1. Mamoun Ahram, PhD Second semester, 2015-2016
Molecular Biology (1)
Mamoun Ahram, PhD
Second semester,

2. Pay attention to this…
You are allowed a total absence of 15% of total credit hours (7 days) without excuses and 20% (9 days) with excuses.
If you exceed this limit, you will fail the class regardless of the 10% score. The two are independent of each other.
No points will be subtracted from your 10% for the first 3 absences.

3. Content for molecular biology
Nucleic acid structure
Restriction endonucleases, RFLP, DNA fingerprinting, recombinant technology, and DNA cloning
DNA replication
DNA sequencing and PCR
DNA mutations
DNA repair
RNA transcription and its regulation
Protein synthesis and its regulation
Cancer: a molecular perspective

4. Resources This lecture
Campbell and Farrell’s Biochemistry, Chapter 9, 13 (pp )

5. Nucleic acids
The primary structure of nucleic acids is the order of bases in the polynucleotide sequence.
The secondary structure is the three-dimensional conformation of the backbone.
The tertiary structure is specifically the supercoiling of the molecule.

6. Chemical composition and bonds
Glycosidic bond
This is why they are acidic
Ribose vs. deoxyribose
Positively charged ions (Na+ or Mg2+) and peptides with positively charged side chains can associate with DNA
Eukaryotic DNA, for example, is complexed with histones, which are positively charged proteins, in the cell nucleus.

7. Nitrogenous bases
Glycosidic bond

8. Nucleotides vs. Nucleosides

9. Nucleotides vs. Nucleosides

10. Nucleic acid polymer
A letter d can be added to indicate a deoxyribonucleotide residue.
for example, dG is substituted for G.
The deoxy analogue of a ribooligonucleotide would be d(GACAT).

11. DNA structure Specific base-pairing Complementary A double helix
A = T; G = C; Pur = pyr
Complementary
A double helix
Backbone vs. side chains
Antiparallel
Stable
Flexible
Groovings
Stability vs. flexibility

12. Base pairing

13. DNA forms B-DNA The principal form of DNA. Right-handed
Base pairs are perpendicular.
A-DNA
11 base pairs per turn
Base pairs lie at an angle
Right-handed.
Wider than B-DNA
Z-DNA
Left-handed
Occurs when alternating purine–pyrimidine and sequences with methylated C
Narrower than B-DNA

14. DNA structure Specific base-pairing Complementary A double helix
A = T; G = C; Pur = pyr
Complementary
A double helix
Backbone vs. side chains
Antiparallel
Stable
Flexible
Groovings
Stability vs. flexibility

15. DNA coiling
The bacterial circular DNA can be coiled by rotating it clockwise (negative supercoiling) or counterclockwise (positive supercoiling).
Supercoils can be added or removed by enzymes called topoisomerases:
Class I topoisomerases cut the phosphodiester backbone of one strand of DNA.
Class II topoisomerases cut both strands of DNA.
In bacteria, it is called DNA gyrase.

16. In eukaryotes…
In eukaryotes, DNA coiling is utilized for DNA packaging and regulation of gene activity.
Eukaryotic DNA is complexed with a number of proteins.
Chromatin = DNA molecule + proteins
The principal proteins in chromatin are the histones.

17. Nucleosomes
The protein core is an octamer (two molecules of histones H2A, H2B, H3, and H4).
A linker DNA/spacer region connects the octamer-DNA complexes.
A nucleosome consists of DNA wrapped around a histone core.
H1 is bound to the the octamer and wrapped DNA (a chromatosome).
Histones are positively cahrged facilitating DNA interaction and charge neutralization.

18. Light absorbance of nucleic acids
Aromatic pyrimidines and purines can absorb UV light
The peak absorbance is at 260 nm wavelength
The absorbance of 260 nm (A260) is constant
dsDNA: A260 of 1.0 = 50 ug/ml
ssDNA: A260 of 1.0 = 30 ug/ml
ssRNA: A260 of 1.0 =40 ug/ml
Reason for ss vs. ds absorbance:
Stacked bases, vs. unstacked bases
hyperchromicity
What is the concentration of a double stranded DNA sample diluted at 1:10 and the A260 is 0.1?
DNA concentration = 0.1 x 10 x 50 µg/ml
= 50 µg /ml

19. Observation of denaturation
The transition temperature, or melting temperature (Tm).
Factors influencing Tm
G·C pairs
Hydrogen bonds
Base stacking pH
Salt and ion concentration
Destabilizing agents (alkaline solutions, formamide, urea)

20. Central dogma of biology
Genetic information must be preserved via DNA replication.
Information must be translated into action makers (proteins) via transcription and translation.
RNA Sequence is dictated by DNA sequence.

21. RNA
Consist of long, unbranched chains of nucleotides joined by phosphodiester bonds between the 3’-OH of one pentose and the 5’-OH of the next
The pentose unit is β-D-ribose (it is 2-deoxy-D-ribose in DNA)
The pyrimidine bases are uracil and cytosine (they are thymine and cytosine in DNA)
In general, RNA is single stranded (DNA is double stranded).

22. Types of RNA

23. Hybridization
DNA from different sources can form double helix as long as their sequences are compatible (hybrid DNA)

24. Techniques
Gel electrophoresis
Hybridization techniques

25. Gel electrophoresis - - +
The length and purity of DNA molecules can be accurately determined by the gel electrophoresis
wells - -
Direction DNA travels +

27. Resources http://www.personal.psu.edu/pzb4/electrophoresis.swf

28. Detection
The DNA molecules of different lengths will run as "bands“
DNA is stained (that is, colored) with a dye (ethidium bromide) or radioactively labeled (32P)
Size Standard
Sample 1
Sample 2 -
1000 bp
850 bp
750 bp
600 bp
200 bp
100 bp +

29. Hybridization techniques
Hybridization reactions can occur between any two single-stranded nucleic acid chains provided that they have complementary nucleotide sequences
Hybridization reactions are used to detect and characterize specific nucleotide sequences

30. Probes
A probes is a short sequence of single stranded DNA (an oligonucleotide) that is complementary to a small part of a larger DNA sequence
Hybridization reactions use labeled DNA probes to detect larger DNA fragments

31. Southern blotting
This technique is a combination of DNA gel electrophoresis and hybridization
Used to detect:
the presence of a DNA segment complementary to the probe
the size of the DNA fragment

33. 1 2 1 2 1 2
: probe
Electrophoresis
Southern blot

34. Restriction endonucleases, RFLP, and gene cloning

35. Resources This lecture
Campbell and Farrell's Biochemistry, Chapter 13, pp ;

36. Endonucleases
Enzymes that degrade DNA within the molecule rather than from either end (exonucleases)
Restriction endonucleases: Enzymes that recognize and cut (break) the phosphodiester bond between nucleotides at specific sequences (4- to 8-bp restriction sites) generating restriction fragments.
Type II restriction endonucleases: Always cleave always at the same place generating the same set of fragments
EcoRI (isolated from E. coli) cuts at 5'-GAATTC-3‘
Some enzymes cut DNA at related sites
HinfI (from Haemophilus influenzae) recognizes 5'-ANTC-3' ('N' is any nucleotide)
Cuts at 5'-AATC-3', 5‘-ATTC-3', 5‘-AGTC-3' and 5'-ACTC-3’

37. Biological purpose of restriction endonucleases
They are present in bacteria to protect them from bacteriophages that infect bacteria by transferring their DNA into them restricting their growth.

38. Types of cleavages Restriction enzymes cut DNA in two different ways:
Blunt: enzymes cut at the same position on both strands giving a blunt ended fragments
Staggered (off-center): enzymes cut the two DNA strands at different positions generating sticky or cohesive ends
The DNA fragments have short single-stranded overhangs at each end.

39. 5’ vs. 3’ overhangs

40. Palindromic sequence
The sequences recognized by restriction endonucleases—their sites of action—read the same from left to right as they do from right to left (on the complementary strand).

41. DNA ligase Covalently joins DNA ends (example, restriction fragments)
Catalyzing the formation of 3’→ 5’ phosphodiester bonds between the 3-hydroxyl end of one strand and the 5-phosphate end of another strand

42. Advantage of restriction endonucleases
Restriction fragment length polymorphism (RFLP)
Cloning

43. DNA polymorphisms
Individual variations in DNA sequence may create or remove restriction-enzyme recognition sites generating different restriction fragments
Remember: our cells are diploid (alleles can be homozygous or heterozygous)
What is an allele?

45. Restriction fragment length polymorphism
The presence of different DNA forms in individuals generates a restriction fragment length polymorphism, or RFLP.
These can be detected by
Gel electrophoresis
Southern blotting

46. Example

47. RFLP in the clinic RFLP can be used as diagnostic tools.
For example, if a mutation that results in the development of a disease also causes the generation of distinctive RFLP fragments, then we can tell
if the person is diseased as a result of this mutation
from which parent this allele is inherited

48. Disease detection by RFLP

49. Think!! What would you see in a gel?

51. Example 1: Disease detection by RFLP (sickle cell anemia)

52. Normal/
carrier
Normal
Diseases
Father
Mother
Son1
Son2
Son3

53. Example 2: Disease detection by ASO (Cystic fibrosis)
ASO: Allele-specific oligonucleotide

54. Example 3: Paternity testing

55. Example 4: Forensics

56. Cloning Cloning means that you make several copies of one thing.
A clone is a genetically identical population, whether of organisms, cells, viruses, or DNA molecules.
Every member of the population is derived from a single cell, virus, or DNA molecule.

57. How do we clone a DNA molecule?
a DNA fragment of interest is inserted into a DNA carrier (called a vector) that can be replicated.
The resulting DNA molecule is what is known as a recombinant DNA molecule.

58. Using plasmids as vectors
Bacterial plasmids are considered excellent vectors.
These are bacterial circular DNA that is not part of the main circular DNA chromosome of the bacterium.
A plasmid exists as a closed circle and replicates independently of the main bacterial genome.

59. Features of plasmids Can replicate
Most plasmid vectors contain at least three essential parts required for DNA cloning:
Can replicate
Can be selected for/against by an internal drug-resistance gene (selectable marker)
Can inset a foreign DNA fragment

60. Making of recombinant DNA
Both DNA fragments (the DNA to be cloned and a vector) are cut by the same restriction endonuclease that makes sticky-ended DNA fragments
When mixed, they will bind to each other

62. Cell clones
Cloned DNA