The development of human craniofacial complex is an
extraordinary combination of evolutionary genetics,
embryogenesis and precise disposition of 4 germ
layers, morphogenesis, organogenesis and maturation.
Of the 25,000 genes in human genome around 17,000
genes are implicated in craniofacial development.
Hence a thorough
knowledge of genetics is
important to know the intricacies of craniofacial
growth and development.
“The genetic messages encoded within our DNA
molecules will provide the ultimate answers to the
chemical underpinnings of human existence and in the
21st century all biology will be gene based and all
biologists will be geneticists”
Science of genetics:
Concerned with inheritance of traits, whether
normal or abnormal and with the interaction of
genes and the environment.
The branch of genetics that deals with hereditary
transmission and variation on the molecular level.
A threadlike linear
strand of DNA and
in the nucleus of
eukaryotic cells that
carries the genes
and functions in the
According to position of
• A consistent numbering system is essential for
mapping chromosomes. In Paris 1971 a mapping
system known as the International System for
Cytogenetic Nomenclature (ISCN) was established.
• THE RULES OF THE ISCN
1. Numbering of a chromosome begins at
2. Chromosomes are assigned a long arm
and a short arm, based on the
position of their centromere. The
shorter arm of the chromosome is
known as the p, or petite arm, from
the French word for "small." The
longer arm is known as the q.
Chromosomal regions that are
present on the short arm will begin
with the designation p, while regions
on the long arm will begin with q.
3. By convention, the p arm of the
chromosome is always shown at the
top in a karyotype.
Arm Region Band Subband
4. Each arm of the chromosome is divided into
regions. The numbers assigned to each region
gets larger as the distance from the
centromere to the telomere increases
5. Depending on the resolution of the staining
procedure, it may also be possible to detect
additional bands within each region, which are
designated by adding a digit to the number of
the region, increasing in value as the distance
from the centromere increases.
Ideogram of the medium-sized sub-metacentric chromosome 12 (400)
• The position of the centromere separates the p
and q arms (hatched area).
This ideogram describes the pattern of Giemsa
staining at a fairly low resolution (about 400
total number of bands in a karyotype).
• At this resolution, the long q arm of chromosome
12 is subdivided into two main regions: 12q1 and
12q2. Region 12q1 is further subdivided into five
sub-regions: 12q11 through 12q15, each of which
corresponds to a band detected by Giemsa
• We refer to these subdivisions as "12q one-one"
through "12q one-five" (not as "12q eleven"
through "12q fifteen"). The more distal 12q2
region is subdivided into sub-regions 12q21
through 12q24. Sub-region 12q24 is further
subdivided into regions 12q24.1 through 12q24.3.
Ideogram of the medium-sized sub-metacentric
chromosome 12 at higher resolution
• When chromosomes that are in
prometaphase are stained a higher
resolution is obtained, because
prometaphase chromosomes are slightly
less condensed than metaphase
• At this higher level of resolution
approximately 850 bands are
distinguishable in a karyotype.
Either of two parallel filaments
joined at the centromere which
make up a chromosome, and which
divide in cell division, each going
to a different pole of the
dividing cell and each becoming a
chromosome of one of the two
• An autosome is a nonsex chromosome. It is
an ordinarily paired type
of chromosome that is
the same in both sexes
of a species. For eg, in
humans, there are 22
pairs of autosomes.
chromosomes are usually
referred to as sex
Pair of chromosomes, one
inherited from each parent,
that have corresponding
gene sequences and that
pair during meiosis
The smallest human chromosome is
chromosome 21 and the largest one is
chromosome 1. This is one reason why Down‟s
syndrome (trisomy 21) is the most common
By comparison to the X chromosome, the much smaller Y
chromosome has only about 26 genes and gene
families. Out of 26 only 9 gene families are involved in
sperm production .
Only one of the Y chromosome genes, the SRY gene, is
responsible for male anatomical traits.
When any of the 9 genes involved in sperm production are
missing or defective the result is usually very low sperm
counts and subsequent infertility.
The molecule that
information in the
nucleus of cells. It
and behaviour of the
A polymeric constituent of all living
cells and many viruses, consisting
of a long, usually single-stranded
chain of alternating phosphate and
ribose units with the bases
adenine, guanine, cytosine, and
uracil bonded to the ribose. The
structure and base sequence of
RNA are determinants of protein
synthesis and the transmission of
A segment of a DNA molecule that
It is the biological unit of heredity
transmitted from parent to progeny.
Different forms of genes at
the same locus or position on
– If both copies of genes
– If the two copies of genes
The position that a
given gene occupies
on a chromosome
The largest gene identified so far is the
dystrophin gene (responsible for Duchenne‟s
muscular dystrophy) . It has 80 coding regions
and encodes only a 3,700 amino acid-long protein.
Chromosome 1 has the most genes (2968), and the Y
chromosome has the fewest (231).
• Mode of inheritance:
• If the trait of the disease manifests itself
when the affected person carries only one copy
of the gene responsible – along with one normal
• If two copies of defective genes are required
for expression of trait
– Genetic constitution
of an individual, and
refers to specified
gene loci or to all loci
– Specified character
or to all observable
The process by which a cell divides and produces two
daughter cells identical to parent cell.
The process of cell division in sexually reproducing
organisms that reduces the number of chromosomes
in reproductive cells from diploid to haploid, leading
to the production of gametes in animals and spores in
1. Any genetically determined characteristic.
2. A distinctive behavior pattern.
Autosomal Dominant Disorders
In this type of disorder, the mutant gene is
located on one chromosome of the autosomal
chromosome pair and the comparable gene or the
(partner chromosome) is normal e.g. Treacher Collin
Autosomal Recessive Disorders
Autosomal recessive disorders are inherited
from clinically .normal parents who have the same
mutant gene on one chromosome of the homologus
chromosome pair (i.e. each not only a single dose
of the mutant gene).
MUTANT GENE NOT EXPRESSED
X-linked Recessive Disorder
Genes located in the X-chromosomes represent only
in a single dose in the XY males.
These are X-linked recessive disorder, is fully
expressed in a male who has only a single X-linked
A female with the single X-linked recessive gene is
phenotypically normal carrier because of the presence of
a normal partner gene at the same locus on the second Xchromosome.
Therefore with some exception only males are affected
with X-linked disorders.
The mutant gene is either transmitted from a carrier
mother to her affected son, or the affected male represent a
fresh mutation. Eg. Haemophilia.
X-linked Dominant Disorders
In this group the X-linked gene is a
dominant gene, expressing itself in a single
dose. Therefore both females and males in
each generation will be affected.
• Gregor Johann Mendel (18221884) was an Augustinian
priest and scientist, and is
often called the father of
genetics for his study of the
inheritance of traits in pea
plants. Mendel showed that
the inheritance of traits
follows particular laws, which
were later named after him.
– Studied segregation of
traits in the garden pea
(Pisum sativum) beginning in
– Presented his paper on
“Experiments with Plant
Hybridization” in 1866
Why Mendel chose pea plant?
The garden pea was easy to cultivate and had
relatively short life cycle
The plant had distinguishing characteristics such as
flower color and pea texture
Because of its anatomy, pollination of plant was easy
to control and cross fertilization could be
Focused on 7 well-defined garden pea traits by
crossing different phenotypes one at a time
Counted offspring of each phenotype and analyzed
the results mathematically
Mendel‟s first law:
The Principle of
The two members of a heredity
factor pair segregate from each
other in the formation of gametes.
Two members = alleles
Hereditary factor = gene
Mendel‟s second law:
The Principle of Independent Assortment:
During gamete formation, members of one heredity factor
pair segregate into gametes independently from other
heredity factor pairs.
Two members = alleles
Hereditary factor = gene
The phenotype of the
between those of the
two homozygotes. The
phenotype shows the
ratio of 1:2:1
AREAS OF GENETICS
Mitosis & Meiosis
Structure of DNA
Chemistry of DNA
DNA cloning & genomics
DNA mutation & repair
Hardy Weinberg equilibrium
Assumptions of equilibrium
STEPPING INTO ERA OF MOLECULAR
• Evidence for DNA as
In 1928 E.Griffith in his
which converted nonlethal strains to lethal
research that supported
the notion that DNA is
RNA as genetic material
Many of the viruses carry RNA as genetic material
This RNA can be:
Single stranded-measles ,rubella ,polio
Retroviruses-HIV1 & HIV2
• A prion is a nonliving, self-replicating infectious agent made
of protein. It can reproduce with the aid of its host's
biological machinery, like a virus. "Prion" is short for
"proteinaceous infectious particle.“
Prions found in animals exclusively infect the brain, are fatal
• Prions are responsible for the outbreak of Mad Cow Disease
in Britain .
• The prion protein was not isolated until 1982, when Stanley B.
Prusiner discovered it and coined the term.
• In 1953 Watson & Crick
a paper in
Nature which suggested
the structure of DNA.
This paper which first
put forth the correct
structure of DNA is a
milestone in modern era
of molecular genetics
• DNA is made of chains
• It is a double helix with
outside and the bases on
• The diameter of helix is
• The two strands of DNA
are antiparallel to each
5‟phosphate and other
has 3‟hydroxyl group
Nucleotides are made of 3 components:
• A nucleotide is formed
when base attaches to
the 1‟ end of the carbon
phosphate attaches to
the 5‟ of same sugar
• Nucleotides take their
present in it
together by phosphodiester
bond formed between the 5‟
C of one nucleotide & 3‟ OH
group of adjacent molecule
• Sugar+ base=nucleoside
thymine with 2 hydrogen
guanine with 3 hydrogen
There is 1:1 correspondence between the purines and
pyrimidines i.e. amount of adenine equals thymine and
cytosine equals guanine
RNA does not follow this rule that is amount of
adenine & uracil are not equal neither the amount of
cytosine and guanine
• Why not 2 purines or pyrimidines
Not enough space (20 Å) for two
purines to fit within the helix and
pyrimidines to get close enough to
each other to form hydrogen
bonds between them.
• But why not A with C and G with
Only with A & T and with C & G
between them. The ability to
form hydrogen bonds makes the
• Types of DNA
• Most common
• Right handed helix
• Bases are perpendicular
to main axis
• 10 bases per turn
• Helix diameter 20A
If the water in DNA
increases to 75% it
changes to A form
• Bases tilt wrt long axis
• 11 bases per turn
• Helix diameter 23A
• Left handed helix
• Backbone formed zigzag
hence called Z
• Found in conditions when
cytosine is methylated or
there is high salt
• Helix diameter 18A
• 12 bases per turn
• The process of DNA replication provides an answer of
how genetic information is transmitted from one
generation to other
• The synthesis of both complimentary DNA strands
occurs from 5‟-3‟ end
• 5 main enzymes involved in this process
-DNA polymerase 1
-DNA polymerase 3
• Continuous replication takes place on the 3‟-5‟ end and
this strand is called leading strand
• Discontinuous form of replication takes place on the
5‟-3‟ end and this strand is referred as lagging strand
• The discontinuous replication takes place in small
fragments called Okazaki fragments (after R.Okazaki
who first saw them)
Why RNA is used to prime DNA synthesis?
Probably, making use of RNA primers lowers the
error rate of DNA replication because priming is
basically an error prone process since nucleotides are
initially added without a stable primer configuration.
Therefore a RNA primer is first put and removed.
Resynthesis by polymerase I is in a much more stable
primer configuration and thus makes very few errors
• Origins of replication
1. Replication Forks: hundreds of Y-shaped
regions of replicating DNA molecules
where new strands are growing.
Parental DNA Molecule
• Origins of replication
2. Replication Bubbles:
Hundreds of replicating bubbles
Single replication fork (bacteria).
• Strand Separation:
1. Helicase: enzyme which catalyze the
unwinding and separation (breaking HBonds) of the parental double helix.
2. Single-Strand Binding Proteins:
which attach and help keep the separated
• Strand Separation:
3. Topoisomerase: enzyme which relieves
stress on the DNA molecule by allowing
rotation around a single strand.
1. RNA primers: before new DNA strands can
form, there must be small pre-existing
primers (RNA) present to start the addition of
new nucleotides (DNA Polymerase).
2. Primase: enzyme that polymerizes
(synthesizes) the RNA Primer.
• Synthesis of the new DNA Strands:
1. DNA Polymerase: with a RNA primer in place,
DNA Polymerase (enzyme) catalyze
synthesis of a new DNA strand in the 5’ to 3’
2. Leading Strand: synthesized as a
single polymer in the 5’ to 3’ direction.
3. Lagging Strand: also synthesized in the 5’ to 3’
direction, but discontinuously against overall
direction of replication.
4. Okazaki Fragments: series of short
segments on the lagging strand.
5. DNA ligase: a linking enzyme that
catalyzes the formation of a covalent bond
from the 3’ to 5’ end of joining stands.
Example: joining two Okazaki fragments together.
Okazaki Fragment 1
Okazaki Fragment 2
The process of making
an identical copy of a
using existing DNA as a
synthesis of new DNA
both the strands are
act as template for new
One strand of DNA act
as template for the
formation of daughter
of daughter DNA
Some parts of parental
DNA are conserved and
other are new daughter
UPDATED VERSION OF CRICKS CENTRAL DOGMA
It is the process whereby
the DNA sequence in a
gene is copied into mRna.
It is the first step in
Types of RNA
• mRNA:It carries the information from the DNA to
the ribosomes in cytoplasm
• tRNA: It brings the amino acids to the ribosomes
where protein synthesis takes place
• rRNA:It is a structural and functional part of
• The process of transcription is controlled by RNA
polymerase. It adds up the appropriate
complementary ribonucleoside to the 3‟ end of the
• The transcribed mRNA strand is called sense strand
and the DNA template strand is called antisense
• The DNA region that RNA polymerase associates
immediately before beginning transcription is known
as promoter.It contains information for transcription
initiation and are the sites where the gene expression
• The polymerase moves down the DNA until the RNA
polymerase reaches a stop signal or terminator
These are segments of DNA within genes that are
transcribed into RNA but are not translated into
They are removed from the RNA before its transport
Discovered by Sharp & Roberts in 1977
The segments between introns are called exons
• Posttranscriptional modifications
The non coding introns are excised & the non
continuous exons are spliced together
Methylated guanine nucleotide is added to the 5‟
end of the molecule to facilitate mRNA transport to
the cytoplasm & its attachment to ribosome.
The cleavage of the 3‟ end of the mRna molecule
from the DNA involves the addition of approximately
200 adenylate residues,the so called polytail, after
cleavage of the RNA .It facilitates transport of mrna
• The formation of
proteins from RNA is
• All proteins are
synthesized from only
20 amino acids
• Amino acids consist of:
• Transfer RNA is shaped
like a clover leaf with
three loops. It contains
an amino acid
attachment site on one
end and a special section
in the middle loop called
the anticodon site. The
anticodon recognizes a
specific area on a mRNA
called a codon
Attachment of amino
acid to Trna
The function of Trna is to
ensure that each AA
incorporated into a protein
corresponds to a particular
codon in Mrna
The amino acid attach to
Trna by enzyme known as
aminoacyl-Trnasynthetases and such RNA
is said to be „charged‟
• Translation can be divided into 3 stages
Initially there is formation of initiation complex :
30S subunit of RNA
Charged methionine(AUG) Trna
Initiation factors(IF-2 & IF-3)
The 30S subunit has 3 sites present in it where the
A site (aminoacyl site)-First step
P site(peptidyl site)
E site(exit site)
Now the first tRna moves to the P site and 2nd tRna
gets attached to the Asite
Next imp step is peptide bond formation between the
amino acids attached to 2 tRnas
Peptidyl transferase in the 50S subunit acts as an
enzyme for the formation of bond between carboxyl
end of one & amino end of other amino acid
The next step is translocation in which an enzyme
called translocase which physically moves the mRna &
its associated tRna. So the first attached RNA moves
to E site & new one to A site
Termination of protein synthesis occur when one of
the 3 nonsense codons appear at A site. These are:
When a nonsense codon enters A site a release
factor(RF-1 or RF-2) recognizes it and cause
hydrolysis of bond between peptide chain & tRna at
After the release factor act, the ribosome has
completed the task of translating mrna to
polypeptide. Finally release of all factors and
dissociation of 2 subunits takes place
• The genetic code is the set of
rules by which information
encoded in genetic material is
translated into proteins
The unit of information is
CODON = genetic 'word„ a
triplet sequence of nucleotides
3 nucleotides = 1 codon = 1
Characteristics of genetic code
Reading in Frames
Codon is defined by the initial nucleotide from which
translation starts. For example, the string
GGGAAACCC, if read from the first position, contains
the codons GGG, AAA and CCC; and if read from the
second position, it contains the codons GGA and AAC.
Every sequence can thus be read in reading frames,
each of which will produce a different amino acid
The genetic code is degenerate that is a given amino
acid may have more than one codon.The genetic code
has redundancy but no ambiguity. For example,
although codons GAA and GAG both specify glutamic
acid (redundancy), neither of them specifies any
other amino acid (no ambiguity).
The genetic code is almost universal.
When mitochondrial mRNA from animals or
microorganisms is placed in a test tube with the
cytosolic protein-synthesizing machinery (amino acids,
enzymes, tRNAs, ribosomes) it fails to be translated
into a protein.
The reason: mitochondria use UGA to encode
tryptophan (Trp) rather than as a chain terminator.
When translated by cytosolic machinery, synthesis
stops where Trp should have been inserted.
It is a process of combining DNA of 2 different
Also called as gene cloning & genetic engineering
Recombinant DNA is a tool in understanding the
structure, function, and regulation of genes and their
Purpose of rDNA
Identification of mutations & diagnosis of affected
and carrier states for hereditary diseases
Production of large quantities of a gene product
(protein or RNA) for easier study of those molecules
Isolation of large quantities of pure protein
Insulin, factor VIII, factor IX, growth hormone
Increased production efficiency for commercially
made enzymes and drug
Correction of genetic defects in complex organisms,
Cloning describes the processes used to
create an exact genetic replica of another
cell, tissue or organism. The copied material,
which has the same genetic makeup as the
original, is referred to as a clone.
There are 3 different types
• Gene cloning, which creates
copies of genes or segments
• Reproductive cloning, which
creates copies of whole
• Therapeutic cloning, which
creates embryonic stem
cells. Researchers hope to
use these cells to grow
healthy tissue to replace
injured or diseased tissues
in the human body.
• Ian Wilmut with his
colleagues working on a
project on July 5 1996
cloned the first mammal
a sheep named Dolly
• Dolly took 277 tries to
create, and other labs
were unable to
reproduce the results.
Celebrity Sheep Has Died at Age 6
Dolly, the first mammal to be cloned
from adult DNA, was put down by lethal
injection Feb. 14, 2003. Prior to her
death, Dolly had been suffering from
lung cancer and crippling arthritis.
• Although most Finn Dorset sheep live to
be 11 to 12 years of age, postmortem
examination of Dolly seemed to indicate
that, other than her cancer and
arthritis, she appeared to be quite
• Losing the diversity of genes
• The great diseases and leading
• Ethical issue
• Process with loopholes
All the countries except
for South Africa have
In these countries only
therapeutic cloning is
DEVELOPMENTAL GENE FAMILIES
Paired-box genes (PAX)
Zinc finger genes
Signal transduction („Signalling‟) genes
Homeobox genes (HOX)
Insect bodies consist of series of repeated body
segments which differentiate into particular
structures according to their position.
Three main groups of segmentation determining
genes have been classified on the basis of their
(A) Gap mutants – delete groups of adjacent
(B) Pair-rule mutants – delete alternate
(C) Segment polarity mutants – cause portions
of each segment to be deleted and duplicated
on the wrong side.
1) Hedgehog (Vertebrates)
Hedgehog morphogens are involved in the control of
left-right asymmetry, the determination of polarity in
the central nervous system, somites and limbs, and in
both organogenesis and the formation of the skeleton.
In humans, Sonic hedgehog (SHH) plays a major role
in development of the ventral neural tube with lossof-function mutations resulting in a serious and often
lethal malformation known as holoprosencephaly where
the facial features shows eyes close together and
there is a midline cleft lip due to failure of normal
PAIRED-BOX GENES (PAX)
The mammalian Pax gene family consists of nine members
that can be organized into groups based upon sequence similarity,
structural features, and genomic organization. The four groups
Pax1 and Pax9
Pax2, Pax5, and Pax8
Pax3 and Pax7 and
Pax4 and Pax6
ZINC FINGER GENES
The term zinc finger refers to a finger-like loop projection which
is formed by a series of four amino acids which form a complex
with a zinc ion. Genes, which contain a zinc finger motif, act as
transcription factors through binding of the zinc finger to DNA.
SIGNAL TRANSDUCTION GENES
Signal transduction is the process whereby
extracellular growth factors regulate cell division
and differentiation by a complex pathway of
Mutations in many of the genes involved in signal
abnormalities. Fibroblast growth factor receptors
(FGFRs) belong to the category of signal
HOMEOBOX GENES (HOX) AND ITS IMPORTANCE
Since their discovery in 1983, the homeobox genes were
originally described as a conserved helix-turn-helix DNA
motif of about 180 base pair sequence, which is believed
to be characteristic of genes involved in spatial pattern
control and development.
The protein domain encoded by the homeobox, the
homeodomain, is thus about 60 amino acids long. Proteins
from homeobox containing, or what are known as HOX
genes, are therefore important transcription factors
which specify cell fate and establish a regional
• Begun formally in 1990, the U.S. Human Genome
Project was a 13-year effort coordinated by the U.S.
Department of Energy and the National Institutes of
Project goals were to
identify all the approximately 20,000-25,000 genes in
determine the sequences of the 3 billion chemical
base pairs that make up human DNA,
store this information in databases
improve tools for data analysis
transfer related technologies to the private sector
address the ethical, legal, and social issues (ELSI)
that may arise from the project.
In the spring of 2000 J. Craig Venter CEO of Celera
Genomics & Francis Collins Director of National Institute
Of Health‟s Human Genome Research jointly announced
the working draft of human genome .
The project originally was planned to last 15 years, but
rapid technological advances accelerated the completion
date to 2003
There are 3 billion base pairs in human genome
Around 25000-30000 genes
• Molecular medicine
• Energy sources and environmental applications
• Risk assessment
Anthropology, evolution, and human migration
• DNA forensics (identification)
• Agriculture & livestock breeding
Mutations are change in the base pair sequences of a
Causes of Mutation
Most amino acids are encoded by several different
codons. For example, if the third base in the TCT
codon for serine is changed to any one of the other
three bases, serine will still be encoded. Such
mutations are said to be silent because they cause
no change in their product and cannot be detected
without sequencing the gene .
With a missense mutation, the
new nucleotide alters the codon
so as to produce an altered
amino acid in the protein
Eg: sickle-cell disease
The replacement of A by T at
the 17th nucleotide of the gene
for the beta chain of
hemoglobin changes the codon
GAG (for glutamic acid) to GTG
(which encodes valine).
With a nonsense mutation, the new nucleotide changes
a codon that specified an amino acid to one of the
STOP codons (UAA, UAG, or UGA). Therefore,
translation of the messenger RNA transcribed from
this mutant gene will stop prematurely.
The earlier in the gene that this occurs, the more
truncated the protein product and the more likely
that it will be unable to function.
Indels involving one or two base pairs (or multiples)
can have devastating consequences to the gene
because translation of the gene is "frameshifted". by
shifting the reading frame by one nucleotide, the
same sequence of nucleotides encodes a different
sequence of amino acids. The mRNA is translated in
new groups of three nucleotides and the protein
specified by these new codons will be worthless.
TOOLS FOR MOLECULAR BIOLOGY
• 1) Restriction enzymes :
Genomic DNA can be cut into a number of
fragments by enzymes called restriction enzymes
which are obtained from bacteria. Eg. : Enzyme
• 2) Gel electrophoresis :
As DNA is negatively charged molecule, the
genomic DNA that has been digested with a
restriction enzyme can be separated according to
size and charge by electrophoresing DNA through
• Pulsed field gel electrophoresis.
• 3) Southern blotting and DNA probes :
Southern blotting allows the visualization of
individual DNA fragments.
DNA probes are useful to indicate where the
fragment of interest lies.
• 4) Northern blotting and western blotting :
Northern blotting is used to visualize RNA
fragments on to membrane.
Western blotting is used to visualize proteins.
• 5) Polymerase
chain reaction :
of DNA can be
amplified over a
within a few
hours using this
6) DNA cloning
foreign DNA into
can be used to
• 7) DNA libraries
These are pools of isolated and cloned DNA
sequences that form a permanent resource for
2 types of libraries :
– Genomic libraries -contains almost every
sequence in the genome.
– cDNA libraries
- contain sequences derived
from all mRNAs expressed in that tissue.
• 8) DNA sequencing :
Used to identify the exact nucleotide sequence
of a piece of DNA.
Polymerase Chain Reaction is an in vitro technique for the
amplification of a specific sequence of DNA Which is used for
Kary Mullis (1987)
Cetus Corporation (A Biotech Company of United States)
Nobel Prize 1993
Components of the reaction mixture
Primers (forward and reverse)
Taq DNA Polymerase
Sterile deionized water
It contains the DNA region to be amplified
Range - 1-2 µl ( for a total reaction mixture of 10 µl)
Short Single stranded oligonucleotides
They are complementary to the 5' or 3' ends of the
Range - 1 µl ( for a total reaction mixture of 10 µl)
TTAACGGCCTTAA . . . TTTAAACCGGTT
AATTGCCGGAATT . . . . . . . . . .>
<. . . . . . . . . . AAATTTGGCCAA
TTAACGGCCTTAA . . . TTTAAACCGGTT
PCR Primer Design Guidelines
Optimal length of PCR primers is 18-22 bp
Primer Melting Temperature: (Tm)
Temperature at which one half of the DNA
duplex will dissociate to become single
stranded and indicates the duplex stability.
Range - 52-58 C
Tm = 4 (G+C) + 2 (A+T)
(GCAT no. of respective nucleotides in
Presence of G or C bases within the last five bases
from the 3' end of primers
Promotes specific binding at the 3' end due to
the stronger bonding of G and C bases
Steps in PCR
Extension / Elongation
Heating the reaction to a temperature of 94-96°C
for 1-9 minutes.
94-98°C for 20-30 seconds.
Denaturation of DNA template by disrupting the
hydrogen bonds between complementary bases of
the DNA strands, yielding single strands of DNA.
50-65°C for 20-40 seconds
Stable DNA-DNA hydrogen bonds are formed
The polymerase binds to the primer-template
hybrid and begins DNA synthesis.
At this step the DNA polymerase synthesizes a
new DNA strand complementary to the DNA
template by adding dNTPs in 5' to 3' direction.
70-74°C for 5-15 minutes
To ensure that any remaining single-stranded
DNA is fully extended.
4-15°C for an indefinite time
short-term storage of the reaction
ALLELE SPECIFIC PCR
• Selective PCR amplification of the alleles to
detect single nucleotide polymorphism (SNP)
• Selective amplification is usually achieved by
designing a primer such that the primer will match
or mismatch one of the alleles at the 3‟ end of the
• It is used for DNA sequencing
• The two primers are used in the 100:1 ratio so
that after 20-25 cycles of amplification one
primer is exhausted thus single stranded DNA is
produced in the next 5-10 cycles
REAL TIME PCR
• Quantitative real time PCR (Q-RT PCR)
• It is used to amplify and simultaneously quantify a
target DNA molecule
HELICASE DEPENDENT AMPLIFICATION
Constant temperature is used rather than cycling
through denaturation and annealing/extension
DNA Helicase, an enzyme that unwinds DNA, is
used in place of thermal denaturation.
INTERSEQUENCE SPECIFIC PCR ISSP
A PCR method for DNA fingerprinting that
amplifies regions between some simple sequence
repeats to produce a unique fingerprint of
amplified fragment lengths.
A method used to allow PCR when only one internal
sequence is known.
This is especially useful in identifying flanking
sequences of various genomic inserts.
• When sequence of only one end of the desired
segment of gene is known,the primer
complimentary to the 3' strand of this end is used
to produce several copies of only one strand of the
RT-PCR (REVERSE TRANSCRIPTION PCR)
It is used to amplify, isolate or identify a known
sequence from a cellular or tissue RNA.
RT-PCR is widely used in expression profiling, to
determine the expression of a gene or to identify
the sequence of an RNA transcript.
Used to obtain 3' and 5' end sequence of cDNA transcripts
Comparison PCR - Polymerase Chain Reaction and Gene Cloning
Selective amplification of
Selective amplification of
In vitro and in vivo
segment from complex DNA
Quantity of starting material
Biological reagents required
Ligase, vector. bacteria
Time for a typical experiment
APPLICATION OF PCR
Cloning a Gene encoding a known protein
Amplification of old DNA
Amplifying cloned DNA from Vectors
Rapid Amplification of cDNA ends
Detecting Bacterial or Viral Infection
● AIDS infection
Diagnosing inherited disorders
Haemophilia A and B
Sickle cell anaemia
Blood group typing.
Problems with PCR
• Polymerase errors
Polymerase lacks exonuclease activity
• Size limitations
PCR works readily with DNA of lengths two to
three thousand basepairs
• Non specific priming
BANDAGE FOR GENETIC WOUNDS
• It is a technique for correcting defective genes that
are responsible for disease development
• There are four approaches:
1. A normal gene inserted to compensate for a
2.An abnormal gene traded for a normal gene
3.An abnormal gene repaired through selective
4.Change the regulation of gene pairs
• In the lab, a virus is stripped of its disease-causing
genes, while the genes that enable it to infect cells
• A therapeutic gene is inserted into this virus. This
allows the virus to "infect" cells with the therapeutic
gene. This viral vector is injected into the specific
• The virus attaches to the diseased cells and gets
sucked into the cell by process called endocytosis.
• The virus breaks apart inside the cell and the genetic
material from the virus enters the nucleus of the cell.
If the procedure is successful, the cell begins to
produce the proteins encoded by the newly delivered
Gene of the Moment: p53
Cancer is a major focus for gene therapy research at
The role of p53 is to keep a check on the cell cycle.
If anything damages the DNA in a cell, p53 stops the
cell cycle and tries to repair it, or if the damage is
beyond repair, it induces apoptosis. If the gene for
p53 has been mutated and no longer works, damaged
DNA and cells can go unchecked. In the case of
cancer, tumours can develop.
Many cancer gene therapies focus on repairing or
adding the p53 gene into the cancer cells in order to
Advantages of Gene Therapy
• Enabling people to have children where natural
conception is impossible (a more effective treatment
• The potential for discovering cures for incurable
diseases - leading to less pain and suffering.
• Sex selection to prevent genetic diseases.
• Increased availability of organs for transplant.
• Increased procreative autonomy (choice over some of
the genetic characteristics that one's future child
• Short Lived
– Hard to integrate therapeutic DNA into genome of
rapidly dividing cells which prevent gene therapy
from long time action
– Would have to have multiple rounds of therapy
• Viral Vectors
– patient could have toxic, immune, inflammatory
– also may cause disease once inside
• Multigene Disorders
– Heart disease, high blood pressure, Alzheimer‟s,
arthritis and diabetes are hard to treat because
you need to introduce more than one gene
EVOLUTION OF GENETICS IN
• Charles de Lourde, of England, who wrote in
“Irregularity is due . . . to heredity, where
the child inherits the jaws of one parent
and the teeth of another.”
AJO 1957 …
A STUDY OF THE FAMILY-LINE TRANSMISSION
OF DENTAL OCCLUSION
MILTON B. ASBELL, CAMDEN, N. J.
• Heredity may be an important factor in many forms of malocclusion, a
project was undertaken to determine the genetic backgrounds in a
series of cases of malocclusion in white boys, aged 9 to 14 years.
Three types of transmission may be noted, which are as
(1) Repetitive trait: This type of transmission is characterized by the
recurrence of a single morphologic trait within the family line over
2) Discontinuous or assortative trait: This type of transmission is
characterized by the recurrence of a single morphologic trait, within
the family line over several generations. This indicates a hereditary
endowment arising in either the maternal side or the paternal side,
plus the shift or possible gene recombination within the family line of
the opposite sex.
3) Mixed trait: This type of transmission is characterized by different
morphologic traits within either family line of several generations;
AJO FEB 1958 …
A REVIEW OF THE GENETIC INFLUENCE ON
MALOCCLUSION - HAROLD J. NOYES,
• I am intrigued with the science of genetics and
impressed with its tremendous growth in the
past few years, I feel that as yet it is essentially
academic with respect to the clinical practice of
orthodontics and of only occasional value as a
tool in the diagnosis and treatment of
• Only in prognosis does it have a measure of value,
and here one must be cautious when predicting
anything that could not be forecast from
comprehensive orthodontic records.
AJO AUG 1966 …
Genetic and environmental factors in dento facial morphology
FRANS ,VAN DER LINDEN
• The interaction between genetic and environmental factors starts at
conception and continues until the end of life.
• During fetal life the contact of the genetic composition with the
environment is rather limited. On the other hand, all components
outside the genes (the protoplasm of the ovum, for example) are
• A permanent interaction between genetic and environmental factors,
both of a continually altering nature, determines the dento facial
morphology in every moment of life.
• Genetic factors seem to have the greatest influence, and
environmental factors appear to be of minor importance.
• The stability of the result of orthodontic treatment depends mainly on
a new state of balance in the interaction between genetic and
AJO MAY 1972 …
Effect of molecular genetics and genetic engineering on the
practice of orthodontics
J. A. Salzmann
• Genetic engineering has opened a new approach to the
diagnosis, prevention, and control of diseases and
malformations. This holds forth great promise for the
future of orthodontics.
• Techniques of genetic engineering include amniocentesis,
chromosome karyotyping, recognition of chromosome
aberrations and their relation to specific dentofacial
anomalies and malocclusion, the aborting of harmful
genes, and the introduction of desirable genes into the
early forming embryo.
• These techniques eventually will make possible the
prevention of many antenatal, congenital, and postnatal
genetically induced dentofacial anomalies, including dental
• Malocclusion and jaw relation of genetic origin
can be successfully treated orthodontically, except
in extreme cases that involve the over-all
morphology of the bones of the face and require
surgical intervention, We modify the direction of
dentofacial growth when we correct dental
malocclusion and, therefore, can change or
forestall abnormalities of genetic origin.
• When the orthodontist corrects a malocclusion
he is, in effect, changing the genetic expression
of his patient.
Hereditary factors in the craniofacial morphology of Angle’s
Class II and Class III malocclusions
• Attempted to assess the role of heredity in the development
of Angle’s Class II and Class Ill malocclusions by comparing
craniofacial morphologic differences between parents with Class
II offspring and those with Class Ill offspring and by analyzing
the parent-offspring correlatfons within each Class II and Class
III malocclusion group.
• There appears to be a strong familial tendency in the
development of Class II and Class Ill malocclusions.
• We conclude that the hereditary pattern must be taken
into consideration in the diagnosis and treatment of
patients with these classes of malocclusion
AJODO MAR 1997 …
A heritable component for external apical root resorption in
patients treated orthodontically
• External apical root resorption (EARR) is a common and occasionally
critical problem in orthodontic patients.
Mechanical forces compress the periodontium, leading to localized
resorption of cementum that exposes dentin to destruction by
clastic activity. Factors controlling occurrence and extent of EARR
are poorly understood, but there may be a familial (genetic) factor
• Heritability estimates were fairly high, averaging 70% for three
roots, although low for the mandibular incisor, probably because
of little variation. No evidence was found for a sex or age difference
• Quantification of a transmissible component suggests it would be
useful to search for the biochemical factors controlling the familial
differences in susceptibility.
AJODO JUNE 2000…
The genetics of human tooth agenesis: New discoveries for
understanding dental anomalies-Heleni Vastardis
• The important role of genetics has been increasingly
recognized in recent years with respect to the understanding
of dental anomalies, such as tooth agenesis. The lack of any
real insight into the cause of this condition has led us to use a
human molecular genetics approach to identify the genes
perturbing normal dental development.
• With the use of “the family study” method, evidence is
produced showing that other genetic defects also contribute
to the wide range of phenotypic variability of tooth agenesis.
Identification of genetic mutations in families with tooth
agenesis or other dental anomalies will enable preclinical
diagnosis and permit improved orthodontic treatment.
AJODO APR 2011…
Incidence and effects of genetic factors on canine
impaction in an isolated Jewish population
• Introduction: The etiology of palatal canine impaction is
multifactorial and includes a genetic contribution. The aim
of this study was to find the incidence and effects of
genetic factors on palatally impacted canines in a genetically isolated community of ultraorthodox Hassidic Jews
of Ashkenazi decent
• Our results imply that genetics plays a signi ficant role in
maxillary canine palatal impaction.
• A genetically isolated Hassidic Jewish community can be a
useful group to study the effects of genetic factors on
various dental anom-alies, including palatally displaced
GENE THERAPY IN ORTHODONTICS
Different studies done on rats by Rabie et al have
demonstrated that use of functional appliances
causes transient upregulation of a number of genes
like PTHrP, Ihh, Runx2, collagen typeX, VEGF
Mandibular Appliance Modulates Condylar Growth through
Marques et al JDR 2008
Test the hypothesis that chondrocytes respond to
forces generated by a mandibular propulsor appliance
by changes in gene expression, and that integrins are
important mediators in this response
Immunohistochemical analyses demonstrated that the
use of the appliance for different periods of time
modulated the expression of fibronectin, integrin
subunits, as well as cell proliferation in the cartilage
confirming that force itself modulates the growth of
the rat condylar cartilage, and that integrins
participate in mechanotransduction.
Gene therapy to enhance condylar growth using rAAVVEGF(recombinant adeno associated virus- vascular
endothelial growth factor)
Dai and Rabie AO 2008
To test the hypothesis that the introduction of
specific vascular growth inducing genes would
favorably affect mandibular condyle growth in rats
over a limited experimental period
Enhancement of mandibular condyle growth occurred
in backward and upward direction in VEGF group
rather than control group
Expression of Vascular Endothelial Growth Factor and the Effects
on Bone Remodeling during Experimental Tooth Movement
Kohno et al JDR 2003
To investigate the effect of rhVEGF injection on the rate of
tooth movement and the comparison of the numbers of
osteoclasts induced by the injection of rhVEGF and rhM-CSF.
• The amount of tooth movement in the rhVEGF injection group
was larger than that in controls. The reason may be because a
large number of osteoclasts induced by rhVEGF appeared
and produced a large amount of bone resorption, leading to a
increase in tooth movement
Local RANKL gene transfer to the periodontal tissue accelerates
orthodontic tooth movement
Kanzaki.H et al Gene Therapy 2006
To test that local RANKL gene transfer into the
periodontal tissue would accelerate tooth movement
It was demonstrated that transfer of the RANKL gene
to the periodontal-tissue activated osteoclastogenesis
and accelerated the amount of experimental TM. Local
RANKL gene transfer might be a useful tool not only for
shortening orthodontic treatment, but also for moving
ankylosed teeth where teeth, fuse to the surrounding
Orthodontics 2047- Genetically Driven Treatment Plans
Gene therapy for sutural growth disturbances:
• Mutations in FGFR2 have been linked to several human
craniosynostosis disorders, including Pfeiffer, Apert, and
• In cases of craniosynostosis involving mutations in
FGFR2, temporarily blocking FGFR2 signaling in the
preosteoblasts within the sutural mesenchyme or
providing a different anti proliferation signal to these
cells would allow normal sutural growth without
Gene therapy for mandibular growth.
• studies of rats by Hagg and colleagues have demonstrated
that the use of functional appliances causes transient upregulation of a number of genes ( PTHrP, Indian hedgehog,
Runx2, collagen type X, and VEGF) in the mandibular
• Identification of the specific genes involved in patients‟
response to functional appliances will be able to help the
orthodontist predict an appliance‟s chances of success in a
• The genes responsible for mandibular growth and safe
methods of transducing genes into tissues, gene therapy
may become the standard of care for the treatment of
Gene therapy for orthodontic tooth movement.
• Two elegant studies by Kanzaki and col-leagues have
used gene therapy with OPG and RANKL to accelerate
and inhibit orthodontic tooth movement in a rat
• The authors concluded: “Local RANKL gene trans-fer
might be a useful tool not only for shortening
orthodontic treatment, but also for moving ankylosed teeth where teeth fuse to the surrounding
Genetic counseling is the process by which patients or
relatives, at risk of an inherited disorder, are advised
of the consequences and nature of the disorder, the
probability of developing or transmitting it, and the
options open to them in management and family
planning in order to prevent, avoid or ameliorate it.
Obtaining a full and careful history.
Establishing an accurate diagnosis.
Drawing a family tree is essential.
Estimating the risk of a future pregnancy
being affected of carrying a disorder.
Continued support and follow up.
Genetic screening – includes prenatal
diagnosis, carrier detection.
• Patients with a great variety of diseases and syndromes
are now referred for evaluation and counseling.
• Genetic evaluation and counseling is a team affair & is a
practical method of calculating risk figures, intended for
information regarding the unborn,.
• The decision taken by the parents after the counselling
session must leave them satisfied instead of placing them
in a state of dilemma
The past 40 years have seen rapid biomedical
advances leading to treatment modalities that could
not have predicted decades ago. Clinically relevant
discoveries in orthodontics during that period have
occurred mainly in material science and appliance
Although progress in those fields will continue to
affect orthodontic profession advances in genetic
testing, gene therapy, pharmacogenomics,
mechanogenomics and stem cell therapy are likely to
produce most dramatic changes in orthodontic
treatment in next 40 years.
• Although genetic screens for various diseases currently
exist, future progress in identifying the functions of
genes in facial development & the mutations that affect
these functions could change orthodontic practice
• For eg. the analysis of genetic background of “responders”
to growth modification would allow orthodontists to apply
appropriate treatment methods judiciously thus reducing
treatment time for average patient
Principles of genetics:Robert H Tamarin
The Heritability of malocclusion I:BJO 1999
The heritability of malocclusion II:BJO 1999
Orthodontics in year 2047 genetically driven
treatment plans:JCO 2007
• Currents concepts in biology of orthodontic tooth
• Genetics of cleft lip and palate: syndromic genes
contribute to the incidence of non-syndromic
clefts:Human molecular genetics 2004
• Gene Therapy to Enhance Condylar Growth Using
• Construction of modern head current concepts in
craniofacial development :JO 2000
• Expression of Vascular Endothelial Growth Factor and
the Effects on Bone Remodeling during Experimental
Tooth Movement:JDR 2003
• Local RANKL gene transfer to the periodontal tissue
accelerates orthodontic tooth movement : Gene
• McNamara, J.A. and Bryan, F.A.: Long-term
mandibular adap-tations to protrusive function: An
experimental study in Macaca mulatta, Am. J. Orthod.
• Rabie, A.B.; Tang, G.H.; Xiong, H.; and Hägg, U.: PTHrP reg-ulates
chondrocyte maturation in condylar cartilage, J. Dent. Res.
• Tang, G.H.; Rabie, A.B.; and Hägg, U.: Indian hedgehog: A
mechanotransduction mediator in condylar cartilage, J. Dent.
Res. 83:434-438, 2004.
• Tang, G.H. and Rabie, A.B.: Runx2 regulates endochondral
ossification in condyle during mandibular advancement, J. Dent.
Res. 84:166-171, 2005.
• Kanzaki, H.; Chiba, M.; Arai, K.; Takahashi, I.; Haruyama, N.
Nishimura, M.; and Mitani, H.: Local RANKL gene transfer to the
periodontal tissue accelerates orthodontic tooth movement,
Gene Ther. 13:678-685, 2006.
• Kanzaki, H.; Chiba, M.; Takahashi, I.; Haruyama, N.;Nishimura,
M.; and Mitani, H.: Local OPG gene transfer to periodontal tissue
inhibits orthodontic tooth movement, J. Dent. Res. 83:920-925,