School- Diagnosis of Genetic Disorders
-
Upload
boobesh-venkata-ramanan -
Category
Documents
-
view
222 -
download
0
Transcript of School- Diagnosis of Genetic Disorders
-
8/2/2019 School- Diagnosis of Genetic Disorders
1/33
Diagnosis of Genetic Diseases
-
8/2/2019 School- Diagnosis of Genetic Disorders
2/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
3/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
4/33
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.
-
8/2/2019 School- Diagnosis of Genetic Disorders
5/33
Recombinant DNA Technology
( Genetic Engineering)
-
8/2/2019 School- Diagnosis of Genetic Disorders
6/33
Recombinant DNA Technology( Genetic Engineering)
Techniques for cutting
and joining DNA
-
8/2/2019 School- Diagnosis of Genetic Disorders
7/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
8/33
Requirements for DNA technology
Restriction endonucleases
Vectors
Probes
Other enzymes
e.g ligases,
Taq polymerases
Primers
NTPs
Special chemicals and
equipment
DNA
-
8/2/2019 School- Diagnosis of Genetic Disorders
9/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
10/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
11/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
12/33
Sources of DNA
Genomic
DNA
Synthesis of DNA
cDNA
Using DNA synthesiser
Synthesised from
mRNA using reversetranscriptase
DNA extracted
from cells
-
8/2/2019 School- Diagnosis of Genetic Disorders
13/33
DNA S th i
cDNA Synthesis
-
8/2/2019 School- Diagnosis of Genetic Disorders
14/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
15/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
16/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
17/33
-
8/2/2019 School- Diagnosis of Genetic Disorders
18/33
-
8/2/2019 School- Diagnosis of Genetic Disorders
19/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
20/33
Hybridization
-
8/2/2019 School- Diagnosis of Genetic Disorders
21/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
22/33
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.
-
8/2/2019 School- Diagnosis of Genetic Disorders
23/33
-
8/2/2019 School- Diagnosis of Genetic Disorders
24/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
25/33
-
8/2/2019 School- Diagnosis of Genetic Disorders
26/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
27/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
28/33
Southern Blotting
Pathogenesis of-Thalassaemia
-
8/2/2019 School- Diagnosis of Genetic Disorders
29/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
30/33
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
-
8/2/2019 School- Diagnosis of Genetic Disorders
31/33
-
8/2/2019 School- Diagnosis of Genetic Disorders
32/33
-
8/2/2019 School- Diagnosis of Genetic Disorders
33/33