This document discusses diagnostic techniques for genetic disorders, including cytogenetic tests like fluorescence in situ hybridization (FISH) and karyotyping, as well as molecular tests like polymerase chain reaction (PCR), DNA microarray, DNA sequencing, and restriction fragment length polymorphism (RFLP). It provides examples of how each technique can be used to detect genetic abnormalities associated with diseases like cystic fibrosis, cancer, and HIV/AIDS. Real-time PCR is also described as a method for quantifying gene expression levels and monitoring viral load during HIV treatment.

1. Diagnostic techniques for genetic disorders
&
Recombinant DNA Technologies
Dr Sehrish ahmed

2. Objectives
CAUSES OF GENETIC PROBLEMS TYPES OF DIAGNOSTIC TECHNIQUES
UNDERSTANDING DIFFERENT
TECHNIQUES AND THEIR
IMPLICATION IN CLINICAL PRACTICE

3. • Genetic diseases are usually categorized into
• Single-gene (mendelians disorder)
• Chromosomal (chromosomal disorders)
• Multi-factorial disorders (mix with
environmental factor)

4. Categories of genetic testing
cytogenetic
testing
molecular
testing

5. CYTOGENIC TESTS:
FLUORESCENCE IN SITU
HYBRIDIZATION (FISH)
01
SPECIAL KARYOTYPING
02

6. FISH

7. 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).

8. Karyotyping
Easier interpretation and better
resolution
Types of chromosome painting:
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)

9. Philadelphia
chromosome
rearrangement (9,22)
involved in chronic
myelogenous leukemia

10. Match the Following
(based on the type of
chromosome painting)
1. Chromosome enumeration probes
2. Whole chromosome painting probe
3. Chromosome arm painting probes
4. Chromosome terminal band painting
probes
A B
C D

11. Molecular
testing

12. 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

13. PCR
The Polymerase
Chain Reaction

14. Overview of PCR
1. Temperature Cycling
• Denaturation 94°
• Annealing 55°
• Extension 72°
2. Every Cycle DNA between
primers is duplicated

15. Thermo-stable polymerase
Taq Polymerase

16. Link to Dolan DNA Learning Center's PCR Animation

17. PCR and
Disease
Some diseases
that can be
diagnosed with
the help of
PCR:
Huntington's disease
cystic fibrosis
Human immunodeficiency
virus

18. Cystic Fibrosis
(CF)
CF is caused by mutations in
the cystic fibrosis
transmembrane conductance
regulator (CTFR) gene.
In non-CF individuals, the
CTFR gene codes for a protein
that is a chloride ion channel
and is involved in the
production of sweat, digestive
juices and mucus.
In CF individuals, mutations in
the CTFR gene lead to thick
mucous secretions in the
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.

20. Real-Time PCR
in Gene
Expression
Analysis
Example: BRCA1 Expression Profiling
BRCA1 is a gene involved in tumor suppression.
BRCA1 controls the expression of other genes.
In order to monitor level of expression of BRCA1,
real-time PCR is used.
DNA
mRNA
Protein
BRCA1

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

22. Microarray
Perform genetic analysis of many
genes at one time

23. DNA Sequencing
Dye-labeled dideoxynucleotides are used to generate DNA fragments of different lengths

24. Restriction
fragment length
polymorphism
(RFLP)

25. RECOMBINANT DNA TECHNOLOGY

28. 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”).