"The 16S rrna gene is a bacterial ribosomal gene and a part of the subunit of the 1930s, which is used in the identification, characterization and classification of several bacteria."
16S rRNAThe gene builds the 16s rrna subunit that binds to the bright-dalgarno sequence present in the bacteria genome.Although the gene is highly preserved of evolution and, therefore, is often known as a ""Molecular fossil".
In particular, the gene has two different domains, a larger domain and a hypervariable region.
Throughout the solar system, our land has a life with two more common ways;PROCRKARIOTS AND EUCARIOTAS.Procryciones are individual cell organisms such as viruses or bacteria, while eukaryotes are multicellular and have different degrees of cellular organization.
It was believed that every current way of life has evolved from primitive prokaryotic organisms.Bacteria are the most common prokaryotic form of life, some are harmful and others are useful.
For example,Lactobaciloand some intestinal bacteria are useful, while some strains ofE. colimiC. Bottle linesThey are harmful to us.
Using microbiological techniques, several types of bacteria can be identified.However, conventional culture and microbiology methods require a lot of time, are prone to pollution and less precise.It takes a few days to grow bacterial cells.
In addition, some bacterial strains are difficult to cultivate and others are highly infectious and lethal to us.
Thanks to the recent genetic advances, the use of the state of molecular art techniques, bacterial identification, classification and other characteristics can be studied well.
Methods such as different forms of PCR and DNA sequencing are precise, efficient, rapid, economic and contaminated compared to conventional microbiological methods.In addition, in a few hours we can obtain results.
This article is more valuable for a microbiology student than for others.However, this article will definitely help all biotechnology students.In this article, I will try to understand what the 16S RRNA gene is done and how the sequencing is carried out.
Related article:Microbial genetics.
In 1977, Carl Woese and George and Fox used the 16S RRNA gene in the bacterial phylogenetic analysis.This was the first attempt to do so.Genetic techniquesHe had revolutionized the field of microbiology.
As I explained, it is a subunit of a ribosome RNA of the 1930s, plays a notable role in bacterial translation.It is, the ribosomal assembly cannot work properly and ribosome cannot function efficiently.
Let's look at some of the main functions of 16s in a biological system:
Interact with the other ribosomal subunits, such as 23s, and facilitates the connections of ribosomal subunits 50 and 30.
As is a great structural support for ribosome, it helps establish the functioning of ribosome.It also provides structural support to ribosomal protein, which works as a scaffolding.
The end 3 'also helps at the beginning of protein synthesis, interacting with the S1 and S21 protein.
With the end of 3 ', 16S RRNA has some special types of sequences known as anti-thalgarno sequences that have the ability to connect in the AUG.
In the ribosome, it stabilizes the correct pairing of the codon-anton.
In conclusion, this RNA plays a very vital role in the synthesis of prokaryotic protein.
16S gene rrenr:
It is evident that some genes form 16s.But immediately a question arises in your mind that it is a RNA type and protein genes, how is it possible?
According to the traditional definition of a gene, "a functional unit of a genome that forms the protein is known as the gene."
But according to recent findings, genes form not only protein, but also build several RNAs.These types of RNAs are RNA, arnt, Mirna, Sirna, Shrna and other small non -functional RNAs.
In particular, these RNAs cannot be translated into proteins, unlike RNM, but plays an essential role inTranscription and translationRegulation.Therefore, these RNAs are more involved in gene regulation.
The RRNA gene has two important domains, one is the preserved domain, present in all bacterial species and remains unchanged.While the other domain is a hypervariable region.See the image below,
Hyperviable regions provide exceptional diversity to bacteria.Therefore, it is variable, different bacteria have different changes, which makes each bacteria unique.9 The different hypervariable regions V1 A V9 are generally present in a gene.
As the hypervariable region is a type of plan for all bacteria, it is widely used in identification, classification, detection, comparison and phylogenetic analysis of several bacteria.
From 5 to 10 copies of the 16S RRNA gene are present in a single bacterial cell, which makes the sensitivity of detection.The gene has approximately 50 functional domains with a total length of up to 1550 bp.
For detection, the set of universal initiators is built based on the preserved domain information (because it is present in all bacteria and remains unchanged in evolution), while detecting specific species of bacteria or bacteria, probe or otherSet of initiators is projected based on based on sequence information of variable regions.
[EPCL_BOX TYPE = "WARNING"] The 16S RRNA gene is a type of "molecular clock" used for the phylogenetic analysis of several bacterial species and arches of the sea. [/Epcl_box]
Now different sets of initiators to make PCR are available using 16S RRNA, however, the set of 27F and 1492R initiators used by Weisburg et al are the most common set of initiators used in the study.
Related article:What is a gene?- Definition, structure and function
However, to reduce the complexity of the reaction, we can project our own set of initiators to create shorter PCR amplifications.Some of the common initiators sets used for PCR -based detection are ready in the table below,
|Primer name||Sequence (5 ' - 3')|
|8f||Aga gtt tga tcc tgg ctc ag|
|skull||Aga gtt tga tcMETROTGG CTC AG|
|Romper||So there as well as known|
|Perfume||ACT GCT GCS YCC CGT AGG AGT CT|
|1100r||GGG TTG CGC TCG TTG|
|907 R.||CCG TCA Para CCT TTR AGT TT|
|Elevator||GGA TTA GAT ACC CTG GTA|
|805r||GAC TAC CAG GGT ATC TAA TC|
|Disgusting||GTG CCA GCC GCC GCG GTA A|
|518R||GTA TTA CCG CGG CTG CTG G|
|1492R||CGG TTA CCT TGT TAC GAC TT|
PCR and sequencing are two common methods that scientists are using to detect bacterial species.Polymerase chain reaction) It has some detection limitations, we will talk about this at the end of this section.
PCR is a temperature -dependent amplification method that synthesizes DNA sequences.The denaturation of the model, the initiator's wear and the extension of the sequence occur in three different steps, adhesive, prosecution and extension, respectively.
Here, a set of specific initiators for the preserved domain of a bacterial gene with other PCR ingredients, such as PCR buffer, DNTP, TAQ polymerase DNA and nucleas -free water, are used to amplify the target gene.
Once the amplification is completed, the reaction is carried out from the agarose gel to detect the amplification.The presence of amplicones or DNA bands indicates the presence of bacterial voltage.Although no amplicon indicates the absence of bacteria.
In general terms, it gives us an idea about the presence and absence of bacteria in any sample.However, new strains or bacterial species can be detected using PCR.
If we have sequence information or a goal to study, we can do it, otherwise we must make DNA sequencing.
In addition, PCR can also be used to quantify the bacterial load.The amount of infection, bacterial load or number of bacteria can be determined using theReal -time PCR method.
The 16S rrna gene is also used to know the relationship of organisms and the evolutionary distance between organisms.
DNA sequencing is a method used to identify the DNA sequence present in any biological sample.Here we are using nucleotides or marked probes to know the DNA sequence.
In particular, instead of all bacterial genomic DNA, the amplified 16S RRNA gene is used as a model for sequencing.To the extent that the sequence is linked to the sequence, a detector detects the fluorescence issued by it.
Once the complete sequence is completed, using a comparative analysis, the sequence of the 16S RRNA gene is compared with other sequences to distinguish any variation, if any.In doing so, we can create a phylogenetic tree of several bacteria.
We can sequence the entire 1500BP region of a gene, but, curiously, the first 500 bp region is powerful enough to establish differences between several bacterial or taxa species.APPX.66% of the variations of the 16S RRNA gene are in the first 500 PBP (studied inBordetellaspecies).
Read more about DNA sequencing:DNA sequencing: history, steps, methods, applications and limitations.
CARNR 16S Detection: Full Process
The first step in the detection process is the collection of samples and the extraction of DNA.Routine DNA extraction protocols such as PCI or K Proteinase cannot work here, we need to use a standard bacterial DNA extraction protocol or the DNA of the extraction kit ready to use to obtain bacterial DNA.
In the next step, quantify the DNA and verify the purity of extracted DNA.Only good quality DNA, if not obtained, repeat DNA extraction.
In the next step, project or select the pair of initiators for the amplification of PC.gerally, a unique set of specific initiators for the 16S RRNA gene is sufficient to amplification.Only the 500 bp amplification region is very small, we can also expand the entire gene.
The reaction preparation process and the amount of each ingredient are provided in the figure below.In addition, cyclic PCR conditions are provided in the figure below.
PCR protocol for PCR 50 microlitrer reaction.
- Genomic DNA: 50 to 100ng
- First: 1 micromol
- 1x reaction buffer (tris-kcl)
- mezclar dntp: 0,2 mm
- Dn Politrasa Taq: 5u/microliter
- MGCL2: 2 mm (if necessary)
PCR cycle conditions:
- Initial denaturation: 94c for 2 minutes
- Denatural: 94c per 1 minute
- Application: 55c for 2 minutes
- Stretching: 72c for 2 minutes
- Final extension: 72c for 5 minutes
- PCR for 30 cycles.
Once the reaction is completed, confirm complications in gel electrophoresis.Use only 2% or 1.8% agarose to do it.
- Electrophoresis in Candarose Gel
Now, in the next step, purify the amplifiers again using the DNA purification kit ready to use and send -D for DNA sequencing.
Using a set of initiators and probe, the sequencing machine identifies the complete sequence of a 16S RRNA gene.After completing the sequencing process, the data is sent to the bioinformatics laboratory to evaluate the results.
Now in the computational analysis, the complete sequence of the 16S RRNA gene is compared to the available sequence.A specialist identifies changes or variations in the hypervariable region of a gene and tries to discover its relationship with other sequences available.
[EPCL_BOX TYPE = "INFORMATION"] Genebank has 90,000 sequences of 16s RRNA genes 16s of several bacterial strains.[/Epcl_box]
16S RRNA applications:
- Identification of bacteria species
- Taxonomic analysis
- Characterization of a bacterial population
- Bacterial diversity evaluation
PCR and DNA sequencing helps scientists identify precision and quickly microbial identification.Currently, several kits based on the PCR amplification are available to increase the automation process.However, the use of the manual method can increase knowledge and reduce the cost of the reaction.
- OTA-TSUZUKI C, BRUNHEIRA AT, MAYER MP.PCR protocol based on the 16S RRNA region for the identification and subtipation of Micra Plvimon.Braz j microbiol.2008;39 (4): 605-607.
- Widmer F, Seidler RJ, Gillevet PM, Waprud LS, Di Giovanni GD.A highly selective PCR protocol to detect the 16S RRNA genes of the genus Pseudomonas (sensu stricto) in environmental samples.Microbiol application.1998;64 (7): 2545-2553.