Advanced molecular biology in Dental

Research: a clinician's perspective

from sample to DNA, RNA & Proteins

Dr Rahul Pandey, PhD

Scientist, DHR-MRU
KG Medical University
What?
It is application based science where the
advances in parallel field of sciences like physics,
chemistry is used to study the composition,
structure and interactions of cellular molecules –
such as nucleic acids and proteins – that carry out
the biological processes essential for the
cell's functions and maintenance
Why?
Cell growth, migration, and differentiation in body (here
craniofacial structures) are controlled by the genetic
constitution of an individual.

Any abnormality in genetic constitution or its functioning

results in pathology known as genetic disorders.

Interference with DNA is utilized for the benefit of human

health and needs.
RNA
is Ribonucleic acid, a biomolecule found
both outside and inside cell and virus, and
useful in and as :
1. Transcription and Translation
2. Genetic Material
3. Enzyme
4. Regulator of various cell
5. processes

Types of RNA?
Messenger RNA (mRNA): the coded instructions by the mRNA
Transfer RNA (tRNA) is responsible for bringing amino acids.
Ribosomal RNA (rRNA) is a component of the ribosome factory
Central Dogma of Biology
Central Molecular Biology Technologies :

1. Polymerase Chain Reaction

2. Centrifugation

3. Hybridization

4. Site Directed Nuclease Activity

5. Gel Electrophoresis

6. Spectrophotometry

7. Sequencing

8. Flowcytometry
Polymerase Chain Reaction (PCR)

1. It is behind applications such as genome sequencing,

gene expressions in recombinant systems, the study of
molecular genetic analyses, including the rapid
determination of both paternity and the diagnosis of
infectious disease.

2. PCR enables the in vitro synthesis of nucleic acids through

which a DNA segment can be specifically replicated in a
semi-conservative way.
Types of PCR
Hybridization

The annealing/binding of two complimentary molecules to each other

Examples
Important Applications
FISH
CGH
ELISA
ChIP
SNP arrays
Methylations arrays
Centrifugation
The centrifuge works using
the sedimentation principle, where the
centrifugal acceleration causes denser
substances and particles to move
outward in the radial direction. At the
same time, objects that are less dense
are displaced and move to the centre.

Gel Electrophoresis

Separates :
Large molecules from small
Based on electrical charge

DNA molecules are negatively charged

hence move towards anode
Sequencing
is the process of determining the exact sequence of nucleotides within a nucleic
acid molecule
FlowCytometry
Technology that is used to analyse the physical and chemical
characteristics of particles in a fluid as it passes through at least one
laser.
 APPLICATIONS
1. Gene Therapy

Can be Direct or
Indirect

Through:
Virus
Liposomes
Direct DNA

Tried in :
Salivary glands to
correct systemic single
gene disorders.
I. Eg GH, Insulin, H3,
Aquaporins
II. Sjogren’s Syndrome
III. Pain
IV. P53 gene
2. Immunization

 They are generally DNA Vaccines

 Delivered via Plasmids

Examples:
I. Plasmids containing fimbrial gene for P. Gingivalis leadinding to
production of antibodies. Potentially useful in Periodotitis and
Dental Caries
3. Recombinant DNA technology

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory

methods of genetic recombination (such as molecular cloning) to bring together
genetic material from multiple sources, creating sequences that would not
otherwise be found in the genome.

Example
I. Production of transgenic strains of Streptococcus mutans which lack the
specific gene required to produce decay.

4. Pre-Natal & Post Natal Diagnosis/Identification

I. Cleft Lip/Palate

II. Malocclusion

III. Periodontal Diseases

Cleft Lip & Palate

1) Autosomal dominant syndromes: Apert Cleidocranial dysostosis Hay-Wells

Treacher Collins Vander Woude Oculodentodigital
2) Autosomal recessive syndromes: Cerebro-costo-mandibular Dubowitz Mohr
Robert
3) X-linked inheritance: Oro-facial-digital Oto-palato-digital
4) Chromosomal disorders: Mutation in 3p arm, 5p arm, 9p arm and 18q arm
Trisomy 4p, 9p Trisomy 13 Trisomy 18

5. FORENSIC ODONTOLOGY

1. Identification/DNA profiling
2. Sex Determination
3. Patternity/Family Tree
4. Anthropological studies as Ethnicity, Migration patterns
Genetics in Periodontal Diseases
RECENT ADVANCES

1) Biochips

2) Human cloning

3) Transcriptome analysis

4) Metabolomics

5) Proteomics

6) Nutrigenomics
BIOCHIPS/KITS

MyPerioPath®
a. Detects (from bacterial DNA) the specific bacteria known to cause
periodontal inflammation and destruction
b. Eleven species fall within this group
c. Sub–divides these bacteria into “risk” groups based on known
risk/virulence properties: high, moderate, low
d. Determines concentration/bacterial load. (e.g., inflammatory burden).
e. Helps to determine therapy options based on bacterial risk assessment

MyPerioID® PST®
a. Detects (from human DNA) genetic variation/polymorphism within the IL–1
gene
b. IL–1 is a major inflammatory mediator
c. 30% to 35% of the U.S. population has this genetic variation
d. IL–1 positive individuals tend to have more aggressive and more severe
infections
e. Determines patients that are most susceptible to severe disease, especially if
the patients smoke
f. This genetic variation can increase risk for severe disease or tooth loss by 2 to
7 times when present
OraRisksm HPV
a. HPV (Human Papilloma Virus) is now a known risk factor for oropharyngeal
carcinoma of the mouth and throat
b. HPV can cause cancer of the mouth and throat regions
c. Provides information to triage patient for high risk HPV without or with
lesions
d. Detects those with HPV infections
e. Genotypes HPV based on known risk: high, low, unknown
f. Should be included in risk assessment for oral cancer in conjunction with
clinical exam, adjunctive tests, and history of alcohol and tobacco.
Kits for Measuring Caries Susceptibility/Activity