Diagnosis of Genetic Diseases

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Diagnosis of Genetic Diseases

Family History*

ClinicalPresentation*

Estimation of Haematological

parameters

Estimation of Biochemical Parameters

ChromosomalAnalysis

RecombinantDNA

Technology

Determination ofEnzyme Activity

or Specific Protein

* Important for all genetic diseases 2

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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D d

D

DD

Dd

d

Dd

dd

Punnet Square for Autosomal Dominant Inheritance

D d

D

DD

Dd

d

Dd

dd

25% Affected

50% NormalCarriers

25% Normal Not Carriers

Punnet Square for Autosomal Recessive Inheritance

25% Normal& 75% Affected

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2. Clinical PresentationCertain 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; Adenosine deaminase (ADA)

deficiency is an inherited disorder that damages the immune system and causes severe combined immunodeficiency (SCID)

• Emphysema - 1 anti-trypsin deficiency.• Hypercholesterolaemia – familial hypercholesterolaemia.• Delayed blood coagulation – Haemophilia (decrease in factor VIII or IX).• Mental retardation – Fragile syndrome (in X chromosome) or phenylketonuria

(PKU).• Muscular weakness and degeneration – Duchenne muscular dystrophy.

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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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Requirements for DNA technology

Restriction endonucleases

Vectors

Probes

Other enzymese.g ligases,

Taq polymerases

Primers

NTPs

Special chemicals and equipment

DNA

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• 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, if read, from 5’ or 3’ end is the same.

• 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, if read, from 5’ or 3’ end is the same.

Restriction Endonuclease

5’-GAATTC-3’3’-CTTAAG-5’ 9

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 DNASources of DNA

GenomicDNA

Synthesis of DNA

cDNA

Using DNA synthesiser

Synthesised from mRNA using Reverse transcriptase

DNA extracted from cells

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cDNA Synthesis

cDNA 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 or yeast.

• 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 or yeast.

• 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.

VectorsVectors

Cloning vesiclesCloning vesicles

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TypeI. Plasmid : circular, double stranded

cytoplasmic DNA in procaryotic e.g. PBR 3 of Ecoli.

II. Bacteriophage lambda: a bacterial virus infects bacteria.

III. Cosmids: a large circular cytoplasmic double stranded DNA similar to plasmid.

IV. Yeast Artificial Chromosomes (YAC)

TypeI. Plasmid : circular, double stranded

cytoplasmic DNA in procaryotic e.g. PBR 3 of Ecoli.

II. Bacteriophage lambda: a bacterial virus infects bacteria.

III. Cosmids: a large circular cytoplasmic double stranded DNA similar to plasmid.

IV. Yeast Artificial Chromosomes (YAC)

Types of vectorsTypes of vectors

Insert size• <5-10 kb.

•Up to 20kb.

• Up to 50kb.

•~100-1000kb.

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Cloned or synthetic nucleic acids used for DNA:DNA or DNA:RNA hybridization reactions to hybridize to DNA of interest.

• DNA or RNA.

• cDNA.

• Labeling of probes:• 3H Radioactive• 32P

Cloned or synthetic nucleic acids used for DNA:DNA or DNA:RNA hybridization reactions to hybridize to DNA of interest.

• DNA or RNA.

• cDNA.

• Labeling of probes:• 3H Radioactive• 32P

ProbesProbes

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Hybridization

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DNA cloningDNA cloning

Recombinant DNA TechnologyRecombinant DNA Technology

Polymerase chain reaction

Polymerase chain reaction

Amplification of DNA Study of DNA structureand functions

DGGEDGGE

RT PCRRT PCR

Dot blot analysisDot blot analysis

ARMSARMS

DNA sequencingDNA sequencing

OthersOthers

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A technique for detecting, analyzing, and identifying proteins, similar to the western blot technique but differing in that protein samples are not separated electrophoretically but are spotted through circular templates directly onto the membrane or paper substrate.

Concentration of proteins in crude preparations (such as culture supernatant) can be estimated semi-quantitatively by using “Dot Blot” method if you have both purified protein and specific antibody against it.

Dot blot analysisDot blot analysis

DGGEDGGE: Denaturing gradient Gel Electrophoresis (DGGE) is used for detection of single

base changes polymorphisms genomic cloned and amplified DNA.

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Amplification Refractory Mutation System (ARMS)Amplification Refractory Mutation System (ARMS)

It is a way to detect mutations in DNA.

Amplification Refractory Mutation System (ARMS), also called allele-specific polymerase chain reaction (ASP) and polymerase chain reaction amplification of specific alleles (PASA).

Is a method used to detect single base pair mutation.

The PCR-based technique can be used to analyze a wide variety of germ-line and somatic mutations, such as sickle-cell anemia.

ARMS has the ability to isolate low levels of a mutant sequence in a background of wild-type DNA. The system depends on the specificity of a primer for the normal sequence and another primer for the mutation.

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Principles of Molecular Cloning

Involves:

• Isolation of DNA sequence of interest.

• Insertion of this DNA in the DNA of an organism that grows rapidly and over extended period e.g. bacteria.

• Growing of the bacteria under appropriate condition.

• Obtaining the pure form of DNA in large quantities for molecular analysis.

Involves:

• Isolation of DNA sequence of interest.

• Insertion of this DNA in the DNA of an organism that grows rapidly and over extended period e.g. bacteria.

• Growing of the bacteria under appropriate condition.

• 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.

• Based on Enzymatic amplification of DNA fragment flanked by primers i.e. short oligonucleotides fragments complimentary to DNA. Synthesis of DNA initiates at the primers.

• Method to amplify a target sequence of DNA or RNA several million folds.

• Based on Enzymatic amplification of DNA fragment flanked by primers i.e. short oligonucleotides fragments complimentary to DNA. Synthesis of DNA initiates at the primers.

Polymerase Chain Reaction (PCR)

5’ ATCAGGAATTCATGCCAAGGTTGATCGATGATCGATCGATCGATTGAT 3’ 3’AGCTAGCTAGCT 5’

DNA

Primer

• Diagnosis of genetic disease by amplification of the gene of interest, followed by

detection of mutation.

• Detection of infectious agent e.g. bacteria and viruses.

• DNA sequencing.

• In forensic medicine.

• Diagnosis of genetic disease by amplification of the gene of interest, followed by

detection of mutation.

• Detection of infectious agent e.g. bacteria and viruses.

• DNA sequencing.

• In forensic medicine.

Application of PCRApplication of PCR

1. Clinical Chemistry:

• Diagnosis of disease e.g. sickle cell anaemia.

• Prenatal diagnosis,• Premarital “• Presymptomatic “• Neonatal screening

1. Clinical Chemistry:

• Diagnosis of disease e.g. sickle cell anaemia.

• Prenatal diagnosis,• Premarital “• Presymptomatic “• Neonatal screening

Application of Recombinant Application of Recombinant DNA TechnologyDNA Technology

Southern Blotting

BglII

BglII

BamHI BglII

14.5Kb

7.0Kb12.5Kb

BamHI

1 2

L R

Pathogenesis of -Thalassaemia

Extract DNA

Treat with BglII

Electrophoresis

Visualize

Withdrawblood

Southern Blotting

2. Human Genetics• Mutations in genes causing hereditary

3. Analysis of stains of blood, semen.

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

8. Transgenic animal production

2. Human Genetics• Mutations in genes causing hereditary

3. Analysis of stains of blood, semen.

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

8. Transgenic animal production

⇝ ⇝ Application of Recombinant Application of Recombinant DNA TechnologyDNA Technology