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DNA BASED DIAGNOSIS OF GENETIC DISEASES

By, Chinmayi Upadhyaya I.M.Pharm (Pharmacology)

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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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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.

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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.

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cells positive for a chromosomal rearrangement

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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.

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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.

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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.

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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.

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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.

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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.

Technique :The known DNA molecule is cut in to fragments by RestrictionEndonucleases Fluorescent marker are attached to these DNA fragments

Allowed to react with probes of DNA chip

Target DNA fragments with complementary sequences bindto DNA probes select

Wash remaining DNA fragments

Target DNA pieces can be identified by their fluorescenceemission by passing a laser beam

Computer recorded the pattern of fluorescence emission andDNA identification. 26

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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.

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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.

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• 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.

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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.

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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).

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• 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.

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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.

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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.

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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.

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5) FRAGILE X SYNDROME:• 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.

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6) ALZHEIMER’S DISEASE:• 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.

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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.

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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.

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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.

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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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