Diagnostic techniques for genetic disorders

& Dr Sehrish ahmed

Recombinant DNA Technologies
 CAUSES OF GENETIC PROBLEMS TYPES OF DIAGNOSTIC TECHNIQUES

Objectives

UNDERSTANDING DIFFERENT
TECHNIQUES AND THEIR
IMPLICATION IN CLINICAL PRACTICE
• Genetic diseases are usually categorized into
• Single-gene (mendelians disorder)

• Chromosomal (chromosomal disorders)

• Multi-factorial disorders (mix with

environmental factor)
Categories of genetic testing

cytogenetic molecular
testing testing
 CYTOGENIC TESTS:

01 02
FLUORESCENCE IN SITU SPECIAL KARYOTYPING
HYBRIDIZATION (FISH)
FISH
Applications
• To detect chromosomal aberrations (additions, deletions, insertions,
inversions, copy number, translocations).
• Used in prenatal diagnosis (of Down syndrome etc.) and cancer
diagnosis (to detect TATAs and TSTAs).
 Karyotyping

Easier interpretation and better Types of chromosome painting:

resolution
1. Whole chromosome painting
probe (WPP)
2. Chromosome arm painting
probes (APP)
3. Chromosome terminal band
painting probes (TPP)
4. Chromosome enumeration
probes (CEP; paints near
centromere)
Philadelphia
chromosome
rearrangement (9,22)
involved in chronic
myelogenous leukemia
Match the Following
(based on the type of
chromosome painting)

1. Chromosome enumeration probes A B

2. Whole chromosome painting probe
3. Chromosome arm painting probes
4. Chromosome terminal band painting
probes

C D
Molecular
testing
TECHNIQUES
• It combines laboratory medicine with molecular genetics to
develop DNA/RNA-based analytical methods for monitoring
human pathologies.
• A wide range of methods has been used for mutation detection
• Known mutation
• PCR
• DNA Microarray
• DNA Sequence
• Unknown mutation
• SSCP
• DGGE
• RFLP
 PCR
The Polymerase
Chain Reaction
Overview of PCR
1. Temperature Cycling

• Denaturation 94°
• Annealing 55°
• Extension 72°

2. Every Cycle DNA between

primers is duplicated
Thermo-stable polymerase
Taq Polymerase
Link to Dolan DNA Learning Center's PCR Animation
 Some diseases
PCR and that can be
diagnosed with
Huntington's disease
cystic fibrosis

Disease the help of

PCR:
Human immunodeficiency
virus
 CF is caused by mutations in
the cystic fibrosis
transmembrane conductance
regulator (CTFR) gene.

In non-CF individuals, the

Cystic Fibrosis CTFR gene codes for a protein
that is a chloride ion channel
In CF individuals, mutations in
the CTFR gene lead to thick
mucous secretions in the
and is involved in the
(CF) production of sweat, digestive
juices and mucus.
lungs and subsequent
persistent bacterial infections.

The presence of CTFR mutations in a individual can be detected by

performing PCR and sequencing on that individual’s DNA.
Real-Time PCR Example: BRCA1 Expression Profiling
in Gene
Expression BRCA1 is a gene involved in tumor suppression.
Analysis BRCA1 controls the expression of other genes.
In order to monitor level of expression of BRCA1,
real-time PCR is used.

DNA
BRCA1
mRNA

Protein
Real-Time Example: HIV Treatment
PCR in
Disease Drug treatment for HIV infection often depends on
Management monitoring the “viral load”.
Real-Time PCR allows for direct measurement of the
amount of the virus RNA in the patient.

Virus
RNA
 Microarray
Perform genetic analysis of many
genes at one time
 DNA Sequencing
Dye-labeled dideoxynucleotides are used to generate DNA fragments of different lengths
 Restriction
fragment length
polymorphism
(RFLP)
RECOMBINANT DNA TECHNOLOGY
Some cool PCR links
• The Dolan DNA Learning Center:
Link to Dolan DNA Learning Center's PCR Animation
This site provides a nice step by step guide to how DNA is copied in PCR reaction.
• DNA Interactive:
Link to DNAi
This site is FULL of cool stuff! Two 3-D animation videos relevant to this Powerpoint presentation are:
• The DNA Replication animation (to get to this, click on the above link, then click on “code”, then
click on “copying the code”, then click on “putting it together”, and finally click on “replication”).
• The PCR animation (to get to this, click on the above link, then click on “manipulation”, then
click on “techniques”, the click on “amplifying” and then finally click on “PCR animation”).