Biomedical Engineering Reference
In-Depth Information
Reverse
transcriptase
in test tube
isolate from
cells and purify
PCR
mRNA
with nointrons
(many copies)
mRNA
in test tube
cRNA
no introns
much shorter
Original gene
(two copies)
Many copies
FIGURE 14.5 Method of producing cDNAs.
14.4.3. Cloning Genes into a Plasmid
Once a desired gene is isolated or created, it can be cloned by inserting it into a plasmid as
shown in Fig. 14.6 .
14.4.4. Polymerase Chain Reaction
Genes can be cloned by cloning the bacterial cells that contain them, but this requires quite
a lot of DNA in the first place. Polymerase chain reaction (PCR) can clone (or amplify) DNA
samples as small as a single molecule. It is a newer technique, having been developed in 1983
by Kary Mullis, for which discovery he won the Nobel prize in 1993. The PCR is simply DNA
replication in a test tube. If a length of DNA is mixed with the four nucleotides (A, T, C,
and G) and the enzyme DNA polymerase in a test tube, then the DNA will be replicated
many times ( Fig. 14.7 ).
Each original DNA molecule has now been replicated to form two molecules. The cycle is
repeated from step 2 and each time the number of DNA molecules doubles. This is why it is
called a chain reaction, since the number of molecules increases exponentially, like an explo-
sive chain reaction. Each cycle the PCR is run, the number of DNA molecules doubles. For an
n-PCR cycles, the increase in DNA molecules would be 2n times. See if we start out from one
DNA strand, each successive PCR cycle would lead to an increase by 100%, or 2, 2 2
4, 2 3
¼
¼
8,
2 4
¼
16. In just 10 cycles, the number of DNA strands becomes 1024. In 20 cycles, the number of
DNA strands becomes 1,048,576. Typically, PCR is run for 20 e 30 cycles as shown.
14.4.5. Vectors and Plasmids
Once a desired gene is isolated or created, it is inserted into a carrier DNA called vector.
Most often plasmids are used as vectors, although viruses can also be used for carrying the
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