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.

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

4. https://ww
w.researchg
ate.net/figu
re/Bacterial
-16S-rRNA-
secondary-
structure-E-
coli-
sequence-
Each-
hairpin-
loop-in-
a_fig6_3032
86740

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.

7. https://www.
clinisciences.
com/en/buy/c
at-
conventional
-pcr-
3473.html

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

11. https://www.nature.co
m/articles/s41467-019-
08942-3

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.

14. https://www.onli
nebiologynotes.
com/southern-
blotting-
principle-
procedure-
application/

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.

20. Reference
https://www.sciencedirect.com/topics/medicine-and-
dentistry/complementary-dna
https://www.gbiosciences.com/GET-AGAROSE-DNA
http://vlab.amrita.edu/?sub=3&brch=76&sim=1421&cnt=1
https://en.wikipedia.org/wiki/DNA_sequencing