Diagnosis of Genetic Diseases

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Diagnosis of Genetic Diseases
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Diagnosis of Genetic Diseases
Family History
Estimation of Haematological parameters
Chromosomal Analysis
Determination of Enzyme Activity or Specific
Protein
Recombinant DNA Technology
Clinical Presentation
Estimation of Biochemical Parameters
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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d D
Dd DD D
dd Dd d
d D
Dd DD D
dd Dd d
25 Affected 50 Normal Carriers 25 Normal
Not Carriers
25 Normal 75 Affected
Punnet Square for Autosomal Dominant Inheritance
Punnet Square for Autosomal Recessive Inheritance
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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
  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
Primers
Vectors
NTPs
Probes
DNA
Other enzymes e.g ligases, Taq polymerases
Special chemicals and equipment
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Restriction Endonuclease

• 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.

5-GAATTC-3 3-CTTAAG-5
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Restriction Endonuclease
Produce either Blunt Ends or Staggered ends
5-GAATTC-3 3-CTTAAG-5
5-GAA TTC-3 3-CTT AAG-5
Blunt Ends
or
5-G AATTC-3 3-CTTAA G-5
5-GAATTC-3 3-CTTAAG-5
Staggered Ends
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Uses of Restriction Endonuclease

• 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

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Sources of DNA
cDNA
Genomic DNA
Synthesised from mRNA using Reverse transcriptase
Synthesis of DNA
DNA extracted from cells
Using DNA synthesiser
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cDNA Synthesis
Poly A tail AAAAAAAAA
mRNA
Viral reverse transcriptase
AAAAAA
TTTT
Hair pin loop
NaOH( Hydrolysis of RNA)
DNA polymerase
dNTP
DNA nuclease (single-strand specific)
Double strand cDNA
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Vectors
Cloning vesicles

• 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.

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Types of vectors

• Type
• Plasmid circular, double stranded
  cytoplasmic DNA in procaryotic e.g. PBR 3 of
  Ecoli.
• Bacteriophage lambda a bacterial virus infects
  bacteria.
• Cosmids a large circular cytoplasmic double
  stranded DNA similar to plasmid.
• Yeast Artificial Chromosomes (YAC)

• Insert size
• lt5-10 kb.
• Up to 20kb.
• Up to 50kb.
• 100-1000kb.

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Probes

• Cloned or synthetic nucleic acids used for
  DNADNA or DNARNA hybridization reactions to
  hybridize to DNA of interest.
• DNA or RNA.
• cDNA.
• Labeling of probes
• 3H
• Radioactive
• 32P

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Hybridization
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Recombinant DNA Technology
Amplification of DNA
Study of DNA structure and functions
Others
Polymerase chain reaction
DNA cloning
ARMS
DNA sequencing
DGGE
RT PCR
Dot blot analysis
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Dot blot analysis
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.
DGGE 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)

• 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.

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Polymerase Chain Reaction (PCR)

• 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.

DNA
5 ATCAGGAATTCATGCCAAGGTTGATCGATGATCGATCGATCGATTGA
T 3 3AGCTAGCTAGCT 5
Primer
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Application of PCR

• 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.

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Application of Recombinant DNA Technology

• 1. Clinical Chemistry
• Diagnosis of disease e.g. sickle cell anaemia.
• Prenatal diagnosis,
• Premarital
• Presymptomatic
• Neonatal screening

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Southern Blotting
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Pathogenesis of ?-Thalassaemia
7.0Kb
12.5Kb
Withdraw blood
14.5Kb
Extract DNA
BglII
?1
?2
BglII
BglII
BamHI
BamHI
Treat with BglII
L
R

Electrophoresis
Southern Blotting
Visualize
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? Application of Recombinant DNA Technology

• 2. Human Genetics
• Mutations in genes causing hereditary
• Analysis of stains of blood, semen.
• Virology
• Detection of viral diseases e.g. hepatitis
• Microbiology
• Using specific gene probes for detection of
  E.coli
• Cytology, Histology and Pathology
• Used in detection of tumor.
• Synthesis of protein in bacterial
• Insulin
• GH
• Somatostatin
• Interferon
• 8. Transgenic animal production

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