DNA, being the site for the storage of all the genetic in-formations,can be used for the diagnosis of the genetic diseases at the earliest .

1. DNA BASED
DIAGNOSIS OF
GENETIC DISEASES
By,
Chinmayi
Upadhyaya
I.M.Pharm
(Pharmacology)
1

2. CONTENTS:
• Introduction.
• Principles involved in diagnosis of genetic diseases
by DNA analysis.
• Methods of DNA Assay.
• Some of the important genetic diseases for which
DNA analysis used.
• References.
2

3. INTRODUCTION:
• Diagnosis of diseases due to pathogens or due to
inheritant genetic defects is necessary for appropriate
treatment .
• Traditional diagnostic methods for genetic diseases
includes the procedures such as estimation of
metabolites (blood/urine) & enzyme assays.
• DNA, being the genetic material of the living organisms,
contains the information which contributes to various
characteristic features of the specific organism. Thus,
the presence of a disease-causing pathogen can be
detected by identifying a gene or a set of genes of the
organism. Inherited genetic defect can be diagnosed by
identifying the alterations in the gene.
3

4. PRINCIPLES INVOLVED IN THE DIAGNOSIS OF
GENETIC DISEASES:
1) COMPLEMENTARY NATURE OF DNA:
• The rules of base pairing-“Guanine pairs with
Cytosine while Adenine pairs with Thymine"-forms
the basis for the accurate replication of DNA.
• This same complementarity facilitates the
molecular analysis of DNA by allowing pieces of
DNA to be used as probes for their complementary
sequences. Even short pieces of DNA are relatively
unique.
4

5. 2) NATURE OF RESTRICTION ENDONUCLEASES:
• Restriction endonucleases’s key feature is the ability
to recognize specific sequences in the DNA and
then cut the DNA in a predictable manner.
• This is important as Restriction Endonucleases
permits the cleavage of DNA into well-defined
fragments in a controlled manner.
• Their relevance to genetic diagnostics results from
the sequence variation in and around a gene.
Alterations of DNA sequences can cause the loss or
gain of cleavage sites, resulting in fragments of
different sizes.
5

6. 3) SIZE SEPERATION THROUGH ELECTROPHORESIS:
• When a molecule is placed in an electric field, it will
migrate towards the electrode of opposite charge.
• DNA, because of its Phosphate moieties, carries a
net negative charge, and consequently will migrate
towards the anode(+).
• Size separation can be carried out by the migration
of DNA through a solid matrix composed of Agarose
or Polyacrylamide.
• DNA fragments migrate through these gels at a
velocity inversely proportional to size; hence, small
fragments migrate faster than large, resulting in the
effective resolution of the fragments. 6

7. METHODS OF DNA ASSAY:
The identification of specific DNA sequence can be
achieved by employing:
1) Nucleic acid hybridization.
2) DNA probes.
3) DNA chip – Microarray of gene probe.
4) Southern blot analysis.
7

8. 1) Nucleic acid hybridization:
• Nucleic acid hybridization is the process of establishing a
non-covalent , sequence-specific interaction between two
or more complementary strands of nucleic acids into a
single hybrid .
• Though a double-stranded DNA sequence is generally
stable under physiological conditions, changing these
conditions in the laboratory will cause the molecules to
separate into single strands.
• These strands are complementary to each other but may
also be complementary to other sequences present in their
surroundings. Lowering the surrounding temperature
allows the single-stranded molecules to anneal or
“hybridize” to each other.
8

9. PRINCIPLE:
• Single stranded DNA molecule recognize and specifically
bind to a complementary DNA strand in a mixture of other
DNA strands.
• This is comparable to a specific key and lock relationship.
BASIC PROCEDURE:
• Single stranded target DNA is bound to a membrane
support.
• DNA probe labeled with detector substance is added.
• DNA probe pairs with the complementary target DNA.
• wash unbound DNA probes.
• Sequence of nucleotide in the target DNA can be
identified.
9

10. 10

11. a) Radioactive detection system:
• The DNA probe tagged with a radioactive isotope
(commonly phosphorus 32) target DNA, is purified &
denatured, mixed with DNA probe Isotope labeled DNA
molecules.
• Specifically hybridizes with the target DNA.
• Presence of radioactivity in the hybridized DNA, detected
by autoradiography.
• Non – hybridized probe DNA is washed away.
Disadvantages:
• Isotopes have short half life.
• risks in handling requiring special laboratory equipments.
11

12. 12

13. 13

14. b) Non – radioactive detection system:
Principle:
Detection is based on enzymatic conversion of a
Chromogenic (colour producing) or Chemiluminescent
(light emitting) substrates.
• Mainly Biotin-labeled (Biotinylated) nucleotides are
incorporated into DNA probe.
Advantages:-
• Biotin-labeled DNA is quite stable for about 1 year.
• Chemiluminescence detection is very sensitive than
chromogenic detection system.
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15. 15

16. FLUORESCENCE IN SITU HYBRIDIZATION:
• Uses Fluorescent probes that bind to only those parts of
the chromosome with a high degree of sequence
complementarity.
• used to detect and localize the presence or absence of
specific DNA sequences on chromosomes.
• Fluorescence microscopy can be used to find out where the
fluorescent probe is bound to the chromosomes.
16

17. Preperation and Hybridization process:
• First, a probe is constructed. The probe is tagged directly
with Fluorophores or with Biotin. Tagging can be done in
various ways, such as Nick translation, or PCR using tagged
nucleotides.
• Then, an interphase or metaphase chromosome
preparation is produced. The chromosomes are firmly
attached to a substrate, (usually glass). Repetitive DNA
sequences must be blocked by adding short fragments of
DNA to the sample.
• The probe is then applied to the chromosome DNA and
incubated for approximately 12 hours while hybridizing.
• Several wash steps remove all unhybridized or partially
hybridized probes. The results are then visualized and
quantified using a microscope that is capable of exciting the
dye and recording images. 17

18. 18

19. 19
cells positive for a chromosomal rearrangement

20. 2) DNA PROBE/GENE PROBE:
• Synthetic single stranded DNA molecule that can
recognize and specifically bind to a target DNA by
complimentary base pairing in a mixture of bio
molecules. DNA probes are either long (>100
nucleotides) or short (<50 nucleotides) Bind to the total
or a small portion of the target DNA. Most important
requirement is their specific & stable binding with target
DNAs.
20

21. Mechanism of action:
• Basic principle is (Hybridization of DNA) i.e.
Denaturation & Renaturation. When a dsDNA
molecule is subjected to physical or chemical
changes, the H-bonds break & complementary
stands get separated. Under suitable conditions
(i.e. temp., pH, salt conc.), the two separated
single DNA strands can reassemble to form the
original ds DNA.
Methods used to obtain DNA probes: Majority of
DNA probes are chemically synthesized in the
laboratory.
21

22. i. Isolation of selected regions of genes:-
– The DNA is cut by restriction enzymes.
– The DNA fragment is cloned in vectors.
– DNA probes are selected by screening.
ii. Synthesis of DNA probes from mRNA:-
– mRNA from specific DNA is isolated.
– Treat with R. transcriptase.
– cDNA molecules are synthesized and used as
probes.
22

23. PCR in the use of DNA probes:
• The polymerase chain reaction (PCR) is a technology used
to amplify a single copy or a few copies of a piece of DNA
across several orders of magnitude, generating thousands
to millions of copies of a particular DNA sequence.
• Detection of target sequence becomes quite difficult if the
quantity of DNA is very low. Therefore, Polymerase Chain
Reaction is first employed to amplify the minute quantities
of target DNA & is identified by a DNA probe.
 The two strands of the DNA double helix are physically
separated at a high temperature in a process called DNA
melting.
 The temperature is lowered and the two DNA strands
become templates for DNA polymerase to selectively
amplify the target DNA. 23

24. DNA probes & signal amplification:
• It is an alternative to PCR for the identification of minute
quantities of DNA by using DNA probes. In PCR, target DNA
is amplified, while in signal amplification, the target DNA
bound to DNA probe is amplified.
Two general methods to achieve signal amplification.
 Separate the DNA target – DNA probe complex from the
rest of the DNA molecules & then amplify it.
 Amplify the DNA probe (bound to target DNA) by using a
second probe. The RNA complementary to the DNA probe
can serve as the second probe. The RNA-DNA complex can
be separated & amplified. The O-beta replicase which
catalyses RNA replication is used.
24

25. 3) DNA chip – Microarray of gene probe:
• DNA chip or Genechip contains thousands of DNA probes
(4000,000 or even more) arranged on a small glass slide of
the size of a postage stamp. Thousands of target DNA
molecules can be scanned simultaneously.
Advantages:
Very rapid, Sensitive &
Specific & Simultaneous
analysis of many DNAs
are possible.
25

26. Technique :
The known DNA molecule is cut in to fragments by Restriction
Endonucleases

Fluorescent marker are attached to these DNA fragments

Allowed to react with probes of DNA chip

Target DNA fragments with complementary sequences bind
to DNA probes select

Wash remaining DNA fragments

Target DNA pieces can be identified by their fluorescence
emission by passing a laser beam

Computer recorded the pattern of fluorescence emission and
DNA identification.
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27. 27

28. Applications:
• Presence of mutations in a DNA sequence is identified.
Genechip probe array has been successfully used for the
detection of mutations in the p53 & BRCA 1 genes (involved
in cancer). Scientists are trying to develop Genechips for
the entire genome of an organism.
28

29. 4. SOUTHERN BLOT ANALYSIS:
• DNA strands are cut into smaller fragments.
• The DNA fragments are then Electrophoresed on an Agarose gel to
separate them by size.
• If alkaline transfer methods are used, the DNA gel is placed into an
alkaline solution ( sodium hydroxide) to denature the double-
stranded DNA. The denaturation in an alkaline environment may
improve binding of the negatively charged Thymine residues of DNA
to a positively charged Amino groups of membrane, separating it
into single DNA strands for later hybridization to the probe.
• A sheet of Nitrocellulose membrane is placed on top of the gel.
Pressure is applied evenly to the gel to ensure good and even
contact between gel and membrane. Buffer transfer by capillary
action from a region of high water potential to a region of low water
potential is then used to move the DNA from the gel onto the
membrane; ion exchange interactions bind the DNA to the
membrane. 29

30. • The membrane is then baked in a vacuum or regular oven
at 80 °C for 2 hours or exposed to ultraviolet radiation to
permanently attach the transferred DNA to the membrane.
• The membrane is then exposed to a hybridization probe.
The probe DNA is labelled so that it can be detected.
• After hybridization, excess probe is washed from the
membrane, and the pattern of hybridization is visualized on
X-ray film by autoradiography in the case of a radioactive or
fluorescent probe, or by development of colour on the
membrane if a chromogenic detection method is used.
30

31. 31

32. Some of the important genetic diseases for which
DNA analysis is used:
1) CYSTIC FIBROSIS:
• It is due to a defect in cftr gene(located on chromosome
that encodes Cystic Fibrosis Transmembrane Regulator
protein. cftr gene is located on chromosome 7. DNA
probes have been developed to identify this gene.
• It is now possible to detect CF genes in duplicate in the
fetal cells obtained from samples of amniotic fluid.
32

33. 2) SICKLE-CELL ANEMIA:
• It occurs due to a single nucleotide change(A-T) in the β-Globin
gene of coding strand. In the normal β-Globin gene the DNA
sequence is CCTGAGGAG, while in Sickle-cell anemia, the
sequence
is CCTGTGGAG.
• DNA for analysis is
isolated from
peripheral blood
leukocytes and from
the fetal-derived
Amniocytes. The DNA
samples are
then amplified by the
polymerase chain
reaction (PCR). 33

34. • This results in the 2,00,000-fold amplification of the specific f-
globin DNA sequences containing the potential site of the sickle-
cell mutation.
 Allele-specific oligonucleotide (Aso) probing.
• In this procedure two short synthetic DNA probes, 19
nucleotides in length are used, one complementary to the
normal human 3-globin gene (PA) and the other complementary
to the sicklecell globin gene(Ps). The amplified DNA iS made
single stranded and spotted on a membrane filter (a "dot blot").
• The membrane is then placed in a hybridization solution
containing the radioactively labelled probes. Under
appropriately stringent hybridization conditions the PA allele-
specific probe will hybridize only to the normal allele.
• Similarly, the ,Ps allele-specific probe will hybridize only to the
Ps allele.
• The radioactive probe produces a spot on an autoradiogram
that is readily interpreted, indicating the presence of a specific
allele. 34

35.  Oligonucleotide restriction analysis.
• Short radioactively labelled synthetic oligonucleotides are
hybridized to the amplified DNA.
• The hybrids are subsequently digested with the appropriate
Restriction Endonucleases.
• The digested DNA is then electrophoretically separated by
size on a Polyacrylamide gel.
• The fragment pattern is detected by autoradiography will
be diagnostic.
35

36. 3) HUNTINGTON’S DISEASE:
• The gene responsible for this disease lies on chromosome 4
and is characterised by excessive repetation of the base
triplet CAG(42-66 times).
• The abnormal protein causes the death of cells in the Basal
ganglia.
• It can be detected by the analysis of RFLPs in blood related
individuals.
36

37. 4) DUCHENNE’S MUSCULAR DYSTROPHY:
• The patients with DMD lack muscle protein, Dystrophin due
to absence of gene encoding Dystrophin.
• For diagnosis, a DNA probe to identify a segment of DNA
that lies close to defective gene is used.
• This DNA segment is
referred to as
restriction fragment
length Polymorphism
(RELP) serves as a
marker and can detect
DMD.
37

38. 5) FRAGILE X SYNDROME:
38
• Is due to genetic defect in X chromosome.
• They have 3 nucleotide bases(CGG) repeated again and
again.
• These trinucleotide repeats blocks
the Transcription process resulting
in a protein deficiency.
• Direct DNA analysis has become
available with the isolation of
DNA probes that detect the
unstable DNA sequence containing
CGG repeat.

39. 6) ALZHEIMER’S DISEASE:
39
• AD patients are found to have mutations in gene: those
encoding AMYLOID PRECURSSOR PROTIEN (APP) . Most
mutations in the APP increase the production of a small
protein called Aβ42, which is the main component
of SENILE PLAQUES.
• Specific gene on chromosome 21 is believed to be
responsible for familial AD.
• DNA probe has been developed to locate the genetic
marker for the AD.

40. 7) OBESITY:
• The gene ‘ob’ is located on chromosome 6.
• The DNA of ob gene encodes a protein with 167
aminoacids in adipose tissue.
• This protein is responsible to keep the weight under
control.
• The genetically obese patient has mutated ob gene.
40

41. 8) DIABETES:
a) TYPE II DIABETES:
• The Glucokinase gene(chromosome 7) from normal and
diabetes patients were cloned and scanned with DNA
probes.
• It was found that a single base mutation of the gene led to
a defective Glucokinase production that is largely
responsible for Type II diabetes.
b) TYPE I DIABETES:
• Researchers have identified at least 18 different
chromosome regions linked with this.
• These DNA sequences are located on chromosome 6,11
and 18.
41

42. 9) CANCER:
P53 GENE:
• It encodes for a protein that helps DNA repair and
suppresses cancer development.
• It binds to DNA and blocks replication.
• The mutation in this gene leads to Cancer development.
GENES OF BREAST CANCER:
• Genes namely BRCAI and BRCAII are implicated in
hereditary forms of Breast cancer.
42

43. References:
1. Dr.U Satyanarayana, Dr.U Chakrapani. Biochemistry.
Elsevier publications;4:599-610.
2. S N Jogdand. Gene biotechnology. Himalaya publishing
house.2009;3:79-89.
3. Internet source.
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44. THANK YOU
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