132
UNIT ONE
4.2
FROM SCIENCE TO TECHNOLOGY
Nucleic Acid Amplif
cation
The polymerase chain reaction (PCR) is a pro-
cedure that borrows a cell’s machinery for DNA
replication, making many copies of a gene of
interest. Developed in 1983, PCR was the F
rst of
several technologies called nucleic acid ampliF
-
cation. Starting materials for PCR include:
two types of short DNA pieces known to
bracket the gene of interest, called primers
a large supply of DNA bases
the enzymes that replicate DNA
Here’s how it works. ±irst, heat is used to sepa-
rate the two strands of the target DNA—such as
bacterial DNA in a body ²
uid sample from a per-
son who has symptoms of an infection. Next, the
temperature is lowered, and the two short DNA
primers are added. The primers complementary
base pair to the separated target strands. The
third step adds DNA polymerase and bases. The
DNA polymerase adds bases to the primers and
builds a sequence complementary to the target
sequence. The newly synthesized strands then
act as templates in the next round of replica-
tion, which begins by raising the temperature.
All of this is done in an automated device called a
thermal cycler that controls the key temperature
changes. The DNA polymerase can withstand the
temperature shifts because it comes from a bac-
terium that lives in hot springs.
The pieces of DNA exponentially accumu-
late. The number of amplified pieces of DNA
equals 2
n
where n equals the number of temper-
ature cycles. After just twenty cycles, 1 million
copies of the original sequence are in the test
tube. PCR has had many diverse applications,
from detecting moose meat in hamburger to
analysis of insect larvae in decomposing human
corpses.
PCR’s greatest strength is that it works on
crude samples of rare and short DNA sequences,
such as a bit of brain tissue on the bumper of a car,
which in one criminal case led to identiF
cation of
a missing person. PCR’s greatest weakness, ironi-
cally, is its exquisite sensitivity. A blood sample
submitted for diagnosis of an infection contami-
nated by leftover DNA from a previous run, or a
stray eyelash dropped from the person running
the reaction, can yield a false positive result. The
technique is also limited in that a user must know
the sequence to be ampliF
ed and that mutations
can sometimes occur in the amplified DNA not
present in the source DNA.
The invention of PCR inspired other nucleic
acid ampliF
cation technologies. One, which cop-
ies DNA into RNA and then amplifies the RNA,
does not require temperature shifts and produces
100 to 1,000 copies per cycle, compared to PCR’s
doubling.
RECONNECT
To Chapter 3, A Composite Cell, page 82.
Protein synthesis is economical. A molecule of mRNA
usually associates with several ribosomes at the same time.
Thus, several copies of that protein, each in a different stage
of formation, may be present at any given moment. As the
polypeptide forms, proteins called
chaperones
fold it into its
unique shape, and when the process is completed, the poly-
peptide is released as a separate functional molecule. The
tRNA molecules, ribosomes, mRNA, and the enzymes can
function repeatedly in protein synthesis.
ATP molecules provide the energy for protein synthesis.
A protein may consist of many hundreds of amino acids and
the energy from three ATP molecules is required to link each
amino acid to the growing chain. This means that a large
fraction of a cell’s energy supply supports protein synthesis.
Table 4.3
summarizes protein synthesis.
The number of molecules of a particular protein that a
cell synthesizes is generally proportional to the number of
corresponding mRNA molecules. The rate at which mRNA is
transcribed from DNA in the nucleus and the rate at which
enzymes (ribonucleases) destroy the mRNA in the cytoplasm
therefore control protein synthesis.
Proteins called transcription factors activate certain
genes, moving aside the surrounding histone proteins to
expose the promoter DNA sequences that represent the start
of a gene. These actions are called “chromatin remodeling,”
The genetic code speci± es more than enough information.
Although only twenty types of amino acids need be encoded,
the four types of bases can form sixty-four different mRNA
codons. Therefore, some amino acids correspond to more
than one codon
(table 4.2)
. Three of the codons do not have a
corresponding tRNA. They provide a “stop” signal, indicating
the end of protein synthesis, much like the period at the end
of this sentence. Sixty-one different tRNAs are speci± c for the
remaining sixty-one codons, which means that more than one
type of tRNA can correspond to the same amino acid type.
The binding of tRNA and mRNA occurs in close asso-
ciation with a ribosome. A ribosome is a tiny particle of
two unequal-sized subunits composed of
ribosomal RNA
(rRNA)
and protein molecules. The smaller subunit of a ribo-
some binds to a molecule of mRNA near the ±
rst codon. A
tRNA with the complementary anticodon brings its attached
amino acid into position, temporarily joining to the ribo-
some. A second tRNA, complementary to the second mRNA
codon, then binds (with its activated amino acid) to an adja-
cent site on the ribosome. The ±
rst tRNA molecule releases
its amino acid, providing the energy for a peptide bond to
form between the two amino acids (see ±
g. 4.24). This pro-
cess repeats as the ribosome moves along the mRNA, add-
ing amino acids one at a time to the extending polypeptide
chain. The enzymatic activity necessary for bonding of the
amino acids comes from ribosomal proteins and some RNA
molecules (ribozymes) in the larger subunit of the ribosome.
This subunit also holds the growing chain of amino acids.
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