Environmental Microbiology of Aquatic and Waste Systems

3.2 The Polymerase Chain Reaction 37

In summary, during translation, the ribosome binds
to the mRNA at the start codon (AUG) that is recog-
nized only by the initiator tRNA. During this stage,
complexes, composed of an amino acid linked to tRNA,
sequentially bind to the appropriate codon in mRNA by
forming complementary base pairs with the tRNA anti-
codon. The ribosome moves from codon to codon along
the mRNA. Amino acids are added one by one, trans-
lated into polypeptidic sequences dictated by DNA and
represented by mRNA. At the end, a release factor
binds to the stop codon, terminating translation and
releasing the complete polypeptide from the ribosome.
Elongation terminates when the ribosome reaches a
stop codon, which does not code for an amino acid and
hence not recognized by tRNA.
After protein has been synthesized, the primary
protein chain undergoes folding; secondary, tertiary,
and quadruple folding occurs. The folding exposes
chemical groups which confer their peculiar properties
on the protein. Protein folding is the process by which
a protein assumes its functional shape or conforma-
tion. All protein molecules are simple unbranched
chains of amino acids, but it is by coiling into specific
three-dimensional shapes that they are able to perform
their biological functions.

3.2 The Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) is a technology

used to amplify small amounts of DNA. The PCR

technique was invented in 1985 by Kary B. Mullis

while working as a chemist at the Cetus Corporation, a

biotechnology firm in Emeryville, California. So use-

ful is this technology that Muillis won the Nobel Prize

for discovery in 1993, 8 years later. It has found use in

a wide range of situations, from the medical diagnosis

to microbial systematics and from courts of law to the

study of animal behavior.

The requirements for PCR are:

(a) The DNA or RNA to be amplified

(b) Two primers

(c) The four nucleotides found in the nucleic acid

(d) A heat stable DNA derived from the thermophilic

Archaebacterium, Thermus aquaticus, Taq

polymerase

The Primer: A primer is a short segment of nucleotides

which is complementary to a section of the DNA which

is to be amplified in the PCR reaction.

Primers are annealed to the denatured DNA tem-

plate to provide an initiation site for the elongation of

U C A G

U UUU = Phe UCU = Ser UAU = Tyr UGU = CysU

UUC = Phe UCC = Ser UAC = Tyr UGC = CysC

UUA = Leu UCA = Ser UAA = StopUGA = StopA

UUG = Leu UCG = Ser UAG = StopUGG = TrpG

C CUU = Leu CCU = Pro CAU = His CGU = ArgU

CUC = Leu CCC = Pro CAC = His CGC = ArgC

CUA = Leu CCA = Pro CAA = Gln CGA = ArgA

CUG = Leu CCG = Pro CAG = Gln CGG = ArgG

A AUU = Ile ACU = Thr AAU = AsnAGU = SerU

AUC = Ile ACC = Thr AAC = AsnAGC = SerC

AUA = Ile ACA = Thr AAA = LysAGA = ArgA

AUG = Met ACG = Thr AAG = Lys AGG = ArgG

G GUU = Val GCU = AlaGAU = AspGGU = GlyU

CUC = Val GCC = Ala GAC = AspGCG = GlyC

GUA = Val GCA = AlaGAA = GluGGA = GlyA

GUG = Val GCG = AlaGAG = GluGGG = GlyG

Note: (i) Most codons for a given amino acid differ only in the last (third) base of the triplet

(exceptions: Leu, Arg, Ser); (ii) One codon (AUG or Met) also signals the START of a poly-

peptide chain; (iii) Three codons (UAA, UAG, and UGA) are used to signal the END of a

polypeptide chain (STOP codons)

AUG start codon; UAA, UAG, and UGA stop (nonsense) codons

Amino Acids: Phe phenylalanine, Leu leucine, Ile isoleucine, Met methionine, Val valine;

Ser serine, Pro proline, Thr threonine, Ala alanine, Tyr tyrosine; His histidine, Gln glutamine,

Asn asparagine, Lys lysine, Asp aspartic acid; Glu glutamic acid, Cys cysteine, Trp tryptophan,

Arg arginine, Gly glycine

Table 3.1 The genetic
code – codons