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By NGS method DNA sequencing is made possible. And Scientist can expound the information of any biological sytem. NGS has transfigured the genomic science. It gives the infinite acumen into the transcriptome, and epigenome. The principle perception overdue NGS is same to CE-sangar sequencing i.e the small fragments of DNA resynthesized as all bases are recognized.
Mandate has never been greater for innovation technologies that deliver reckless, inexpensive and accurate genome information. This challenge has accelerated the advancement of next-generation sequencing (NGS) technologies. The economical manufacturing of great bulk of sequence data is the fundamental benefit over conservative approach. Here, a technical review of template preparation, sequencing, genome arrangement and assembly approaches, and current advances in current and near-term commercially obtainable NGS instruments. Here are drawn some broad range of applications of NGS technologies. [ref 1]
Any kind of disease factors are tend to accuma-ulate and express in next generation influenced by environmental factor. NGS provided novel applications such as, ancient DNA sampling, from which metagenomic investigation is made conceiveable.
Though DNA was discovered in 1953 but it was not possible to analyse even a small fragment of DNA after several decade. First, Genome of Bacteriophage MS2, was analysed and sequenced by Walter Fiers and his co-worker at university of GHENT ( Belgium). (ref3,4,5 of google). Fredrick Sanger developed a rapid DNA sequencing method, the method is based on chain termination technique. Maxam and Gilbert developed another method of DNA sequencing by chemical degradation. First DNA next generation sequencing was developed by Nyren and his co-worker and patient them as a ''PYROSEQUENCING.
The fundamental of this method is the radiolabelling at 5' end. Chemical are used to generate the cleavage. These chemical break them in a proportion of one or two of the four nucleotide like, T, G+C, A, C+T. Purines are treated with formic acid. Guanine and adenine are methylated by dimethylsulfate. The sodium salt usually inhibits the methylation of thymine. This modified fragment is trated with hot Piperidine which make a cut at the site of modified DNA. The concentration of the pipredine is controlled per DNA molecule. Thus chain of fragments are obtained. Their length is from radiolabel site to the first cut site in the DNA molecule.File:Maxam gilbert sequencing.png
Fig1: Diagramatic representation of Maxm Gilbet method.
CHAIN TERMINATION METHOD OR SANGER METHOD:
Sanger method is developed by Frederick Sanger in 1977. This classic methods involves the single stranded DNA i.e dNTP'S and ddNTP's. This ddNTP's lack the 3'-OH end that is necessary for chain elongation as a result chain elongation is stopped. This ddNTP's are radioactively or fluorescently labelled for recognition. These bands may be seen by autodariograph , Uv light or by X-ray film.
CHALLENGES IN THE BASIC METHODS
Advantages and disadvantages of biochemical-based methods to study soil microbial diversity
Method Advantages Disadvantages Selected references
Plate counts Fast Unculturable microorganisms Tabacchioni et al. (2000),
Inexpensive not detected
Bias towards fast growing individuals
Bias towards fungal species that produce large quantities of spores
Community level physiological Fast Only represents culturable Classen et al. (2003), Garland profiling (CLPP) Highly reproducible fraction of community (1996a), Garland and Mills
Relatively inexpensive Favours fast growing (1991)
Fatty acid methyl ester analysis
Differentiate between microbial communities Generates large amount of data Option of using bacterial, fungal plates or site specific carbon sources (Biolog)
No culturing of microorganisms, direct extraction from soil
Follow specific organisms or communities
Only represents those organisms capable of utilizing available carbon sources Potential metabolic diversity, not in situ diversity
Sensitive to inoculum density If using fungal spores, a lot of material is needed
Can be influenced by external factors
Possibility results can be confounded by other microorganisms
Graham et al. (1995), Siciliano and Germida (1998), Zelles (1999)
Advantages and disadvantages of some molecular-based methods to study soil microbial diversity
Method Advantages Disadvantages Selected references
Guanine plus cytosine
Not influenced by PCR
Requires large quantities of
Nusslein and Tiedje (1999), Tiedje et al. (1999)
Includes all DNA extracted Dependent on lysing and
Quantitative extraction efficiency
Includes rare members of community
Coarse level of resolution
Nucleic acid reassociation Total DNA extracted Lack of sensitivity Torsvik et al. (1990a,b,
and hybridization Not influenced by PCR
Sequences need to be in high copy number to be
1996), Cho and Tiedje
Study DNA or RNA detected
Can be studied in situ Dependent on lysing and extraction efficiency
DNA microarrays and DNA
Denaturing and temperature gradient gel electrophoresis (DGGE
Same as nucleic acid hybridization
Thousands of genes can be
If using genes or DNA fragments, increased specificity
Large number of samples can be analyzed simultaneously
Reliable, reproducible and
Only detect most abundant species
Need to be able to culture
Only accurate in low diversity systems
Dependent on lysing and extraction efficiency Sample handling can
influence community, i.e. if
stored too long before extraction, community can change
One band can represent more than one species (co-migration)
Only detects dominant
Hubert et al. (1999), Cho and Tiedje (2001), Greene and Voordouw (2003)
Muyzer et al. (1993), Duineveld et al. (2001), Maarit-Niemi et al. (2001)
Single strand conformation Same as DGGE/TGGE PCR biases Lee et al. (1996), Tiedje polymorphism (SSCP) No GC clamp Some ssDNA can form et al. (1999)
No gradient more than one stable
Amplified ribosomal DNA Detect structural changes in PCR biases Liu et al. (1997), Tiedje
restriction analysis (ARDRA) or restriction fragment length polymorphism (RFLP)
microbial community Banding patterns often too complex
et al. (1999)
Terminal restriction fragment length
Simpler banding patterns than RFLP
Dependent on extraction and lysing efficiency
Tiedje et al. (1999), Dunbar et al. (2000), Osborn et al.
polymorphism (T-RFLP) Can be automated; large PCR biases (2000)
number of samples
Type of Taq can increase variability
Compare differences in Choice of universal primers microbial communities Choice of restriction
enzymes will influence
Ribosomal intergenic spacer analysis (RISA)/automated ribosomal intergenic spacer analysis (ARISA)
Highly reproducible community profiles
Requires large quantities of
Fisher and Triplett (1999)
J.L. Kirk et al. / Journal of Microbiological Methods 58 (2004) 169-188
Small samples of DNA (or RNA) are added to an electrophoresis gel that contains a denaturing agent. The denaturing gel induces melting of the DNA at various stages. As a result of this melting, the DNA spreads through the gel and can be analyzed for single components.
DGGE (Muyzer et al. 1993) analyses are employed for the separation of double-stranded DNA fragments that are identical in length, but differ in sequence.
In practice, the DNA fragments are usually produced via PCR amplification. The DGGE technique exploits (among other factors) the difference in the stability of G-C pairing (3 hydrogen bonds per pairing) as opposed to A-T pairing (2 hydrogen bonds). A mixture of DNA fragments of different sequence is separated by electrophoresis on an acrylamide gel containing a linearly increasing gradient of DNA denaturants (usually urea and formamide). In general, DNA fragments richer in GC will be more stable and remain double-stranded until reaching higher denaturant concentrations. Double-stranded DNA fragments migrate better in the acrylamide gel, while denatured DNA molecules slow down or stop in the gel. In this manner, DNA fragments of differing sequence can be separated in an acrylamide gel. DGGE is commonly performed for partial 16S rRNA gene, but also functional genes may be used. A GC (guanine plus cytosine) rich sequence can be incorporated into one of the primers used in the PCR to modify the melting behaviour of the fragment of interest and to improve the separation of the fragments. The DGGE gels can be stained with DNA binding fluorescent dyes, such as SYBR Green and visualized under UV light. Known standards may be used for comparing the samples on different gels. Ideally one band on the gel corresponds to one species, and therefore the number of bands gives an idea of the diversity of the sample. The gene fragments can be excised from the gel, eluted e.g. into sterile water and amplified for sequencing. The relative abundance of various microorganisms can be estimated by measuring the intensity of their bands relative to the intensity of all bands in the corresponding sample.