This document discusses methods for diagnosing genetic diseases using DNA analysis. It covers DNA probes, hybridization techniques using radioactive and non-radioactive detection, polymerase chain reaction to amplify DNA samples, DNA microarrays, and examples of genetic diseases that can be diagnosed this way such as cystic fibrosis, sickle cell anemia, and Huntington's disease. The key applications of DNA diagnostics are to detect inherited genetic defects or the presence of pathogenic genes through identification of alterations in genes.

1. Prepared by
Abhishek mondal. 2nd pharm
Pes cp. Dept of pharmacology

2.  Methods of DNA assay.
 DNA probes.
 DNA in the Diagnosis of genetic diseases.
 Examples of diagnosis of genetic diseases.

3.  Diagnosis of disease due to pathogens or
due to inherent genetic defects is
necessary for appropriate treatment .
 Traditional diagnostic methods for parasite
infection include microscopic examination
, in vitro culture , & detectionn of ab in
serum .
 And for genetic disease , the procedures
such as estimation of metabolites (blood ,
urine)& enzyme assay are used .
 These laboratory technique are indirect ,
not always specific.

4.  Dna being the genetic material of living
organism , contains the information which
contributes to various characterics
features of specific organism.
 Thus the prensence of diasease 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 alteration in gene .

5.  Definition : a disease or disoder which is
inherited geneticaly .
 Types :1. chromosomal eg down syndrome
 2. single gene ( mendelian or
monogenic}
 3. cancer
 4. Multi factorial ( complex or
polygenic )
 eg . Alzheimer’s disease, arthritis
,diabetis , obesity .
 5. mitochondrial

6.  1. nucleic acid hybridization
 a. radioactive detection system .
 b. non – radioactive detection syst em .
 2. DNA probes
 a. PCR in use of dna probes .
 b. DNA probes & signal amplification
 3. DNA chip – microarray of gene probe .

7.  Hybridization is based on the principle that, a single
stranded DNA molecule recognizes and specifically
binds to complimentary DNA strand.
 This is comparable to a specific key and lock
relationship.
 PROCEDURE:
 The single stranded target DNA is bound to a
membrane support.
 The DNA probe is added.
 At appropriate conditions the DNA probes pairs with
the complimentary target DNA.
 The unbound DNA probe is removed.
 Sequence of nucleotides of the target DNA can be
identified from the known sequence of the DNA
probe.

8. Radio-active detection system:
 The DNA probe is tagged with radioactive isotope
target DNA is purified denatured and mixed with DNA
probe.
 Isotope labeled DNA molecules specifically
hybridizes with the target DNA.
 The non hybridized probe DNA is washed away.
 The presence of radioactivity in the hybridized DNA
detected by autoradiography.
 It reveals the presence of any bound probe molecules
thus the complimentary DNA sequence in the target
DNA.
 Disadvantage : Isotopes have short half-life; Risks in
handling; Requires special lab equipment.

10.  Biotin labeled nucleotides are incorporated in
the DNA probe.
 It is based on the enzymatic conversion of the
chromogenic or chemiluminescent substrates.
 A biotin labeled DNA probe is hybridized to a
largest DNA.
 The egg white protein avidin or its bacterial
analog streptavidin is added to bind to biotin.
 Biotin labeled enzyme such as alkaline
phosphatase, is added which attaches to avidin
or streptavidin.
 These proteins have 4 separate biotin binding
sites.
 Thus a single molecule can bind to biotin
labelled DNA probe, as well as biotin labeled
enzyme.

11.  On the addition of chemiluminescence
substance, the enzyme alkaline phosphatase
acts and converts it into light emitting body
which can be measured.
ADVANTAGES :
 The biotin labeled DNA is quite stable at
room temperature for about 1 yr.
 The detection devices using
chemiluminescence are preferred since they
are as sensitive as radio-isotope detectors.
 Are more sensitive than, the use of
chromogenic detection system.

13.  A DNA probe or a gene probe is a synthetic
single stranded DNA molecule the that can
recognize and specifically bind, to a target
DNA, in a mixture of bio-molecules.
 DNA probes may be long or short.
 It may bind to the total or a small portion of
the target DNA.
 Requirement : Specific and stable binding to
the target DNA.

14. Majority of the DNA probes are chemically
synthesized in laboratory.
 Other ways:
ISOLATION OF SELECTIVE REGION OF GENES:
 DNA from organism cut using restriction
endonucleases DNA fragments cloned
in vectors DNA probes selected by
screening.
DNA PROBE FROM mRNA:
 mRNA molecules specific to a particular DNA
sequence Isolated.
 using reverse transcriptase cDNA
molecules synthesised cDNA used as a probe to
detect target DNA.

15.  The principle is based on denaturation and
renaturation of DNA.
 Double stranded DNA molecule
physical(temp>95 ̊C) or chemical (urea or
formaldehyde) the H-bonds break
complimentary strands gets separated
Denaturation.
 Under suitable conditions (temp, pH, salt
conc) separated single stranded DNA
strands reassemble original double
stranded DNA
 Hybridization or renaturation.

17.  The detection of target sequence becomes difficult if
the quantity of DNA is very low in such a case, the
polymerase chain reaction is first employed to
amplify the minute quantities of target DNA.
 And identified by a DNA probe.
 PCR has many desirable features in a clinical setting.
1.Sample preparation is minimal, only nano-gram
amounts of DNA are needed to begin the reaction.
 Hence, diagnosis can be performed on very small
sample of blood or tissue.
2.Detection of PCR products are much easier than
detecting single copy sequences in genomic DNA.
 Diagnosis can be made in a matter of hrs, rather than
days needed for standard southern procedures.

18.  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, target DNA bound to
DNA probe is amplified.
 2 general methods to active signal
amplification:
Separate the target DNA-DNA probe complex
from the rest of the DNA molecule and amplify
it.
Amplify the DNA probe(bound to target DNA)
by using second probe.

19.  The RNA complimentary to the DNA probe
can serve as the second probe.
 The RNA-DNA-DNA complex can be separated
and amplified.
 The enzyme O-β-replicase which catalyses
RNA replication is commonly used

20.  The DNA chip are gene chip contains 1000’s of DNA probes,
arranged on a small glass slide of the postage stamp size.
 By this recent advanced approach, 1000’s of target DNA
molecules can be scanned simultaneously.
 Technique:
 Known DNA molecule cut into fragments by restriction
endonuclease
 Fluorescent marker are attached to these DNA fragments.
 Allowed to react with probe of DNA chip.
 Target DNA fragments with complimentary sequences bind
to DNA probes selectively.
 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.

22. Application:
 The presence of mutation in a DNA can be
conveniently identified.
 Gene chip probes used to find mutation in
p53 and BRACI gene (involved in cancer).
Advantages:
 Very rapid.
 Sensitive and specific.
 Simultaneous analysis of many DNA is
possible

25.  Cystic fibrosis.
 Sickle cell anemia.
 Duchennels muscular dyatrophy.
 Huntington’s disease.
 Alzheimer’s disease