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Ribosomal RNA Sequencing Techniques
- 1. Ribosomal RNA Sequencing Name :- Avdhesh kumar MSc. I sem Under the guidance of Dr. Swati kujur (Department of Biotechnology) SANT GAHIRA GURUVISHWAVIDYALAYA, SARGUJA, AMBIKAPUR, (C.G.)
- 2. Ribosomes are complex structures found in all living cells which functions in protein synthesis machinery. Basically ribosome’s consists of two subunits, each of which is composed of protein and a type of RNA, known as ribosomal RNA (rRNA). Prokaryotic ribosomes consist of 30S subunit (small sub unit) and 50S subunit (large sub unit) which together make up the complete 70S ribosome, where S stands for Svedberg unit non-SI unit for sedimentation rate. 30S subunit is composed of 16S ribosomal RNA and 21 polynucleotide chains while 50S subunit is composed of two rRNA species, the 5S and 23S rRNAs. The presence of hyper variable regions in the 16S rRNA gene provides a species specific signature sequence which is useful for bacterial identification process. 16S Ribosomal RNA sequencing is widely used in microbiology studies to identify the diversities in prokaryotic organisms as well as other organisms and thereby studying the phylogenetic relationships between them. The advantages of using ribosomal RNA in molecular techniques are as follows Ribosomes and ribosomal RNA are present in all cells. RNA genes are highly conserved in nature. Culturing of microbial cells is absent in the sequencing techniques. Introduction
- 3. Signature sequences are some specific base sequences which are always found in all groups of organisms.These unique DNA sequences are about 5– 10 bases long and found specifically in the 16S rRNA location, and are unique to many major groups of prokaryotic organisms, archaea and Eukarya. The average lengths of the structural rRNA genes are 1,522 bp, 2,971 bp, and 120 bp respectively for 16S, 23S, and 5S rRNAs. https://journals.plos.org/plosone/article?id= 10.1371/journal.pone.0088222
- 5. Methods Library preparation 1. RNA Isolation: RNA is isolated from tissue and mixed with deoxyribonuclease(DNase). DNase reduces the amount of genomic DNA. The amount of RNA degradation is checked with gel and capillary electrophoresis and is used to assign an RNA integrity number to the sample. This RNA quality and the total amount of starting RNA are taken into consideration during the subsequent library preparation, sequencing, and analysis steps. 2. cDNA synthesis: RNA is reverse transcribed to cDNA because DNA is more stable and to allow for amplification (which uses DNA polymerases) and leverage more mature DNA sequencing technology. Amplification subsequent to reverse transcription results in loss of strandedness, which can be avoided with chemical labeling or single molecule sequencing. Fragmentation and size selection are performed to purify sequences that are the appropriate length for the sequencing machine.The RNA, cDNA, or both are fragmented with enzymes, sonication, or nebulizers. Fragmentation of the RNA reduces 5' bias of randomly primed-reverse transcription and the influence of primer binding sites,with the downside that the 5' and 3' ends are converted to DNA less efficiently. Fragmentation is followed by size selection, where either small sequences are removed or a tight range of sequence lengths are selected. Because small RNAs like miRNAs are lost, these are analyzed independently. The cDNA for each experiment can be indexed with a hexamer or octamer barcode, so that these experiments can be pooled into a single lane for multiplexed sequencing.
- 6. Polymerase Chain Reaction PCR is a rapid, automated technique used for the amplification of specific DNA sequences, invented by Kary B Mullis in 1983, and for which he won the Nobel Prize in Chemistry in 1993. PCR has gained over nucleic acid based detection techniques due to its simplicity, specificity, rapidity and sensitivity. In this technique only the DNA of the organism is examined, not the entire viable microorganism, as a result, the pathogenic microorganism can also be evaluated.Valuable genetic information about the microorganisms can be obtained quickly. PCR has become an essential tool in research laboratories and is also creating an impact in diagnostic laboratories.
- 8. Agarose Gel Electrophoresis Electrophoresis is a technique used in the laboratory for separating charged molecules. DNA is negatively charged and it can be moved through an agarose matrix by means of electric current.Shorter molecules migrate more easily and move faster than longer molecules through the pores of the gel and this process is called sieving.The gel might be used to look at the DNA in order to quantify it or to isolate a particular band.The DNA can be visualized in the gel by the addition of ethidium bromide. It is an intercalating agent which intercalates between nucleic acid bases and allows the convenient detection of DNA fragments in gel. When exposed to UV light, it will fluoresce with an orange color.After the running of DNA through an EtBr-treated gel, any band containing more than ~20 ng DNA becomes distinctly visible under UV light.The migration rate of the linear DNA fragments through agarose gel is proportional to the voltage applied to the system.As voltage increases, the speed of DNA also increases. But voltage should be limited because it heats and finally causes the gel to melt. https://www.cleaverscientific.com/applications/agarose-gel- electrophoresis-of-dna/
- 9. Elution of DNA Elution describes the extraction of specific bands of DNA from agarose gels in which they are separated through electrophoresis. The first step in extracting DNA is identifying the DNA band which is to extract, by illuminating under UV light. Recovery of DNA from agarose gels by electrophoresis onto DEAE- cellulose membrane is one of the rapid and effective methods. Electro elution is a rapid method for the successful isolation of DNA especially for larger DNA fragments, where the gel fragment containing the DNA band is cut out of the gel and placed into a dialysis bag with some buffer. The bag is then kept into a gel box, which contains the same buffer, and then subjected to an electric current. The extracted DNA precipitates out from the solution. Low melting point agarose is widely employed for the separation of DNA from agarose. Low melting point agarose melts at a lower temperature than standard agarose since it does not denature DNA structure. https://www.gbiosciences.com/GET- AGAROSE-DNA
- 10. RadiolabelingTechnique The ability to label nucleic acids is one of the most fundamental tools in molecular biology techniques. Radiolabeling is one of the best methods of choice for the most sensitive when it would be difficult to visualize a nonradioactive label, such as the gel mobility shift assay, where the probe remains within the gel matrix. Radioactive tracers have the ability to detect small quantities of substances of interest. In case of radio labeling 16S ribosomal sequence, the specific sequence is in tiny amount compared to the large genomic size of the organism.The direct measurement methods such as ultraviolet absorption, staining with specific dyes are not applicable in most cases due to the limited sensitivities of the methods. Modern techniques such as autoradiography, phosphor imaging and liquid scintillation counting techniques are recently applied for detecting the radioactive tracers
- 12. Restriction Digestion Restriction enzymes are endonucleases which cleave double-stranded DNA at specific oligonucleotide sequences.The specific sites at which they cleave the nucleic acids in order to generate a set of smaller fragments are called restriction sites.The natural function of restriction enzymes in bacteria may be the destruction of foreign DNA that may enter the bacterial cell. But the cells own DNA is not cleaved by these restriction enzymes.This self protection is achieved by the help of the specific DNA methyltransferase enzyme which will methylates the specific DNA sequence for its respective restriction enzymes by transferring methyl groups to adenine or cytosine residues to produce N6-methyladenine or 5- methylcytosine. An interesting feature of restriction endonuclease is that they commonly recognize recognition sequences that are mostly palindromes - they shows the same forward (5' to 3' on the top strand) and backward (5' to 3' on the bottom strand) sequences.The DNA fragments of varying length can be separated by gel electrophoresis and stained with ethidium bromide and can be photographed for future studies.
- 13. Southern Blotting DNA fragments obtained by restriction digestion and separation on gel can be transferred from the gel by blotting to nitrocellulose or nylon membrane that binds the DNA.The DNA thus bound to the nitrocellulose membrane is converted to the single-stranded forms (denaturation) and then treated with radioactive single-stranded DNA probes.These will hybridize with the homologous DNA, if present in the sample, to form radioactive double stranded segments. Finally the bands are visualized by autoradiography with x-ray film or by phosphor imaging techniques.This highly sensitive technique for identifying DNA fragments by DNA-DNA hybridization is called Southern blotting technique.
- 15. Walter Gilbert and Frederick Sanger In both Sanger and Maxam-Gilbert sequencing, the general principle is to reduce the DNA to four sets of labeled fragments.The reaction producing each set is base-specific, so the lengths of the fragments correspond to positions in the DNA sequence where a certain base occurs. For example, for an oligonucleotide with the sequence pAATCGACT, labeled at the 59 end (the left end), a reaction that breaks the DNA after each C residue will generate two labeled fragments: a fournucleotide and a seven- nucleotide fragment; a reaction that breaks the DNA after each G will produce only one labeled, five-nucleotide fragment. Because the fragments are radioactively labeled at their 59 ends, only the fragment to the 59 side of the break is visualized. The fragment sizes correspond to the relative positions of C and G residues in the sequence. When the sets of fragments corresponding to each of the four bases are electrophoretically separated side by side, they produce a ladder of bands from which the sequence can be read directly (Fig. 8–33).We illustrate only the Sanger
- 16. DNA sequencing by the Sanger method.This method makes use of the mechanism of DNA synthesis by DNA polymerases (Chapter 25). (a) DNA polymerases require both a primer (a short oligonucleotide strand), to which nucleotides are added, and a template strand to guide selection of each new nucleotide. In cells, the 39-hydroxyl group of the primer reacts with an incoming deoxynucleoside triphosphate (dNTP) to form a new phosphodiester bond. (b) The Sanger sequencing procedure uses dideoxynucleoside triphosphate (ddNTP) analogs to interrupt DNA synthesis. (The Sanger method is also known as the dideoxy method.) When a ddNTP is inserted in place of a dNTP, strand elongation is halted after the analog is added, because it lacks the 39-hydroxyl group needed for the next step.
- 17. (c) The DNA to be sequenced is used as the template strand, and a short primer, radioactively or fluorescently labeled, is annealed to it. By addition of small amounts of a single ddNTP, for example ddCTP, to an otherwise normal reaction system, the synthesized strands will be prematurely terminated at some locations where dC normally occurs. Given the excess of dCTP over ddCTP, the chance that the analog will be incorporated whenever a dC is to be added is small. However, ddCTP is present in sufficient amounts to ensure that each new strand has a high probability of acquiring at least one ddC at some point during synthesis. The result is a solution containing a mixture of labeled fragments, each ending with a C residue. Each C residue in the sequence generates a set of fragments of a particular length, such that the differentsized fragments, separated by electrophoresis, reveal the location of C residues. This procedure is repeated separately for each of the four ddNTPs, and the sequence can be read directly from an autoradiogram of the gel. Because shorter DNA fragments migrate faster, the fragments near the bottom of the gel represent the nucleotide positions closest to the primer (the 59 end), and the sequence is read (in the 59S39 direction) from bottom to top. Note that the sequence obtained is that of the strand complementary to the strand being analyzed.
- 18. Denature Strategy for automating DNA-sequencing reactions. Each dideoxynucleotide used in the Sanger method can be linked to a fluorescent molecule that gives all the fragments terminating in that nucleotide a particular color. All four labeled ddNTPs are added to a single tube. The resulting colored DNA fragments are then separated by size in a single electrophoretic gel contained in a capillary tube (a refinement of gel electrophoresis that allows for faster separations). All fragments of a given length migrate through the capillary gel in a single peak, and the color associated with each peak is detected using a laser beam. The DNA sequence is read by determining the sequence of colors in the peaks as they pass the detector. This information is fed directly to a computer, which determines the sequence.
- 19. Applications of 16S Ribosomal RNA in Microbiology 1. 16S rRNA gene sequencing has been established as the “gold standard” for identification and taxonomic classification of bacterial species. 2. Comparison of the bacterial 16S rRNA sequence has been emerged as a valuable genetic technique and can lead to the recognition of novel pathogens such as Mycobacterium species. 3. The hyper variable regions of 16S rRNA gene sequences provide species-specific signature sequences useful for bacterial identification. 4. In medical microbiology, 16S rRNA sequencing serves as a rapid and cheap alternative to phenotypic methods of bacterial identification 5. It is also capable of reclassifying bacteria into completely new species, or even genera. 6. The sequencing techniques can be used to describe new species that have never been successfully cultured in laboratories.