CLASSIFICATION OF PATHOGENS ON THE BASIS OF RNA TYPING

Taxonomy
The branch of biology concerned with the classification of organism into groups based on
the similarities of structure and origin
OR
The scientific classification of organism into especially named group on the basis of
shared character or evolutionary relationship inferred from fossil record or genetic
analysis
Phylogeny
A phylogeny is a representation of organisms based on the evolutionary relationship
OR
The evolutionary history of a taxon is called phylogeny
Phylogenetic tree of life
Phylogenetic tree is a branching diagram or tree showing the inferred evolutionary
relationship among various biological species based upon similarities and difference in
their physical and genetic character.
Classification system of organism
Usually there are two classification system
1. Five kingdom system.
Traditional system of classification of organism based on similarities in their
morphological, developmental and nutritional character.
This system was proposed by the Robert Whittaker in 1969 on the basis of
Cell type (simple or complex)
Their ability to make food
The number of cells present in their body
Five kingdom system

Three domain system

Three domain system of classification of living organism was introduced by Carl Woese
in 1977 on the basis of molecular evidences ( small subunit 16s rRNA)
Three domain
system
Archaea domain
(archaebacteria
Kingdom)

Bacteria domain
(eubacteria
kingdom

Protista kingdom

Eukarya domain

Fungi kingdom

Plantae kingdom

Animalia kingdom

Structure of Ribosome
Ribosome is a complex structure composed of rRNAs and proteins and is therefore called
as ribonucleoprotein
Each ribosome has two subunit :
1. Smaller subunit bind to mRNA
2. Larger subunit bind to tRNA and amino acid
Prokaryote
Prokaryote have 70s (Svedberg) ribosome having:
1. small 30s which have SSU 16s rRNA ( 1540 nucleotide and 21 r-protein)
2. Large 50s which have SSU 5s rRNA (120 nucleotide) and 23s rRNA ( 2900
nucleotide) and 31 r-protein
Eukaryotes
Eukaryotes have 80s ribosome that have:
1. Small 40s that have 18s SSU rRNA
2. Large 60s that have SSU 5s and 28s rRNA and 5.8s rRNA
RNA TYPING
RNA typing is a culture independent technique which involved the extraction of DNA or
RNA followed by amplification of 16s rRNA encoding DNA (rDNA) or 16s rRNA for the
identification, classification and phylogenetic diversity.
16S rRNA gene sequence analysis is considered as gold standard in the bacterial
identification
Advantages of 16s rRNA

Universal presence
Easy accessibility
PCR amplification
Rare lateral gene transfer
Broad coverage of taxa between domain and species
High informative nature and constant in function
Not enough variation
Not encode any virulence factor

 Variable rate of change in sequence at different position

Sequence Homology
Homology between protein and RNA or DNA sequence are described in terms of
shared ancestry. Two segments of DNA can have shared ancestry because of
speciation or duplex event.
Sequence Homology is performed on the bases of DNA or RNA
In 1960 Sequence Homology on DNA bases is performed using mol% G+C
content or DNA-DNA hybridization according to which:
1. Two strain of distinct phenotype show >70% hybridization are said to be members
of same specie
2. Two strain of distinct phenotype show <70% hybridization are said to be members
of different specie
Difficulty in conducting DNA hybridization and inability to assess speciation are
draw back of the DNA.
16S rRNA gene homology analysis was performed using the smart gene IDNS
(integrated database network system) and NCBI ( national centre for biotechnology
information) Genbank database. Major variable regions of gene are in 1st 500/1600
bp of 16s rRNA gene
1. Sequence homology of 16s rRNA <99% of isolate to known taxa indicate novel
specie
2. Sequence homology <95%= novel genus
3. Sequence homology <87% = novel family
4. Sequence homology <78%= novel order

Procedure to perform 16s rRNA gene sequence analysis

1. Harvesting: organism can be harvested from plate, broth or non- cultured
organism identification and classification
2. Extraction of genome: using sonification, bead beater and sedimentation
techniques for 16s rRNA

Extraction of total genome

Preparation with shotgun DNA library in bacteriophage
Screening by hybridization with a 16s rRNA specific probe
Sequence determination from clone of 16s rRNA gene

By using the PCR the laborious method of 16s rRNA gene sequencing can be avoided
By using the PCR 16s rRNA gene fragment amplified selectively and avoid mixed
DNA gene library formation
Cloning of cDNA transcribed from 16s rRNA with reverse transcriptase (smaller size,
more rigorous nucleic acid extraction technique )
Formation of chimeras is the draw back of this technique
PCR Amplification
The amplification of 16s rDNA is carried out selectively from genomic DNA of
or after the reverse transcription of rRNA by PCR with oligonucleotide primers
annealing to conserved region of 16s rRNA gene.
Cloning of 16s rDNA PCR product
Cloning of 16s rDNA PCR product is done using the different primer kits

 Reamplification of cloned PCR products with Primers uni 3 and rev 3

Purification of PCR products
Purification of PCR products using Endonuclease and separation on gel
electrophoresis ARDRA( amplified ribosomal DNA restriction analysis)
technique using the enzyme hinf1. this is RFLP analysis of PCR generated r DNA
fragment.
Sequencing of the 16s rRNA
Sequencing of the 16s rRNA is done using a cycle sequencing kit. ARDRA based
purification try to resolve specie difference in the sequence of amplified 16s
rRNA.
Comparative analysis
After sequencing the comparative analysis is performed using the information in
the gene bank. After analysis a nucleic acid accession number is allotted to
sequenced microbe like
(The important thing to remember is that we can isolate the 16s rRNA either from
DNA or directly can extract from rRNA then producing the complementary DNA
for further processing as shown in schematic diagram.)
Pitfall of different step
There are certain pitfall at each step that could lead erroneous result these are:
1. Sample collection: sample collection procedure and handling can cause variation
in sequence analysis
2. Cell lysis and Extraction of DNA
Insufficient disruption bias the composition because DNA not released
Rigorous condition cause fragmented NA
Chimeras formation due to artifact
3. Cell lyses and RNA extraction: for RNA extraction RNase are used that can
cause degradation
4. PCR amplification
Inhibition by co extracted contaminants
Differential amplification ( hybridization efficiencies for mixed 16s rDNA
template )
Formation of artefact(chimerase,mutation)
Separation of amplified 16s rRNA after amplification the separation of amplicon
of same length but different sequence can also cause problem
Analysis of 16s rRNA sequence data the ultimate goal of PCR-mediated analysis
of 16s rRNA molecule is the retrieval of sequence information used in diversity.
The quality of result of comparative 16s rRNA depends on the available dataset
If there is low similarity of 16s rRNA gene to known sequence then classification
is phylogenetic difficult. So this novel organism is placed in new taxa.