school- diagnosis of genetic disorders

8/2/2019 School- Diagnosis of Genetic Disorders

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Diagnosis of Genetic Diseases


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Diagnosis of Genetic Diseases

Family History*

ClinicalPresentation*

Estimation ofHaematological

parameters

Estimation ofBiochemicalParameters

ChromosomalAnalysis

RecombinantDNATechnology

Determination ofEnzyme Activity

or Specific Protein

* Important for all genetic diseases


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1. Family History

Consanguinity of parents.

Presence of other siblings with the same disorder.

Occurrence of the disorder in other members of the family.

Repeated abortions or still births,

mother and fathers ages.

Drawing punnet square helps to determine the mode of inheritance

of the genetic disorders.

Autosomal or X-linked

Dominant or recessive


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2. Clinical Presentation

Certain clinical features are specific for a disease: Chronic anaemia: Haemoglobinopathies Thalassaemia

Other genetic anaemias Acute anaemia, under certain stressful conditions.

G-6-PD deficiency Hypoxia sickle cell disease.

Dependence on blood transfusion - -thalassaemia (major) Severe immune deficiency ADA deficiency.

Emphysema - 1 anti-trypsin deficiency. Hypercholesterolaemia familial hypercholesterolaemia.

Delayed blood coagulation Haemophilia (decrease in factor VIII orIX).

Mental retardation Fragile syndrome (in X chromosome) orphenylketonuria (PKU).

Muscular weakness and degeneration Duchenne musculardystrophy.


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Recombinant DNA Technology

( Genetic Engineering)


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Recombinant DNA Technology( Genetic Engineering)

Techniques for cutting

and joining DNA


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Recombinant DNA

- The DNA created byjoining DNA fromdifferent origins e.ghuman DNAsequence ofinterest andbacterial or other

DNA molecule.- It is capable of

duplication in the

laboratory.

- The DNA created byjoining DNA fromdifferent origins e.ghuman DNAsequence ofinterest andbacterial or other

DNA molecule.- It is capable of

duplication in the

laboratory.

Recombinant DNA


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Requirements for DNA technology

Restriction endonucleases

Vectors

Probes

Other enzymes

e.g ligases,

Taq polymerases

Primers

NTPs

Special chemicals and

equipment

DNA


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Restriction Endonuclease

Endonucleases.

Synthesized by procaryotes. Donot restrict host DNA. Recognize and cut specific base

sequence of 4-6 bases in

double helical DNA. The sequence of base pairs is

palindromic i.e. it has two fold

symmetry and the sequence, ifread, from 5 or 3 end is thesame.

Endonucleases. Synthesized by procaryotes. Do

not restrict host DNA. Recognize and cut specific base

sequence of 4-6 bases in

double helical DNA. The sequence of base pairs is

palindromic i.e. it has two fold

symmetry and the sequence, ifread, from 5 or 3 end is thesame.

5-GAATTC-3

3-CTTAAG-5


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Produce either Blunt Ends or Staggered ends:Produce either Blunt Ends or Staggered ends:

Restriction Endonuclease

5-GAATTC-3

3-CTTAAG-5

5-GAA TTC-3

3-CTT AAG-5

5-G AATTC-3

3-CTTAA G-5

5-GAATTC-3

3-CTTAAG-5

Blunt Ends

Staggered Ends

or


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Obtaining DNA fragments of interest. Gene mapping. Sequencing of DNA fragments. DNA finger printing Recombinant DNA technology Study of gene polymorphism.

Diagnosis of disease. Prenatal diagnosis

Obtaining DNA fragments of interest. Gene mapping. Sequencing of DNA fragments. DNA finger printing Recombinant DNA technology Study of gene polymorphism.

Diagnosis of disease. Prenatal diagnosis

Uses of Restriction Endonuclease


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Sources of DNA

Genomic

DNA

Synthesis of DNA

cDNA

Using DNA synthesiser

Synthesised from

mRNA using reversetranscriptase

DNA extracted

from cells


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DNA S th i

cDNA Synthesis


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cDNA SynthesiscDNA Synthesis

mRNA

Poly A tailAAAAAAAAA

Viral reverse transcriptase

AAAAAA

TTTT

NaOH( Hydrolysis of RNA)

DNA polymerase

Hair pin loop

DNA nuclease(single-strand specific)

Double strand cDNA

dNTP


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DNA molecules.

Can replicate in a host e.g bacterial cells oryeast.

Can be isolated and re-injected in cells.

Presence can be detected.

Can be introduced into bacterial cells e.g. E.coli.

May carry antibiotic resistance genes.

DNA molecules.

Can replicate in a host e.g bacterial cells oryeast.

Can be isolated and re-injected in cells.

Presence can be detected.

Can be introduced into bacterial cells e.g. E.coli.

May carry antibiotic resistance genes.

Vectors Cloning vesicles


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TypeI. Plasmid : circular, double

stranded cytoplasmic DNA inprocaryotic e.g. PBR 3 of Ecoli.

II. Bacteriophage lambda: a bacterialvirus infects bacteria.

III. Cosmids: a large circularcytoplasmic double stranded DNAsimilar to plasmid.

IV. Yeast Artificial Chromosomes (YAC)

TypeI. Plasmid : circular, double

stranded cytoplasmic DNA inprocaryotic e.g. PBR 3 of Ecoli.

II. Bacteriophage lambda: a bacterialvirus infects bacteria.

III. Cosmids: a large circularcytoplasmic double stranded DNAsimilar to plasmid.

IV. Yeast Artificial Chromosomes (YAC)

Types of vectors

Insert size


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Cloned or synthetic nucleic acids used for DNA:DNA orDNA:RNA hybridization reactions to hybridize to

DNA of interest.

DNA or RNA.

cDNA.

Labeling of probes: 3HRadioactive

32P

Cloned or synthetic nucleic acids used for DNA:DNA orDNA:RNA hybridization reactions to hybridize to

DNA of interest.

DNA or RNA.

cDNA.

Labeling of probes: 3H

Radioactive 32P

Probes


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Hybridization


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DNA cloningDNA cloning

Recombinant DNA Technology

Polymerase chainreaction

Polymerase chainreaction

Amplification of DNA Study of DNA structureand functions

DGGEDGGE

RT PCRRT PCR

Dot blot analysisDot blot analysis

ARMSARMS

DNA sequencingDNA sequencing

OthersOthers

Principles of Molecular Cloning


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Principles of Molecular Cloning

Involves:

Isolation of DNAsequence of interest.

Insertion of this DNA inthe DNA of an organismthat grows rapidly and

over extended period e.g.bacteria.

Growing of the bacteria

under appropriatecondition. Obtaining the pure form

of DNA in large quantitiesfor molecular analysis.

Involves:

Isolation of DNAsequence of interest.

Insertion of this DNA inthe DNA of an organismthat grows rapidly and

over extended period e.g.bacteria.

Growing of the bacteria

under appropriatecondition. Obtaining the pure form

of DNA in large quantities

for molecular analysis.


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Method to amplify a target sequence of DNA or RNA

several million folds.

Developed by Saiki et al in 1985.

Based on Enzymatic amplification of DNA fragmentflanked by primers i.e. short oligonucleotides fragments

complimentary to DNA. Synthesis of DNA initiates atthe primers.

Method to amplify a target sequence of DNA or RNA

several million folds.

Developed by Saiki et al in 1985.

Based on Enzymatic amplification of DNA fragmentflanked by primers i.e. short oligonucleotides fragmentscomplimentary to DNA. Synthesis of DNA initiates atthe primers.

Polymerase Chain Reaction (PCR)

5 ATCAGGAATTCATGCCAAGGTTGATCGATGATCGATCGATCGATTGAT 3

3AGCTAGCTAGCT 5

DNA

Primer


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Application of PCR

Diagnosis of genetic disease by amplification of thegene of interest, followed by detection of mutation.

Detection of infectious agent e.g. bacteria andviruses.

DNA sequencing.

In forensic medicine.

Application of PCR

Diagnosis of genetic disease by amplification of thegene of interest, followed by detection of mutation.

Detection of infectious agent e.g. bacteria andviruses.

DNA sequencing.

In forensic medicine.

Application of Recombinant


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1. Clinical Chemistry: Diagnosis of disease e.g.sickle cell anaemia by Mst II.

Prenatal diagnosis

1. Clinical Chemistry:

Diagnosis of disease e.g.sickle cell anaemia by Mst II.

Prenatal diagnosis

Application of RecombinantDNA Technology


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Southern Blotting

Pathogenesis of-Thalassaemia


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BglII

BglII

BamHI BglII

14.5Kb

7.0Kb12.5Kb

BamHI

12

L R

at oge es s o a assae a

Extract DNA

Treat with BglII

Electrophoresis

Visualize

Withdraw

blood

Southern Blotting


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2. Human Genetics Mutations in genes causing hereditary disease e.g.

diagnosis of fibrosis Channes disease.

3. Forensic Medicine Analysis of stains of blood, semin.

4. Virology Detection of viral diseases e.g. hepatitis

5. Microbiology

Using specific gene probes for detection of E.coli

6. Cytology, Histology and Pathology Used in detection of tumor.

7. Synthesis of protein in bacterial Insulin GH Somatostatin Interferon

2. Human Genetics

Mutations in genes causing hereditary disease e.g.diagnosis of fibrosis Channes disease.

3. Forensic Medicine Analysis of stains of blood, semin.

4. Virology Detection of viral diseases e.g. hepatitis

5. Microbiology

Using specific gene probes for detection of E.coli

6. Cytology, Histology and Pathology Used in detection of tumor.

7. Synthesis of protein in bacterial Insulin GH Somatostatin Interferon


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school- diagnosis of genetic disorders

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