to coincidental mixtures in the ecosystem. Thus, these metals pose a great threat to
aquatic fauna, especially to fish, which constitute one of the major sources of
protein-rich food for mankind. Thus, we studied the acute and sublethal toxic effects
of heavy metals in Krishna River sediment, water, and aquaculture using atomic
absorption spectroscopy. The aquatic environment becomes contaminated with a
variety of pollutants generated from diverse sources (industry, agriculture, domestic). Among these pollutants, pesticides, heavy metals, and detergents are the major
causes of concern for the aquatic environment because of their toxicity, persistency,
and tendency to accumulate in organisms. The 19 elements that constitute the heavy
metal group have many similar physical and chemical properties and are remarkably
different from the other 97 known elements. Among these 19 heavy metals, lead,
cadmium, and mercury are extremely toxic. Other metals, such as chromium,
copper, manganese, nickel, tin, and zinc, when once dispersed in the biosphere
cannot be recovered or degraded and thus cause permanent environmental damage.
Metal pollution has harmful effects on biological systems and does not undergo
biodegradation. Toxic heavy metals such as Pb, Co, Cd, and Hg can be differentiated
from other pollutants, because they cannot be biodegraded but can be accumulated in
living organisms, thus causing various diseases and disorders even at relatively
lower concentrations.
4 Molecular Fingerprinting Techniques in Microbial
Identification
Fingerprinting methods rely on sequence variations in the genome of different
organisms. Those differences result in different melting behaviors as well as different restriction enzyme recognition sites. Different species have sites at unique
positions along the whole genome. Two commonly used methods to analyze environmental samples of unknown microbial community composition are denaturing or
temperature gradient gel electrophoresis (DGGE or TGGE) and restriction fragment
length polymorphism (RFLP). Both these methods, which were introduced by
Muyzer et al. (1993), make use of sequence variations of polymerase chain reaction
(PCR) products amplified from environmental DNA on a gradient of either increasing denaturants or temperature. Both methods rely on differences in the sequencedependent melting behavior of double-stranded DNA. For this, the extracted nucleic
acids must be amplified using primers that target specific molecular markers such as
the 16S rRNA gene. For DGGE/TGGE, the use of a 50-GC clamped (30–50
nucleotides) forward primer is essential to avoid complete dissociation of the double
strands. After loading the PCR product onto the gel, an electric current is applied,
pulling the DNA fragments through the gel. Depending on the sequence of the
amplicon, a higher or lower denaturant concentration or temperature is needed for a
complete dissociation of the double strands into single strands, causing the
amplicons to stop moving through the gel at different positions. To determine the
444
S. K. Shekhar et al.
aquatic fauna, especially to fish, which constitute one of the major sources of
protein-rich food for mankind. Thus, we studied the acute and sublethal toxic effects
of heavy metals in Krishna River sediment, water, and aquaculture using atomic
absorption spectroscopy. The aquatic environment becomes contaminated with a
variety of pollutants generated from diverse sources (industry, agriculture, domestic). Among these pollutants, pesticides, heavy metals, and detergents are the major
causes of concern for the aquatic environment because of their toxicity, persistency,
and tendency to accumulate in organisms. The 19 elements that constitute the heavy
metal group have many similar physical and chemical properties and are remarkably
different from the other 97 known elements. Among these 19 heavy metals, lead,
cadmium, and mercury are extremely toxic. Other metals, such as chromium,
copper, manganese, nickel, tin, and zinc, when once dispersed in the biosphere
cannot be recovered or degraded and thus cause permanent environmental damage.
Metal pollution has harmful effects on biological systems and does not undergo
biodegradation. Toxic heavy metals such as Pb, Co, Cd, and Hg can be differentiated
from other pollutants, because they cannot be biodegraded but can be accumulated in
living organisms, thus causing various diseases and disorders even at relatively
lower concentrations.
4 Molecular Fingerprinting Techniques in Microbial
Identification
Fingerprinting methods rely on sequence variations in the genome of different
organisms. Those differences result in different melting behaviors as well as different restriction enzyme recognition sites. Different species have sites at unique
positions along the whole genome. Two commonly used methods to analyze environmental samples of unknown microbial community composition are denaturing or
temperature gradient gel electrophoresis (DGGE or TGGE) and restriction fragment
length polymorphism (RFLP). Both these methods, which were introduced by
Muyzer et al. (1993), make use of sequence variations of polymerase chain reaction
(PCR) products amplified from environmental DNA on a gradient of either increasing denaturants or temperature. Both methods rely on differences in the sequencedependent melting behavior of double-stranded DNA. For this, the extracted nucleic
acids must be amplified using primers that target specific molecular markers such as
the 16S rRNA gene. For DGGE/TGGE, the use of a 50-GC clamped (30–50
nucleotides) forward primer is essential to avoid complete dissociation of the double
strands. After loading the PCR product onto the gel, an electric current is applied,
pulling the DNA fragments through the gel. Depending on the sequence of the
amplicon, a higher or lower denaturant concentration or temperature is needed for a
complete dissociation of the double strands into single strands, causing the
amplicons to stop moving through the gel at different positions. To determine the
444
S. K. Shekhar et al.