Introduction to Nucleic acids General features of nucleic acids structural organization in eukaryotic cells Human Genome variations Single Nucleotide Polymorphisms (SNPs) Nucleic acid detection techniques PCR RT-PCR DNA sequencing Electropherosis RFLP

1. 1
Clinical Laboratory Principle

2. Nucleic Acids Techniques
Nucleic acids
Nucleic acids structure and organization
Eukaryotic cells
Central dogma of Molecular Biology
Nucleic acid sequence variation
Single Nucleotide polymorphism (SNPs)
 Techniques
2

3. Nucleic Acid
• Overall name for DNA and RNA
•Biopolymers/ large biomolecules
•DNA /RNA is synthesized from monomers of
Nucleotides
• Nucleotide Composition
Sugar = Deoxyribose or Ribose
Bases = Purine
Pyrimidine
Phosphate Residue 3

4. Nucleic Acid Structure
• Four nucleotide building blocks of DNA
1- Purines : Two carbon-nitrogen rings
Adenine = (A) , dATP (deoxyadenine-triphosphated)
Guanine =(G), dGTP(deoxyguaninetriphosphate)
2- Pyrimidines : one carbon-nitrogen rings
Cytosine = (C) , dCTP (deoxycytosine-triphosphated)
Thymine = (T), dTTP (deoxythymine-triphosphated)
4

5. Nucleic Acids structure
5

6. Structure of DNA
Adenine Thymine
Cytosine Guanine
C --- G
T --- A
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7. Nucleic acids structure and organization in Eukaryotic cells
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8. Karyogram of a Human Male
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9. Central dogma of molecular biology
The central dogma specifies that biological information is
transferred from DNA to RNA to protein
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10. Organization of the Human Genome
Human Genome
Nuclear Genome
=3000 Mb
Mitochondrial Genome
=16.6 Kb
Genes and Gene-
related sequences
~20%
20,000-25,000 genes
Long Interspersed
Nuclear Elements
LINE
Alu Repeats Microsatellites
Extragenic DNA
~80%
e.g.
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11. Nucleic Acid
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12.  If the DNA of any two individuals is compared, there is on
average one difference every 1250 bases
(i.e., approximately 99.9% of the sequence is identical)
 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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13. 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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14. About 70% of human mutations are SNPs and most of
these are missense or nonsense mutations.
Human Variations
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15.  Only 9% of disease-causing mutations are SNPs that
affect splicing sites and result in altered concatenation of
coding sequences.
 less than 1% of Known disease-causing mutations are
SNPs that affect the regulatory efficiency of transcription
by altering promoter/enhancer regions in introns or the
stability of the RNA transcript.
Human Variations
15

16. Definition:
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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17. 17

18. 18

19. 19

20. DNA in the Cell
Target Region
chromosome
cell nucleus
Double stranded
DNA molecule
Individual
nucleotides
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21. Nucleic Acid Techniques
Polymerase Chain Reaction (PCR)
DNA Sequencing
Restriction Fragment Length Polymorphism
Real-Time PCR
Electrophoresis
Hybridization
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22. Polymerase Chain Reaction
(PCR)
Target Region for PCR
chromosome
cell nucleus
Double stranded
DNA molecule
Individual
nucleotides
22

23. 23

24. 24

25. 25

26. 26

27. 27
Reference:
Tietz Fundamentals of Clinical Chemistry,
Sixth Edition. Chapter 17