protein From a Cloned Gene.
We have previously discussed how a gene (e.g.,
Factor VIII) may be cloned using a cloning vector. After a gene has been cloned, the next step is
to get the cloned gene to produce a protein, that is, be transcribed and translated into the amino
acid sequence of a protein (frequently called gene expression). To do this, a plasmid vector is used
(called an expression vector). Expression vectors differ from cloning vectors in that expression
vectors contain gene promoter DNA sequences and gene regulatory DNA sequences that enable a
nearby protein-coding DNA sequence to be expressed. Many expression vectors have been
designed for use in bacteria, yeast, and mammalian cells. Expression vectors have made an impor-
tant contribution to recombinant DNA technology because they allow the production of any pro-
tein (even rare proteins) in large amounts (e.g., Factor VIII, human insulin, and human growth
hormone).
Figure 14-8A Expression vector. An expression vector containing regulatory and promoter DNA
sequences that drive the expression of the nearby human insulin gene is shown.
• The expression vector is introduced into respective bacteria, yeast, or mammalian cells where the
human insulin gene can be transcribed and translated into human insulin.
• This methodology allows for the production of large amounts of human insulin for use by all
Type I and Type II diabetics in the world. This methodology has replaced the extraction of
insulin from bovine pancreases collected from the slaughterhouse.
Figure 14-8B Expression vectors and nuclear transfer technique. This technique provides an-
other method to produce large amounts of human proteins, but in this method, the human pro-
tein is produced in the milk of large farm animals like transgenic sheep or transgenic cows. Human
Factor IX used in the treatment of hemophilia B has been produced in the milk of transgenic
sheep. An expression vector containing regulatory and promoter DNA sequences that drive the
expression of the nearby human Factor IX gene is shown.
• The expression vector is introduced into ovine fetal (OF) cells that are allowed to grow in cul-
ture to form colonies.
• The OF colony containing the human Factor IX gene is isolated.
• The nucleus is removed from one of the OF cells containing the human Factor IX gene and trans-
ferred into an enucleated ovine oocyte.
• The “new” oocyte is stimulated to undergo cleavage divisions and implanted into a pseudo-
pregnant ewe.
• Transgenic sheep containing human Factor IX gene will be produced at birth. The female trans-
genic sheep will produce human Factor IX protein in their milk.
• Note that the nuclear transfer technique is what is giving medical bioethicists cause for grave
concern because this technique leads directly to the cloning of a human being. As the nuclear
transfer technique is perfected, a nucleus from one of your cells can be removed, transferred into
an enucleated human oocyte, and introduced into a pseudo-pregnant woman. At birth, the off-
spring will be your human clone.
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CHAPTER 14
Breed chimeras
Homozygous
knockout
–/+
–/+
+/+
–/+
–/–
–/+
colony
● Figure 14-9
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MOLECULAR BIOLOGY TECHNIQUES
Site-Directed Mutagenesis and Knockout Animals.
The ability to express a cloned gene
within a cell opened up a new area of medical research. It is now possible to mutate a cloned gene
at specific sites (called site-directed mutagenesis) and then test the function (or lack thereof) of
the mutant gene. The cloned gene can be mutated so that as little as one amino acid in the protein
is changed (gene replacement) or a large deletion can be made so that the protein is nonfunctional
(called gene knockout). The ultimate test of the function of the mutant gene is to insert it into the
genome of an animal (e.g., mouse) and observe its effect on the entire animal. A normal gene is
shown in an expression vector containing regulatory and promoter DNA sequences.
• The normal gene can undergo site-directed mutagenesis so that the expressed protein is changed
as little as one amino acid (gene replacement) or is rendered nonfunctional (gene knockout).
• The mutant gene within the expression vector can be introduced into embryonic stem (ES) cells.
• After culturing the ES cells, one can isolate the particular ES cells that have incorporated the
mutant gene (shaded cells).
• These ES cells are then injected into an early embryo obtained from a 3-day pregnant mouse
(female mouse X). This forms an early embryo containing cells from female mouse X (white
cells) and ES cells with mutant gene (shaded).
• A number of these early embryos are produced and introduced to a pseudo-pregnant mouse. At
birth, a number of chimeric mice (white and shaded areas;
/) are produced. A chimeric
mouse is a mouse produced from a mixture of two different cell types. Because there are two
copies of every gene, the chimeric mice will have one copy of the mutant gene (
) and one copy
of the normal gene (
), that is, heterozygous.
• The chimeric mice are bred and the resultant offspring will have four possibilities:
/, /,
/, and /. The / homozygous knockout mouse has both copies of the gene disrupted.
• Many times, the homozygous knockout can be lethal and the mice will die before birth.
Consequently, it would be ideal if we could delay turning the gene off until later in life. The
Cre-loxP recombination system is useful in this regard.
• Cre is the C-recombination enzyme from the P1 bacteriophage and loxP is the DNA recognition
site for Cre. If Cre finds two loxP sites close together, it chops out the intervening DNA and
recombines the loxP sites. If the gene you wish to knockout is placed between two loxP sites,
when the Cre protein is present, the gene is deleted. So, you now have an off/on switch. When
Cre protein is present, the gene is off. When Cre protein is absent, the gene is on. The only ques-
tion is how do you control Cre protein. This is usually done by placing the cre gene behind a
promoter that can be directly controlled in an experimental situation or behind a promoter of a
gene that is expressed under some experimental condition we can control.
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