Hematological principles

1. Dr. Freddie Bwanga
M.B;Ch.B, M. Med (Microb), Ph.D.
07th Nov 2024
Introduction to PCR Diagnostics
(a.k.a Molecular Diagnostics
or NAATs for Nucleic Acid Amplification Tests)
PCR = Polymerase Chain reaction

2. Definition of
PCR/Molecular Diagnostics
Detection and/or analysis of nucleic
acid molecules (DNA or RNA) to
provide clinical information
RNA = Ribonucleic acid
DNA = Deoxyribonucleic acid

3. DNA discovery
In the late 1940's, the scientific community was aware
that DNA was most likely the molecule of life.
Many were skeptical since this sounded so simple.
They also knew that DNA included the four bases
adenine, thymine, guanine and cytosine (A, T, G and
C), but nobody had the slightest idea of what the
molecule might look like!
DNA = Deoxyribonucleic acid

4. Discovery of the Double Helix DNA Structure
A Scientific Breakthrough

5. Maurice Wilkins
Discovery of the Double Helix DNA Structure
A Scientific Breakthrough
"This structure has novel features which are of considerable biological interest"
may be one of science's most famous understatements!
………..James Watson and Francis Crick, April 1953
1962 - James Watson & Francis Crick shared the Nobel Prize in Physiology/Medicine with Maurice
Wilkins, for solving one of the most important of all biological problems

6. DNA Double Helix Structure

7. Nitrogenous Bases in DNA and RNA

8. Components of RNA Backbone Structure

9. Components of DNA Backbone Structure

10. Discovery of PCR
1983 Kary Mullis invented the polymerase
chain reaction (PCR)
1993 Awarded a Nobel Prize
Kary Mullis

11. PCR
PCR = Polymerase Chain reaction
PCR allows us to amplify a few copies of DNA/RNA
segments in a specimen into millions - billions of
copies within a few hours, which are then detected
…….to confirm presence or absence of the targeted
pathogen or genetic segment

12. 1. Reaction buffer (10x)
2. Magnesium Chloride (25mM)
3. Primers – Forward and reverse oligonucleotides 15-30 bases
4. Thermos table DNA polymerase
5. Deoxynucleotide triphosphates (dNTP’s)
6. Target DNA
Contents are mixed in thin walled PCR tubes and the
reaction is placed in a thermal cycler
PCR Reaction Mix

13. dNTP vS ddNTP

14. What Exactly is detected at
PCR/Molecular Diagnostics?
In PCR/Molecular Diagnostics, we detect;
1. Specific DNA or RNA sequences of a gene.
……..Could be a signature DNA/RNA sequence which
if detected = presence of pathogen or a novel
gene
OR
2. Single nucleotide point mutation(s) that may or may
not be associated with disease/drug resistance

15. Monday, December 9, 2024 15
The average size of a human gene is about 62 kilobases
(kb), median length 24 kb
Gene Detection

16. Mutation Detection

17. Each PCR assay requires 4 basic steps in 4
independent self-contained Lab rooms:
1. PCR Lab 1: Reagent Mix Lab
2. PCR Lab 2: Extraction & Purification of nucleic
acids (Manual/automated)
3. PCR Lab 3: Amplification or making copies of the
nucleic acid of interest (target)
4. PCR Lab 4: Detection of the amplified products in
the PCR reaction tube

18. Reagent Example of
volumes (µl)
per reaction
tube
Buffer
MgCl2
Primer – Forward 5’-3’
Primer – Reverse 3’-5’
Taq Polymerase
Probe (in case of Real Time PCR)
Total vol. per sample 10, or 25 or 50
PCR Lab 1: Reagent mix/Recipe

19. Basic PCR Recipe
• Reaction buffer (Tris-HCl, ammonium ions, KCl), magnesium
ions, bovine serum albumin)
The buffer provides the ionic strength and buffering capacity needed during the
reaction
• MgCl2: 1.5 - 3mM - For optimal functioning of Taq polymerase
• dNTPs: Equimolar ratios, 200 µM each dNTP
• Primers: 0.1 and 0.5 µmol
• DNA polymerase: 1-2 units/25 µl reaction

20. PCR Lab 2: DNA/RNA Extraction

21. Location of DNA/RNA in Cells
HUMAN CELLS
SARS-CoV-2 virus
Hepatitis C virus Hepatitis B virus
HIV virus
PCR Lab 2: DNA Extraction… continued

22. PCR Lab 2: DNA/RNA Extraction….continued
Extraction & Purification of nucleic acid
Kits e.g QUIAGEN
In-house Methods e.g
•Boiling method,
•Phenol-chloroform
Automated e.g. Roche, Hain,
QIAGEN
Note: The Extracted DNA is quantified and added
to PCR reaction reagents/tubes from this lab room

23. Addition of DNA to the PCR Reaction Tube
The extracted DNA/RNA Target is then added to PCR tubes from Lab 2

24. Thermocyclers
PCR tubes with reagents and extracted DNA
ready for Amplification in a thermocycler
PCR Lab 3: DNA / RNA Amplification
….or making copies of the nucleic acid of interest (target)

25. Inside the Thermocycler during a typical PCR reaction:
The 3 repeating steps of PCR
DENATURATION
ANNEALING
EXTENSION
95°C
60°C
72°C
DENATURATION
ANNEALING
EXTENSION
60°C

26. PCR cycle 1
Target region
Flanking region
Primer binding sites

27. PCR cycle 2

28. PCR cycle 3
By around cycle 30: 1,073,741,793 DNA copies in the tube

29. PCR Lab 4
Lab for Detection of Amplified
DNA/RNA products (a.k.a. Amplicons)

30. Methods for Detection of
amplification products
1. Detection on a gel: Gel Electrophoresis
2. Nucleic Acid Hybridization - with probes in solution
or on nitrocellulose membrane. Detects gene/DNA
segments or point mutations e.g Line-probe assays, Dot
blot, etc.
3. Real-time PCR detection – detects amplification products;
either long gene/DNA sements or point mutations in real time
4. Sequencing: Exact mutations are detected. Amplified DNA segment
with mutations is sequenced and compared with the wildtype gene segment

31. Gel Electrophoresis

32. Gel Electrophoresis
Detection of the mecA gene in S. aureus
500bp
162bp mecA
L MP- M+ P+ B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
©Bwanga 2006
S. aureus acquisition of the mecA gene causes MRSA i.e. the S.
aureus bacteria become resistant to all penicillins,
cephalosporins and carbapenenms

33. Nucleic Acid Hybridization
e.g. Line-probe assays for MDR-TB
Presence of point mutations in
the rpoB gene causes
resistance to Rifampicin (Rif)
Point Mutations in the katG
and inhA genes cause
resistance to Isoniazid (INH) in
MTB
Resistance to both Rif and INH
= MDR-TB

34. Real-time PCR Detection

35. DNA Sequencing
Exact Point Mutations are detected

36. PCR/Molecular Diagnostics Applications in day-to-day
Patient/Clinical Diagnostics
1. Diagnosis of infectious diseases
2. Diagnosis of Drug resistance
3. Diagnosis of non-infectious diseases
4. Genetic Typing of Microorganisms
5. Biological relationship Testing e.g.
 DNA Paternity/Maternity Testing
 DNA Relationship Testing
 Forensics
6. GM foods research
7. Drug discovery e.g. insulin
8. Research
8. E.t.c