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DNA BASED DIAGNOSIS OF GENETIC DISEASE
Prepared by Abhishek mondal. 2nd pharm Pes cp. Dept of pharmacology
CONTENTS Methods of DNA assay. DNA probes. DNA in the Diagnosis of genetic
diseases. Examples of diagnosis of genetic
diseases.
INTRODUCTION 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.
CONTINUED….. 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 .
GENETIC DISORDER 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
METHODS OF DNA ASSAY 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 .
NUCLEIC ACID HYBRIDIZATION:
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.
RADIOACTIVE DETECTION SYSTEM 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.
NON-RADIOACTIVE DETECTION SYSTEM: 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.
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.
DNA PROBES :
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.
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.
MOA OF DNA PROBES 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.
PCR IN THE USE OF DNA PROBES 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.
DNA PROBES AND 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, 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.
CONTD….. 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
DNA CHIP MICRO ARRAY OF GENE PROBE 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.
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
SOME OF THE IMPORTANT GENETIC DISEASES FOR WHICH DNA ANALYSIS USED : Cystic fibrosis. Sickle cell anemia. Duchennels muscular dyatrophy. Huntington’s disease. Alzheimer’s disease