1. Characteristics of the Genetic Material
Any substance which form the heriatable material must fulfill some essential requirements and DNA was found to fulfill them all.
1- It is stable .
2- It is able to carry and transcribe information which are required to control the processes which give the organism its specificity .( transcription )
3- It is capable of replicating exactly, so that the genetic determinants are transmitted down from cell to cell and from generation to generation unchanged .
4- It is able to mutate to give more variations .

2. Genome: entire complement of DNA molecules of each organism
Overall function of genome: Control the generation of molecules (mostly RNA & proteins) that will regulate the cell function and structure Transfer the genetic information from cell to cell ( cell division ) and from generation to generation without change .

3. DNA Structure DNA is a nucleic acid.
The building blocks of DNA are nucleotides, each composed of:
a 5-carbon sugar deoxyribose
a phosphate group (PO4)
a nitrogenous base
adenine, thymine, cytosine, guanine

4. Ribose and Deoxyribose Ribose in RNA Deoxyribose in DNA

5. Purines Pyrimidines Uracil CH3 N O NH Thymine N Adenine NH2 NH2 O N NH
(RNA)
CH3 N O NH
Thymine
(DNA) N
Adenine
NH2
NH2 O N NH
Guanine N O
NH2
Cytosine

6. Nucleosides and Nucleotides
Nucleosid is a base+sugar
Nucleotide is nucleoside + phosphate
Nitrogen base is connected on C-1’ position of sugar
Phosphate commonly on C-5’ of sugar

7. DNA Structure
Nucleotides are connected to each other to form a long chain
phosphodiester bond: Covalent bond between adjacent nucleotides
formed between the phosphate group (attached to 5’ carbon) of one nucleotide and the 3’ –OH of the of sugar of next nucleotide
This bond is very strong, and for this reason DNA is remarkably stable.
DNA can be boiled and even autoclaved without degrading
The chain of nucleotides has a 5’ to 3’ orientation.

8. Double Helix of DNA
The clue to the structure of DNA came from two sourses:
the work of Erwin Chargaff and his colleagues in the late 1940s and Rosalind Franklin and Maurice Wilkins work.
Base composition studies of Erwin Chargaff:
1. The base composition of DNA generally varies from one species to another.
2. DNA specimens isolated from different tissues of the same species have the same base composition.
3. The base composition of DNA in a given species does not change with an organism’s age,nutritional state, or changing environment .
4. In all cellular DNAs, regardless of the species, the number of adenosine residues is equal to the number of thymidine residues (that is, A T) ,and the number of guanosine residues is equal to cytosine.
Chargaff’s Rule:
amount of adenine = amount of thymine
amount of cytosine = amount of guanine

9. Chargaff’s Rule

10. Double Helix of DNA Rosalind Franklin and Maurice Wilkins 1950s
Franklin performed X-ray diffraction studies to identify the 3-D structure
discovered that DNA is helical
discovered that the molecule has a diameter of 2nm and makes a complete turn of the helix every 3.4 nm
X-ray diffraction pattern of DNA. The spots forming a
cross in the center denote a helical structure. The heavy bands at the left and right arise from the recurring bases.

11. The Watson - Crick Model Of DNA
1953 propose double helix model
Right-handed double helix
Chains antiparallel
Bases lie flat, perpendicular to long axis of chain
Bases pair by hydrogen bonds, A with T and C with G
Two strands are complementary
10 bases per turn (34 angstroms)
Now known to be 10.4 or 34.6 degrees turn per bp)
Has a major and minor groove
Is 20 angstroms in diameter

12. complementary base pairing involve the formation of two hydrogen bonds between adinine and thymine, three hydrogen bonds between gauine and cytosine.No other paire form in DNA

13. Space-filling model
Schematic model
The double helix consists of:
2 sugar-phosphate backbones
nitrogenous bases toward the interior of the molecule
bases form hydrogen bonds with complementary bases on the opposite sugar-phosphate backbone

14. The DNA Double Helix General structural features
The double-bonded structure is stabilized by
1. Hydrogen bonding between complementary bases
A bonded to T by two hydrogen bonds
C bonded to G by three hydrogen bonds
2. Base stacking
Within the DNA, the bases are oriented so that the flattened regions are facing each other

15. The DNA Double Helix General structural features
There are two asymmetrical grooves on the outside of the helix
1. Major groove
2. Minor groove
Certain proteins can bind within these grooves
They can thus interact with a particular sequence of bases

16. The DNA Double Helix General structural features
Two strands are twisted together around a common axis
There are 10 bases per complete twist
The two strands are antiparallel
One runs in the 5’ to 3’ direction and the other 3’ to 5’
The helix is right-handed
As it spirals away from you, the helix turns in a clockwise direction

17. DNA Double helix Coding strand 5’→ 3’. Non-coding strand 3’ → 5’.
Stores genetic code as a linear sequence of bases.
≈ 20 Å in diameter
Human genome ≈ 3.3 x 109 bp
≈ 25,000 genes

18. DNA Characterization Absorption Spectra
Absorb light in ultraviolet range, most strongly in the nm range
Due to the purine and pyrimidine bases.
Useful for localization, characterization and quantification of samples

19. DNA Characterization Denaturation of DNA
Denaturation involves the breaking of hydrogen bonds
Disrupts the base stacking in the helix and lead to increased absorbance at 260 nm(Hyperchomic effect)
By increasing temperature slowly and measuring absorbance at 260 nm as melting profile can be generated
Temperature for midpoint of denaturation is called the Tm
Melting Temperature (Tm): Temperature at which 50% of the dsDNA is denaturated to ssDNA.

20. Renaturation of DNA HEAT
When ssDNA is cooled graudly hydrogen bonds between bases can reform and the DNA renatures.
During the renaturation, absorbance at 260nm is decreasing
(Hypochromic effect).
TACTCGACATGCTAGCAC
ATGAGCTGTACGATCGTG
HEAT
Denatured DNA
Denaturation
Single stranded DNA
TACTCGACATGCTAGCAC
ATGAGCTGTACGATCGTG
Double stranded DNA
Renaturation
TACTCGACATGCTAGCAC
ATGAGCTGTACGATCGTG
Double stranded DNA

21. Denaturation of DNA
Denaturation Can be monitored by measuring absorption absorbance at 260nm.
When 2 strands are separated, absorbance may increase by 30-40%.
For dsDNA, A260=1.0 for 50 µg/ml
For ssDNA and RNA A260=1.0 for 38 µg/ml

22. DNA Denaturation Factors Affecting Tm G-C content of sample
Increased G+C gives increased Tm
3 vs. 2 hydrogen bonds
Presence of intercalating agents (anything that disrupts H-bonds or base stacking)
Salt concentration
- Increased ionic strength also increases Tm pH
Length of the molecule

23. Determination of GC Content
OD260
1.0
Temperature (oC)
Double stranded DNA
Single stranded DNA
Tm is the temperature at which half the DNA is melted
Relatively low GC content
Relatively high GC content
Tm = 75 oC
Tm = 85 oC