Agriculture Reference
In-Depth Information
6.3.4.7
Single Strand Conformation Polymorphism (SSCP)
The method was originally used for the detection of novel polymorphisms or point
mutations in DNA (Peters et al.
2000
) and has been adapted to analyse the succes-
sion of microbial communities. Similar to DGGE, this method allows the separation
of DNA molecules with the same length and different DNA fragments; however
the separation on polyacrylamide gel is based on the differences in motilities that
originate from the presence of secondary structures (heteroduplex) in the DNA mol-
ecules. The conformational changes in the secondary structure are a unique charac-
teristic of each DNA molecule, and this method has been used to track changes in
bacterial populations throughout the composting process, thereby allowing the as-
sessment of bacterial diversity and also in the identification of important microbes
(Alfreider et al.
2002
).
6.3.4.8
Pyrosequencing
Pyrosequencing determines the order of nucleotides in DNA based on the sequenc-
ing by synthesis principle and relies on the detection of pyrophosphate release on
nucleotide incorporation rather than chain termination with dideoxynucleotides.
This method has several advantages over other molecular methods, as it allows the
determination of the exact sequence by providing the same accuracy as convention-
al sequencing methods. It dispenses with the need for labelled nucleotides, labelled
primers, and electrophoresis and allows the sequencing of large number of samples
in a short period of time. In the context of analysing bacterial diversity, it allows for
the study of a large number of samples from a given population and thus counters
the deficiency associated with limited analysis of a small number of clones and a
few different samples for the determination of a bacterial community (Lauberet
et al.
2009
; Fakruddin et al.
2012
).
6.3.4.9
Illumina-based High Throughput Microbial Community Analysis
In this method, DNA molecules and primers are first attached on a slide and ampli-
fied with polymerase, so that local clonal DNA colonies, or otherwise termed “DNA
clusters” are formed. To determine the sequence, four types of reversible termina-
tor bases (RT-bases) are added and non-incorporated nucleotides are washed away.
This technology supports both single read and paired-end libraries. This method
offers a short-insert paired-end capability for high-resolution genome sequencing
as well as long-insert paired-end reads using the same robust chemistry for efficient
sequence assembly, de novo sequencing, large-scale structural variation detection
etc. The combination of short inserts and longer reads increases the ability to fully
characterize any genome (Caporaso et al.
2012
).
