This document discusses nucleic acid techniques used in clinical laboratories. It begins by introducing nucleic acids, their structure, and their roles in storing and transmitting genetic information. It then describes the structures of DNA and RNA, including their nucleotide composition and base pairing. Various nucleic acid analysis techniques are introduced, including polymerase chain reaction (PCR), DNA sequencing, restriction fragment length polymorphism, real-time PCR, electrophoresis, and hybridization. PCR is discussed in detail, outlining its principles, components, steps of DNA denaturation, annealing and extension, and exponential amplification of target DNA sequences.

1. Nucleic Acids Techniques
Course: Clinical Laboratory Principle (SIMS-443)
ZA School of Medical Technology
1
Dr. Ali Raza
Senior Lecturer
SIMS-SIUT

2. Nucleic Acids Techniques
Nucleic acids
Nucleic acids structure and organization
Eukaryotic cells
Central Dogma of Molecular Biology
Nucleic acid sequence variation
 Techniques
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3. Nucleic Acid:
Main information-carrying molecules
 Makeup the genetic material
Function:
Store information of every living cell
 Express that information inside and outside the cell
nucleus to the interior operations of the cell
 Transmit information to the next generation of
each living organism
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4. Nucleic Acids structure
•Two main classes of nucleic acids are
Deoxyribonucleic acid (DNA)
Ribonucleic acid (RNA)
•Biopolymers/ large biomolecules
• Synthesized from monomers of Nucleotides
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5. 5
Nucleotide Composition
Phosphate Residue
Sugar = Deoxyribose or
Ribose
Bases = Purine
Pyrimidine
Ribose Sugar

6. Purines :
•Two carbon-nitrogen rings
Adenine = (A) , dATP (deoxyadenine-triphosphated)
Guanine =(G), dGTP(deoxyguaninetriphosphate)
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dATP dGTP

7. Pyrimidines :
one carbon-nitrogen rings
Cytosine = (C) , dCTP (deoxycytosine-triphosphated)
Thymine = (T), dTTP (deoxythymine-triphosphated)
Uracil = (U), dUTP (deoxyuridine-triphosphated)
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dCTP dTTP dUTP

8. Nucleic Acid Structure
• Four nucleotide building blocks DNA/RNA
 Purines :
Adenine = (A)
Guanine =(G)
 Pyrimidines :
Cytosine = (C)
Thymine = (T)
Uracil = (U)
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9. 9
Nucleic Acids Structure
DNA is synthesized in the 5'-to-3' direction
Phosphodiester bonds

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Structure of DNA
Purine Pyrimidine
Complementary

11. Structure of DNA
Adenine Thymine
Cytosine Guanine
C --- G
T --- A
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Antiparallel alignment

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Chargaff's rules
“ State that DNA from any species of any organism should
have a 1:1 stoichiometric ratio of purine and pyrimidine
bases”
 Amount of guanine should be equal to cytosine and
the amount of adenine should be equal to thymine.

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Chargaff's rules
20% A = 20% T
30% G = 30% C

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Chargaff's rules
A DNA molecule has 18 nucleotide base pairs.
Calculate the
A= (n )%
G= (n )%
C= (n )%
T= (n )%
Apply Chargaff's rule

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Structure of DNA
 Double helix
A DNA molecule consists of two strands that wind around
each other like a twisted ladder.
 Each strand has a backbone made of alternating
groups of sugar (deoxyribose) and phosphate
groups.
 Attached to each sugar is one of four bases:
adenine (A), cytosine (C), guanine (G), or thymine (T).
 The two strands are held together by bonds between
the bases, adenine forming a base pair with thymine,
and cytosine forming a base pair with guanine.

16. Nucleic acids structure and organization in Eukaryotic cells
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17. Karyogram of a Human Male
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18. Central dogma of molecular biology
The central dogma specifies that biological information is
transferred from DNA to RNA to protein
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19. Nucleic Acid
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20.  Interestingly, approximately 99.9% of the sequence is
identical
 If the DNA of any two individuals is compared, there is on
average one difference every 1250 bases
 Many sequence variants, alterations, and polymorphisms in
the genome do not affect human health and are benign or
silent.
Human Variations
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21.  Any sequence change (compared to a reference
sequence) is called a sequence variant or alteration.
 If a sequence variant or alteration is present at a
frequency of at least 1%, it is a poly- morphism.
 The most common sequence variations are single base
changes, also known as single nucleotide polymorphisms
(SNPs).
Single Nucleotide Polymorphisms
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22. Although an SNP has been identified every 100 to 300 bases,
many of these are not found frequently in the population.
Majority of SNPs (97%) occur in non-coding regions
only 3% of SNPs are associated with exons.
Human Variations
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25. 25

26.  About 70% of human mutations are SNPs
 Missense or nonsense mutations.
Human Variations
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27. Nucleic Acid Techniques
Polymerase Chain Reaction (PCR)
DNA Sequencing
Restriction Fragment Length Polymorphism
Real-Time PCR
Electrophoresis
Hybridization
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28. Polymerase Chain Reaction
(PCR)

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Its principle is based on the use of DNA polymerase
which is an in vitro replication of specific DNA
sequences.
Polymerase Chain Reaction
(PCR)

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 This method can generate tens of billions of copies of
a particular DNA fragment (the sequence of interest,
DNA of interest, or target DNA) from a DNA extract.
(DNA template).
Polymerase Chain Reaction
(PCR)
Target Region for PCR

31. Polymerase Chain Reaction (PCR)
Components
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Thermostable DNA polymerase
(Taq polymerase)
DNA primers (18-22 bases)
PCR Buffer
dNTPs (dATPs, dGTPs, dCTPs, dTTPs)
Cations (Mg++)
Samples (DNA/RNA)

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Steps:
Denaturation
Annealing
Extension
Polymerase Chain Reaction
(PCR)

33. 33
Polymerase Chain Reaction
Thermal Cycler

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35. 35
PCR Machine and Program

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39. 39
 PCR cycles are set 30-40.
 At the end of each PCR
cycle, the PCR product or
amplicon will increase
exponentially because the
newly synthesized DNA
sequences can be used as
templates (in addition to the
original DNA template).
Polymerase Chain Reaction

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Assignments:
What are the applications of PCR techniques

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Reference:
Tietz Fundamentals of Clinical Chemistry,
Sixth Edition. Chapter 17